| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Dipartimento di Fisiopatologia Medica, Università di Roma "La Sapienza," Rome, Italy
 |
Abstract |
|---|
 Top Abstract I. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
/EGF (TNF) |
I. Introduction |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
Due to the cyclic course of spermatogenesis, any given function
of the spermatogenic cells overlaps with an earlier or later generation
to create a constant combination of cells known as cell associations or
stages. The complete sequence of stages or cell associations
constitutes one cycle of the seminiferous epithelium, whose duration
appears to be specific for each species. This continuous progression of
spermatogenic lineages causes profound reciprocal changes of the
environment to which each cell is exposed (Fig. 1
).
|
These data have reinforced the assumption that the testicular physiology is not fully accounted for by traditional endocrine paradigms. Presumably, then, there should be an intratesticular network of regulators, the exquisitely timed and highly regionalized expression of which might participate first in the development of the male gonad and later in the initiation and maintenance of testicular function. This involves intercellular, intracellular, and cellular-environmental communication rather than total reliance on intracellular programming and classic hormonal control. Thus, testicular development can be influenced by a number of variables, including physical parameters, nutrients, extracellular matrix components, cell adhesion molecules, soluble factors, and membrane junctional complexes between apposing cells. In addition to development and differentiation, which are readily evident during embryogenesis, these variables are involved in the fully developed organ in tissue maintenance, cellular renewal, and local control mechanisms. These general rules appear to be particularly important in the testis since in the seminiferous tubules, as in the hemopoietic system, a limited number of multipotent stem cells give rise to a much larger population of functionally mature cells, and thus the processes of proliferation and differentiation must be finely tuned throughout life.
In the last two decades, these considerations have produced a shift
from endocrine to paracrine research, generating a large body of
studies that have been subject to debates and attempts to fit the
available information into hypothetical models (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Among the
substances employed as symbols in the language of intercellular
communication are the polypeptidic factors. At present, we refer to the
polypeptidic factors as regulatory substances released by practically
any type of cells. They can also be referred to with a broader term
such as cytokines (from the ancient greek 
u
o
, cell,
, to stimulate). More than 50 of these substances have
been described and the majority have been cloned. Most of them have
pleiotropic effects, their actions are markedly influenced by the
context in which they operate, and some act synergistically or are
capable of inducing or inhibiting the production of other cytokines.
They form the fourth major class of soluble intercellular signaling
molecules, alongside neurotransmitters, endocrine hormones, and
autacoids. Many of these factors are produced by the various cellular
components of the testis.
This article will attempt to provide an analysis of the available information on the functional interactions between the cells of the male gonad mediated by locally produced regulatory peptides. When dealing with the literature on this topic, there are some focal points that must be kept firmly in mind. The vast majority of the results are generated from in vitro studies, and thus to extrapolate the products in vivo is to force the evidence. It should never be forgotten that in vitro we are out of context, and cellular behavior can be markedly affected by artificial experimental conditions. For example, Sertoli cells or PMC isolated from their normal environment and placed in culture look different and function differently (17, 18). Furthermore, the effect of a regulatory substance can be profoundly influenced by reciprocal interactions of the substance itself and the responding cells with the extracellular matrix and the normal cellular complement (19). Modern techniques now allow us to measure very small quantities, perhaps even below the limit of biological significance. What regulates the testicular cell components in adult life may not be the same as during pre- and early postnatal development or during puberty. Although comparable regulatory interactions exist in mammals, the relative importance of distinct local control systems may vary from species to species.
Crucially important in the identification and validation of peptide factors such as local modulators is the demonstration of local production, expression of specific receptors, and evidence for a biological action on the producing cell or on neighboring cells. The agreement of different methodological approaches gives additional strength to the information. Another important criterion that should be met to define a factor as a local regulator is that local production and action is controlled under physiological conditions or altered in pathological circumstances.
The general characteristics of the substances that will form the focus
of this review are listed in Table 1.
|
|
The advent of transgenic and gene-targeting techniques has allowed us the unique opportunity to test the functions of specific genes directly in vivo. Many of the functions of the regulatory peptides within the context of a complex organism have been elucidated through the use of such experimental models. Thus, we have reported the results generated by these powerful technologies, which may help to clarify the complex functions of the regulatory peptides in the testis.
Due to the enormous amount of information available, in some instances
contradictory and difficult to interpret, the experimental evidence has
been summarized in tabular form to provide an easily understood format,
enabling quick comparison of data. Tables 2
and 3
summarize the reports on the presence of regulatory peptides and their
receptors in the mammalian testicular tissue and in isolated cells,
including cellular source, detection systems, and modulation of
expression/production. Tables 4
and 5 provide a synopsis of the effects
of the gonadal peptides in in vitro and in vivo
models. The pattern of expression of these substances and their
receptors during development and in relation to the stages of the
seminiferous epithelium in the rat is provided in Tables 6 and 7.
Finally, Table 8 summarizes the
characteristics of transgenic mice models with recognizable effects on
male reproduction that are thought to be exerted through direct
testicular action.
|
|
|
|
|
|
|
|
|
|
|
|
|
II. Neurohormones/Neuropeptides |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
1. Expression, localization, and production.
High levels of a
GHRH-like substance in mature rat testis are present both at the level
of protein product and gene transcript with the mRNA substantially
larger than the GHRH transcript from the hypothalamus (23). The
partially purified testicular GHRH is capable of stimulating GH
secretion from cultured anterior pituitary cells in a dose-dependent
manner, and GHRH-like immunoreactivity has been localized to mature
sperm forms in rat testis (24).
Testicular GHRH mRNA and peptide are developmentally regulated. No GHRH mRNA is detected in testes from day 19 fetal rats, but it is present in low amounts on day 2 of life, increasing gradually to day 21 (25). The GHRH gene expression increases more dramatically beginning on day 21 and reaches adult levels by day 40 (25). Immunohistochemical studies have confirmed these findings in that the presence of GHRH-like immunoreactivity has been found in the interstitial cells of the testis from postnatal day 4 and the positively stained cells increase with age (26). Immunoreactive GHRH was also present in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle (26). Accordingly, GHRH mRNA has been localized in developing spermatogenic cells by in situ hybridization and Northern blot analysis (27).
Transgenic mice bearing the fusion gene encoding the promoter region of the mouse metallothionein-1 (MT-1) gene and the coding region of the human GHRH gene have been used as a model for studying the tissue-specific expression and processing of GHRH (28, 29). In the testis of these animals the GHRH gene was clearly expressed (29), and the primary site of GHRH-immunoreactive staining was the Leydig cells (28). In line with these results, authentic GHRH under positive gonadotropin control is actively released from cultured adult rat Leydig cells (30).
Interestingly, the GHRH mRNA found in the rat testis contains an exon 1 sequence different from that found in hypothalamus or placenta, and the initiation of GHRH transcription in the testis begins approximately 700 bp 5' to that in placenta and 10.7 kbp 5' to that in hypothalamus (31). Thus, the GHRH transcripts in each tissue have distinct exon 1 sequences and may use different promoters, suggesting that as yet unidentified spermatogenic-specific transcription factors may bind to the promoter region of testicular GHRH to regulate its expression (31).
Recently, a novel peptide, the putative 30-aa C-terminal peptide of the GHRH precursor, called GHRH-related peptide, has been found in abundance in adult rat germ cells by immunohistochemistry (32). Specific staining predominated in stage IV seminiferous tubules, in pachytene spermatocytes, and in the acrosomes of spermatids (32).
Moretti et al. (33) found immunoreactive GHRH-like material in human testis. They noted intense staining of the interstitial compartment with localization to the Leydig cells. The presence of GHRH-like peptides in human testicular tissue has been further analyzed by means of enzyme-linked immunosorbent assay (ELISA) and Northern blot of adult testis extracts (34). Both methodologies confirmed that the human testis is an extrahypothalamic site of expression for both immunoreactive GHRH-like peptides and GHRH gene.
2. Receptors.
It has been shown that GHRH acts on the Leydig
cells via a vasoactive intestinal peptide (VIP) receptor, directly
stimulating cAMP production (30). Recently, the GHRH receptor mRNA has
been detected in rat Sertoli cells and at lower levels in germ cells
and Leydig cells; moreover, the treatment of Sertoli cells with GHRH
has been found to increase the GHRH receptor expression in a
dose-dependent manner (35).
3. Local functions.
GHRH has direct actions on the Sertoli
cell, including stimulation of cAMP production and c-fos and
steel factor (SLF) mRNA expression (36). Accordingly, GHRH has been
found to increase basal and FSH-stimulated cAMP formation in cultured
adult and pubertal Sertoli cells with a higher potency in pubertal than
in adult cultures (26). Moreover, it has recently been shown that
GHRH-related peptide specifically activates the expression of SLF by
Sertoli cells to a higher extent than GHRH without increasing
intracellular cAMP levels, or transferrin, androgen binding protein
(ABP), or inhibin (INH) -subunit transcripts (32).
Spontaneous and transgenic mutants may help to clarify the roles of the peptide described above. Mice homozygous for the spontaneous little (lit) gene mutation are normally proportioned dwarfs (37) carrying a missense mutation of the gene encoding for the GHRH receptor (38, 39). Both male and female little mice exhibit delayed sexual maturation (37). Although fertility in little males was initially reported to be reduced (37), this has subsequently been shown to be diet related (38), and the allometric reduction of all reproductive organs with body size was not associated with impairment of steroidogenesis or spermatogenesis (40) or with fertility (41). However, direct examination of GHRH and GHRH receptor expression in the gonads of little mice is lacking and, therefore, whether or not this animal model might provide further clues toward understanding the in vivo roles of GHRH in the testis is uncertain.
On the other hand, the expression of human GHRH in transgenic mice results in elevations of serum GH and stimulation of linear growth (42). Both males and females carrying the GHRH fusion gene are fertile and transmit the gene. These data appear to exclude the severe GHRH-mediated consequences for male reproductive physiology. However, whether this lack of testicular effect in both overexpressing and nonexpressing GHRH applies also to animals other than mice is not predictable.
B. Pituitary adenylate cyclase-activating peptide (PACAP)
PACAP is a novel member of the secretin/glucagon/VIP/GHRH family
of peptides that was originally isolated from ovine hypothalamic
tissues based on its ability to stimulate the accumulation of cAMP in
rat pituitary cell culture (43). PACAP exists in two forms: a longer
form of 38-aa residues (PACAP-38) and a shorter one (PACAP-27)
corresponding to the amino-terminal 27 residues of PACAP-38 (44). Both
PACAPs are derived from a 175-aa precursor, which in addition gives
rise to a 29-aa peptide designated PACAP-related peptide (PRP) (45). To
date there is no known function for PRP.
PACAP shares binding sites with VIP in a variety of tissue types. There are three cloned receptors for PACAP, designated PVR1, PVR2, and PVR3. The PVR1 binds PACAP 100- to 1000-fold more potently than VIP and couples, through G proteins, to the activation of both adenylate cyclase and phospholipase C. PVR2 and PVR3 bind PACAP and VIP with approximately equal affinities and are coupled, probably through the G protein Gs, to the activation of adenylate cyclase. Five splice variants of the PVR1 receptor have been described. PACAP does not act as a classic hypophysiotropic factor that stimulates or inhibits anterior pituitary hormone release, but instead modulates the responses to factors such as GnRH directly or indirectly or has more general effects on gene expression or cell growth and differentiation (46).
1. Expression, localization, and production.
Immunoreactive
PACAP-38 has been found in the rat testis (47, 48) that exceeds even
the total amount of immunoreactivity in the entire brain (47). Extracts
of adult rat testes contain all the PACAP precursor-derived peptides:
PACAP-38, PACAP-27, and PRP (49). PACAP mRNA has been found near the
perimeter of the seminiferous tubules in early germ cells by in
situ hybridization (50), and immunohistochemical studies have
shown PACAP-38 staining in spermatids near the lumen at stages VII and
VIII of the seminiferous cycle, in spermatogonia and primary
spermatocytes, but not in mature spermatids or spermatozoa (49). An
unusual PACAP mRNA shorter than that reported in the rat cortex and
hypothalamus has been isolated in rat testis (51, 52). This smaller
form of PACAP mRNA is also present in human, murine, and bovine testis
(52).
Cloning of the PACAP-38 cDNAs showed the expression of the corresponding mRNAs and peptide in human testis (53).
2. Receptors.
The PACAP PVR1 receptor has been found in the
most mature stages of the adult rat germinal cells by autoradiography
(54). Although testicular cell membrane preparations showed some
specific PACAP-27 binding, the rate was too low relative to the protein
content to generate informative displacement profiles (54, 55). The
five spliced variants of PVR1 receptor are coupled differently to
adenylyl cyclase and/or phospholipase C stimulation, and the form
denominated PACAP-R hop is the predominant one in the testis
(56).
Recently, the cloning of the gene encoding the human type I VIP receptor, also called type II PACAP receptor or PVR2, has been achieved (57). The gene is selectively expressed in various human tissues including the testis (57).
3. Local functions.
PACAP stimulates cAMP accumulation in
Sertoli cells from 15-day-old rats, and this property declines with the
increasing age of the donor animals (58). This effect is additive with
submaximal, but not maximal, concentrations of FSH. Furthermore, PACAP
increases the Sertoli cell secretion of lactate, estradiol, and INH in
a concentration-dependent manner (58). The PACAP-mediated stimulation
of the Sertoli cell cAMP accumulation is not altered by a VIP
antagonist, suggesting that PACAP is acting via the PVR1 receptor on
these cells (58). In addition to the effects on Sertoli cells, PACAP
increases the synthesis of both secreted and intracellular proteins in
spermatocytes, but decreases the synthesis of both spermatid-secreted
and intracellular proteins (59).
Despite the distinct possibility of PACAP effects within the testis, direct proof of an in vivo function is still lacking.
C. GnRH
GnRH is a decapeptide synthesized in the cell bodies of
hypothalamic neurons that selectively stimulates the gonadotrope cells
of the anterior pituitary to release LH and FSH (60). The GnRH receptor
is a seven-transmembrane region protein that on activation initiates a
series of events that start with G protein-mediated stimulation of
phosphoinositide (PI) turnover followed by elevations in
[Ca2+]i and activation of protein kinase C (PKC) (61, 62).
1. Expression, localization, and production.
In 1980, Sharpe
and Fraser (63) reported the presence of a factor with GnRH-like
activity in the testicular interstitial fluid of human (h) CG-treated
adult rats (63). Later, it was demonstrated that seminiferous tubules
from the rat and the stumptailed macaque (Macaca arctoides)
and medium conditioned by cultured rat Sertoli cells contained a factor
with GnRH-like receptor-binding and biological activity (64, 65),
immunologically distinct from native GnRH (64). Paull et al.
(66) found a GnRH-like immunoreactivity in the cytoplasm and nuclei of
germ cells using an antiserum directed to the center of the molecule,
but not with antisera directed to the end of the molecule. The partial
isolation and characterization of GnRH receptor-binding activity from
adult rat testis acetic acid extracts revealed the presence of two
factors chemically distinct from the native decapeptide, with
approximate molecular weights of 68,000 and 6,000, respectively (67).
This partially purified material led to a dose-dependent inhibition of
LH-stimulated testosterone production in a mixed Sertoli-Leydig cell
monolayer culture similar to that seen with synthetic GnRH (68). Low
concentrations of GnRH-like factors have been found in adult rat testis
extracts by RIA (69, 70). This low concentration has been partially
attributed to the presence of a testicular GnRH-peptidase associated
with the Sertoli cells, one of the putative sources of rat testicular
GnRH. GnRH-peptidase content has been found to be much higher in adult
testis than in immature testis (71).
Recently, GnRH mRNA has been found at a specific, although not specified, stage of spermatogenesis within the seminiferous tubules of both mature rat and adult human testes (72). In the rat, the expression of the GnRH mRNA was identified in Sertoli cells and spermatogenic cells of some seminiferous tubules. In humans, the GnRH mRNA was localized only in some spermatogenic cells in some seminiferous tubules, suggesting that the mRNA was expressed at specific stages of tubular development (72).
2. Receptors.
GnRH-specific low-affinity binding sites were
originally identified in testicular membrane preparations from adult
rats by Marshall and collaborators (73, 74). Subsequent in
vitro studies demonstrated high-affinity receptors located in the
interstitial cells of the adult rat testis (75, 76, 77, 78). In keeping with
these results, quantitative autoradiography confirmed the presence of
GnRH binding sites distributed on adult rat interstitial cells (79).
Moreover, GnRH-binding sites have been found in the testis of the frog
Rana esculenta (80).
The ontogeny of the GnRH receptors in the rat testis has been described. GnRH receptors were not detectable in homogenates of acutely excised 20.5-day fetal testes or in freshly prepared fetal Leydig cells, but were clearly present starting from 3 days of culture (81). These receptors were also readily detectable postnatally in the testes of 5-day-old rats and increased markedly during maturation (81, 82).
After Leydig cell binding, GnRH stimulates the inositol lipid metabolism, which triggers a cascading mechanism that ultimately results in the generation of increased cytosolic free calcium levels, enhanced PKC activity, and liberation of arachidonic acid (62, 83, 84).
In contrast to what was observed in the rat, GnRH receptors have not been found in adult human (85) or mouse testes (86, 87), leading to the assumption of a species-specific expression in the male gonad.
However, the picture has been further complicated by the recent immunohistochemical finding of GnRH receptors in adult human Leydig cells (72).
3. Local functions.
The first hypotheses of a direct effect of
GnRH and its agonists on testicular physiology came from the
observation that the in vivo administration of
pharmacological doses of GnRH exerted paradoxical inhibitory effects on
male reproductive functions in immature and adult hypophysectomized
rats (88, 89, 90, 91) and inhibited the in vitro gonadotropin
stimulation of androgen production by cultured testicular cells (75, 92, 93, 94). GnRH agonists inhibited LH-dependent steroid production and
abolished the acute testosterone response to hCG in cultured testicular
cells from fetal and neonatal rats (81, 95, 96). This inhibitory effect
of GnRH on testicular basal and LH-stimulated testosterone secretion
has also been confirmed by in vivo experiments in the rat
fetus (97). However, different experimental models gave contrasting
results.
Short-term incubations of adult rat Leydig cells with GnRH resulted in increased phospholipid turnover, prostaglandin E and testosterone production (86, 98, 99, 100), and activation of the cytochrome P450 enzyme (101), whereas chronic exposure decreased the response to hCG (102).
In short-term incubations the GnRH analog buserelin had a direct positive effect on testicular testosterone production by R. esculenta minced testes (103) and decapsulated rat testes between 1 and 60 days of life (82). A number of studies have been conducted on different frog species demonstrating that GnRH-like material acts directly on testes promoting androgen production (104, 105, 106, 107) and primary spermatogonial mitosis (106, 108, 109).
In an elegant experiment, Huhtaniemi et al. (110) examined the functions of the GnRH receptors in the adult and immature rat testis, blocking the receptors by a 7-day in situ infusion of a potent GnRH antagonist. The infusion of the antagonist resulted in a dose-dependent decrease of the testicular GnRH receptors up to 90%; the circulating levels of gonadotropins, PRL, and testosterone were unaffected, but there was a subtle yet significant decrease (16–32%) in the testicular content of testosterone, and of LH, FSH, and lactogen receptors (110). GnRH agonists have been shown to stimulate testicular blood flow in hypophysectomized rats, an effect that is mediated via the Leydig cells and is presumed to reflect one of the actions of testicular GnRH (4, 111).
Initial studies investigating the factors regulating the GnRH receptor expression in the testis indicated that the in vivo administration of GnRH induced an increase in the number of its own receptors in both intact and hypophysectomized rats (77, 112). Furthermore, it has been demonstrated that the administration of LH to hypophysectomized rats prevents the posthypophysectomy increase of GnRH receptors, as well as reducing the high levels of receptors in previously hypophysectomized animals (113). Experimental adult rat unilateral cryptorchidism induces a significant reduction in the number of receptors for LH, FSH, and PRL, but the number of GnRH receptors is unaffected (114).
Studies conducted in humans showed that the administration of a potent
GnRH agonist for 6 days did not inhibit the hCG-induced increase in
plasma testosterone levels or the testicular steroidogenic pathway in
patients with gonadotropin deficiency (115). The chronic treatment of
elderly men with disseminated prostatic cancer with a GnRH agonist
resulted in inhibition of both the 
4 and
5 pathways, with a subsequent decrease in the
intratesticular testosterone concentration (116, 117). The ability of
exogenous hCG to reverse both the reduction in 4 and
5 intratesticular steroid content and the
intratesticular enzyme activities induced by the GnRH analog suggests
that GnRH does not have a direct inhibitory effect on testicular
testosterone biosynthesis in man (116, 117). In support of this
evidence, no effect has been found with high concentrations of a GnRH
agonist on steroid conversion in human testicular tissue in
vitro (118). The lack of local effects of GnRH in human testis has
been confirmed in other primates (119, 120) as well as in other species
(87, 121).
Genetically hypogonadal mice (hpg/hpg), homozygous for a deletion mutation in the gene encoding GnRH and GnRH-associated peptide (122), cannot synthesize or release hypothalamic GnRH and GnRH-associated peptide. This accounts for diminished production of gonadotropins, which is responsible for the arrested development of their gonads and sterility. Thus, these endogenous mutants cannot help to define the importance of GnRH in testicular function, considering also the reported lack of expression of the GnRH receptors in the mouse testis (86, 87).
Based on present evidence, the physiological significance of testicular GnRH-like peptides and of the direct gonadal actions of GnRH and its agonists is as yet unresolved. The data reported above indicate that the local effects of testicular GnRH are subtle and species-specific.
D. CRH
CRH, a 41-aa peptide, is the key hormone controlling
hypothalamic-pituitary-adrenal function. It now appears that the
actions of CRH go beyond its role as a hypothalamic releasing factor.
Through actions in the brain and in the periphery, CRH coordinates the
endocrine, autonomic, behavioral, and immune responses to stress (123).
The CRH receptor is a glycoprotein that belongs to the family of the
seven-transmembrane G protein-coupled receptors, with a higher
molecular weight in peripheral tissues and a lower molecular weight in
the brain (124, 125).
1. Expression, localization, and production.
CRH mRNA is
present in the testis at levels comparable to those found in the
midbrain (126) and is most abundantly distributed in the testis of
MT-CRH transgenic mice (127). Immunoreactive CRH has been found to be
present in the testis of rat, guinea pig, sheep, and man (128, 129) and
localized in the Leydig cells, germ cells, and epididymal sperm (130, 131). In the rat, testicular CRH concentrations fluctuated with age,
showing high levels at 10 days of age, a marked reduction at 20 days, a
significant increase at 60 days, and maximal levels at 90 days (130).
The increase of immunoreactive CRH concentrations at the time of full
spermatogenic activity and sperm production led to the suggestion that,
in adult life, most testicular CRH is localized in germinal cells
(130). However, recent immunohistochemical studies indicate that in
adult rat testis, CRH is mostly localized to interstitial cells, and
that purified Leydig cells show consistent cytoplasmatic immunostaining
for CRH (131). In adult human testis, CRH immunostaining has also been
shown to be mainly localized in the interstitial compartment (132). CRH
was isolated and characterized from testicular extracts and found to
be, apart from a microheterogeneity at position 39, identical in amino
acid sequence to the hypothalamic peptide (128).
Adult rat Leydig cells in culture release a consistent amount of CRH (132). LH/hCG, cAMP, and 5-hydroxytryptamine (5HT) are potent acute stimuli for its production (132, 133). Furthermore, the 5HT2 receptor subtype is found in the Leydig cells and mediates the serotonin stimulation of CRH secretion (133). Extrapolating from other tissues, it is conceivable that the mechanism of action of 5HT on binding to the 5HT2 receptor in Leydig cells involves the activation of PI hydrolysis and stimulation of PKC (133), an intracellular messenger system that appears to mediate the stimulation of CRH release in Leydig cells (132). Studies from the same group showed that 1) serotonin is a more effective stimulus than hCG in stimulating CRH secretion, and 2) gonadotropin-induced CRH release is inhibited by the 5HT2 receptor antagonist ketanserin, indicating that stimulation of CRH by hCG results from the action of endogenously released serotonin (133).
In vivo studies have shown that acute immobilization stress is a stimulus for testicular CRH mRNA expression and transcription into CRH protein (134). This model of stress was also characterized by a marked reduction (80%) in testosterone serum concentration but no change in LH serum levels. Therefore, the involvement of testicular CRH in stress-induced testosterone inhibition was suggested.
2. Receptors.
CRH-binding sites have been demonstrated in
whole testis (135) and isolated adult rat Leydig cells (136).
Adrenalectomy of adult rats induced, after 14 days, an increase of CRH
binding in testis membranes by approximately 215% above sham-operated
controls, suggesting that glucocorticoids may be a regulator of
peripheral CRH receptors (137). Scatchard analysis of CRH binding data
on intact cells and purified Leydig cell membranes revealed a single
class of high-affinity binding sites with a dissociation constant
(Kd) of 0.1 nM, and showed that CRH receptors
are present in low abundance [500/800 per cell vs. 20,000
for LH/hCG and 2,000 for angiotensin II (AT-II)] (136). Subsequent
studies in rat Leydig cells have shown that, in contrast to
corticotropes, CRH receptors interact with a G protein different from
Gs, which is linked to phospholipase C, possibly Gq, G11, or an isoform
not yet described (138).
Recently, two types of hCRH-receptor cDNAs were identified (type I and type II hCRH-R) (125). hCRH-RII is identical to type I except that it contains a 29-aa insert in its first cytoplasmic loop, suggesting that hCRH-RI and hCRH-RII result from alternative splicing of a single gene. Studies on the signaling properties of the two receptors showed that hCRH-RI is coupled to stimulation of cAMP accumulation and PI hydrolysis. In contrast, hCRH-RII is deficient in signaling through both effectors, especially PI turnover in COS-7 cells (139). Since the CRH receptor in rat Leydig cells is mainly coupled to PKC activation, it can be excluded as an CRH-RII subtype. Interestingly, an additional CRH receptor has been identified, which was shown to be expressed at high levels in the heart and at low levels in the brain and lungs, and found to be significantly different (30%) from that of the pituitary gland (140). Functional studies have demonstrated that this "peripheral" CRH-receptor recognizes CRH and the CRH-related amphibian peptide sauvagine and is coupled with Gs and adenylate cyclase (140). Thus, it is conceivable that, in addition to the pituitary type, there might be distinct peripheral CRH receptor subtypes capable of coupling with different intracellular signaling pathways (i.e., adenylate cyclase and/or phospholipase C).
PCR analysis of human tissues revealed CRH receptor transcripts in the brain, pituitary gland, and testis (141).
3. Local functions.
CRH receptors in rat Leydig cells are
coupled to stimulatory actions on ß-endorphin (ßEND) production
(142) and inhibition of LH-induced steroidogenesis (132, 136, 138).
In vitro studies showed that CRH acts rapidly (in minutes)
in the fetal and adult rat Leydig cell to exert highly effective
negative autoregulation of the Leydig cell steroidogenic response to
the LH stimulus (132, 136). Similarly, intracellular and extracellular
cAMP production stimulated by gonadotropin were significantly reduced
by CRH treatment of rat Leydig cells (136, 138). Studies performed in
both purified mouse Leydig cells and in a mouse cell line derived from
a Leydig cell tumor (MA-10 cells) led to different results, showing
that CRH had a stimulating effect on cAMP accumulation and steroid
production (143). In the same studies, experiments performed on
partially purified rat Leydig cells (60–80% Leydig cells) showed that
CRH had no effect on basal and hCG-stimulated steroidogenesis. These
results indicate that mouse Leydig cells respond differentially from
rat Leydig cells to CRH, suggesting that CRH action in the male gonad
is species-specific. Mice and rats might have different forms of CRH
receptors on Leydig cells, which couple to different signal pathways
and have opposite actions on steroidogenesis. This is consistent with
the large number of differences between mouse and rat Leydig cells
found by others (143). In highly purified rat Leydig cells (90–95%
pure), it has been found that the inhibition of hCG-induced
steroidogenesis by CRH was maximal (150–200% reduction) at the
earliest incubation times (30–60 min) and much less evident at later
time points (60–120 min, 20–40% reduction) (136). The marked
reduction of CRH effects on hCG-induced steroidogenesis in rat Leydig
cells after prolonged incubation was related to the temporal
degradation of the peptide in culture, which was complete after 180 min
(136). These observations might explain the above reported
inconsistency of CRH inhibition on hCG-induced steroidogenesis observed
in rat Leydig cells (143). These latter studies were performed in
partially purified Leydig cell populations, which can be contaminated
by high CRH-degrading activity, and CRH actions were assessed after
incubation for only 2 h, a time point at which CRH inhibition has
been reported to be minimal (136). Taken together, these results
strengthen the concept that extreme caution must be used in examining
the actions of a peptide in the testis based solely upon in
vitro methodologies.
To further complicate the picture, in vivo studies performed in neonatal (5-day-old) rats showed that the intratesticular injection of an anti-CRH-antiserum led to a significant decrease in serum testosterone levels (144). These findings indicate that in the neonatal period in the rat, testicular CRH might be a local stimulator of steroidogenesis. A developmental regulation of CRH action in the testis is also indicated by the finding that intratesticular injection of CRH in vivo causes a significant increase of ßEND secretion in interstitial testicular fluid in pubertal but not adult rats (145). In adult unrestrained intact male rhesus macaques, a 4-h infusion of CRH caused a prompt decrease in testosterone levels without significant changes in LH levels (146), leading to the suggestion that CRH may directly inhibit testosterone production by Leydig cells.
Both CRH deficient and transgenic mice have been generated (147, 127). CRH-deficient mice require glucocorticoid for lung maturation during fetal life (147). Despite marked glucocorticoid deficiency, these animals exhibit normal postnatal growth, fertility and longevity, suggesting that the major role of glucocorticoid is restricted to the prenatal period, and that the lack of CRH does not impair reproductive potential.
As pointed out earlier, analysis of CRH mRNA distribution in transgenic mice has revealed that transgene expression is primarily detected in all the classic expression sites of endogenous CRH and in the testis (127). Mapping of CRH within the testis by in situ hybridization revealed hybridization signal over seminiferous tubules and in an interstitial pattern consistent with Leydig cell expression. The CRH-expressing transgenic mice were developed using the mouse MT-1 promoter fused to the rat CRH gene. Therefore, the tissue distribution of rat CRH in these animals may more closely resemble MT-1 expression than CRH, and for this reason the CRH mRNA distribution does not necessarily represent the normal tissue-specific expression of CRH. The transgenic mice developed a Cushing-like syndrome; male animals bred successfully.
In conclusion, CRH actions in the testis may vary depending upon the species and the period of life examined; it becomes apparent that, at least in adult male rats and rhesus macaques, CRH may have direct inhibitory effects on steroidogenesis and locally mediate the detrimental effect of stress on testicular function. However, further studies are needed to verify whether the testicular effects of CRH observed in some animal species in vitro have significant pathophysiological consequences in vivo.
E. Oxytocin (OT)
OT is a nonapeptide involved in parturition and lactation that is
synthesized in the hypothalamus and secreted by the posterior pituitary
(148). The OT gene consists of three exons encoding a preprohormone
that is processed in several mature peptides, including the nonapeptide
hormone (149, 150). Exon I encodes a signal peptide, the OT hormone,
and the N terminus of a carrier molecule, neurophysin-I. Exon II
encodes the bulk of neurophysin-I, and exon III encodes the C terminus
of this molecule. A single class of OT receptors has been characterized
that shows a structure with seven-transmembrane domains typical of the
G protein-coupled receptors (151).
1. Expression, localization, and production.
Immunoreactive OT
was first identified in the human and rat testis in 1984 (152). Since
then, OT immunoreactivity has been found in testes of other mammals
(153, 154) and birds (155). Immunohistochemical studies have shown OT
immunoreactivity in the interstitial tissue, probably the Leydig cells,
of rat and dog testes (156, 157). The depletion of the Leydig cell
population in the adult rat by the drug ethan-1,2-dimethanesulfonate
(EDS) causes a reduction in the levels of OT immunoreactivity in
testicular extracts to undetectable levels by RIA, confirming the
Leydig cell origin of OT in the rat gonad (158). Accordingly,
immunocytochemical studies have revealed OT localization in purified
rat Leydig cells (159). Cultured Leydig cells from adult rats release
significantly more OT into the medium over a 3-day period than was
present in the cells at the beginning of the experiment (160).
Furthermore, the production of OT by these cells is significantly
reduced by treatment with the protein synthesis inhibitor
cycloheximide, providing further evidence for a Leydig cell production
of OT (160). An OT-like peptide is also secreted by purified guinea pig
Leydig cells in culture (161). Interestingly, in the hypogonadal mouse
(hpg/hpg), no OT can be found in the testis, but the
treatment with LH or with testosterone causes the appearance of
testicular OT (162). Using HPLC and specific RIA, authentic OT has been
identified in the testis of the Australian marsupial bandicoot
(Isoodon macrourus) but not in the possum (Trichosurus
vulpecula) testis (163).
OT gene transcripts are not detectable by Northern hybridization of rat testicular extracts, but the authentic hypothalamic-type mRNA can be detected using highly sensitive PCR analysis (164). Normal cattle have relatively high levels of testicular OT mRNA (155). In situ hybridization in bovine testicular tissue sections localized OT transcripts to the seminiferous tubules. It is thought that its expression is localized in Sertoli cells. The same testicular distribution of bovine OT RNAs was also shown by in situ hybridization in a transgenic mouse bearing a bovine OT transgene but not in the normal wild type mice (165, 166). Bovine and sheep testis contain moderate levels of an OT gene transcript as revealed by Northern blot analysis (167). In situ hybridization localized this mRNA within the seminiferous tubules, possibly in the Sertoli cells. Conflicting with this result, in the same study, immunohistochemistry analysis showed that both OT and the syngenic neurophisin I epitopes were clearly restricted to the Leydig cells, being expressed here at low levels. It has been suggested that the absence in the OT protein within the tubules is probably due to a lesion of OT gene expression at the posttranscriptional level, whereas, the low level peptide expression in the Leydig cells can be attributed to the presence of functional transcripts in these cells, which are below the level of significant detection for the in situ hybridization assay (167).
In humans, evidence for OT gene transcription in the testis was found in three of five experiments only by using a highly sensitive assay, based on a modification of the PCR, sufficient to detect one mRNA molecule/cell (168).
The effect of increasing doses of LH (0.001–100 ng/ml) on OT production from highly purified adult rat Leydig cells in culture has been tested (160). Maximal secretion of OT occurred with 0.1 ng/ml LH. Since there is a prolonged delay in the peak rate of OT production relative to the testosterone peak, it has been postulated that OT production could be indirectly regulated by LH through an intermediate factor, probably testosterone. This hypothesis is in line with the reported observation that testosterone alone stimulates high levels of testicular OT in the absence of LH in hpg/hpg hypogonadal mice (162).
2. Receptors.
A high density of OT receptors has been found in
tunica albuginea and the epididymis of prepubertal pigs (169).
Subsequent studies revealed the presence of OT receptors in the adult
rat testis with ligand-binding characteristics similar to mammary and
uterine OT receptors and an autoradiographic localization consistent
with binding to Leydig cells (153, 170, 171).
The apparently straightforward action of OT on the contractility of the seminiferous tubules and the claim of a partial characterization of a tubular receptor (172) contrast with the inability to demonstrate functional OT receptors in rat testicular myoid cells at any stage of development (172, 173).
3. Local functions.
The contractility of the seminiferous
tubules is enhanced by OT (172, 174). This effect is confirmed by the
observation that the tubules from testes in which immunoreactive OT
could not be detected are always quiescent (158, 162).
There are conflicting reports on the influence of OT on steroidogenesis. Adashi and Hsueh (175, 176) showed that OT inhibits the gonadotropin-stimulated androgen biosynthesis in isolated rat Leydig cells through testicular recognition sites similar to those mediating the pressor actions of the neurohypophysial hormones. This was supported by Nicholson et al. (177, 178), who used continual-release OT implants in vivo and the perfused whole-testis model, and Kwan and Gower (179), who reported that OT completely inhibited androgen biosynthesis in incubated microsomal fractions from rat testis. However, Tahri-Joutei and Pointis (180) found that OT stimulated testosterone production by purified murine Leydig cells with an effectiveness more pronounced at puberty, while Sharpe and Cooper (181) found OT to have no effect on testosterone production either in vitro or in vivo in the rat. Using short-term cultures of isolated rat Leydig cells, OT significantly increased basal testosterone production in a dose-dependent manner without affecting LH-stimulated testosterone production (182).
It has been shown recently that transgenic mice, which overexpress OT in the testis, have a 50% decrease in the levels of intratesticular testosterone and dihydrotestosterone (DHT) without detectable effects on testicular morphology, sperm production, or fertility parameters (166). While the results with the transgenic mice seem to be consistent with the in vitro and in vivo results in the rat (175, 176, 177, 178, 179), they contradict the in vitro experiments in the mouse (180). At this point the most reasonable explanation is that unlike the in vitro studies, transgenic models cannot distinguish between direct or indirect effects on Leydig cells and experience different exposure time compared with in vitro experiments (long-term vs. short-term). It is interesting to note that destruction of Leydig cells from adult rat testis using EDS, which results in the loss of OT (158), has very little impact on spermatogenesis, provided that high intratesticular levels of testosterone are maintained by exogenous administration (183). However, what the authors of the latter study observed was a 2- to 3-fold increase in the number of degenerating meiotic spermatocytes at stages XIV-I of the seminiferous cycle, and this was hypothesized to be related to the absence of OT, since active immunization of adult rats against OT caused a similar change.
Taking into account the possible differences between species, the important consistent finding must be that OT modulates androgen levels, possibly via direct action on the Leydig cells themselves, and regulates the extent of germ cell degeneration in the final stages of meiosis without severe physiological consequences to male fertility.
F. Arginine vasopressin (AVP)
AVP is principally an antidiuretic hormone. The substrate for
production of the 9-aa AVP is a 164-aa precursor molecule. This
molecule consists of a signal peptide, AVP itself, a specific
neurophysin, and a glycosylated moiety. After synthesis in neurons of
the hypothalamus, AVP migrates along neuronal axons into the posterior
pituitary.
Two classes of cell surface AVP receptors, which are seven-membrane-spanning G-protein-coupled, mediate the actions of AVP. The V1 receptor occupancy induces an increase in PI hydrolysis and cytosol-free calcium. A subtype V1a receptor is found in the anterior pituitary. The V2 receptor, which is expressed only in the kidney, is coupled through the G-stimulatory protein to adenylate cyclase, cAMP, and a cAMP-dependent protein kinase (184).
1. Expression, localization, and production.
Adult rat and pig
testes contain an immunoreactive AVP-like peptide that behaves like
authentic AVP by chromatographic criteria (185, 186). AVP-like peptides
have also been identified in the testis of the adult homozygous
Brattleboro rat, a genetic mutant deficient in hypothalamic, pituitary,
and circulating AVP, suggesting that the production of hypothalamic and
peripheral AVP may involve different biosynthetic pathways (187, 188).
Immunoreactive AVP has been detected in the interstitial fluid of adult
rat testis, and the disruption of spermatogenesis was associated with a
decrease in AVP concentration (189).
The detection by Northern blotting of a mRNA in the rat testis, which was considerably shorter than that in the hypothalamus and which hybridized to a specific AVP probe, was initially reported by Ivell et al. (190). Structural analysis revealed that while exons II and III of the testicular RNAs are identical to those of the hypothalamic mRNA, the hypothalamic exon I, which encodes the AVP nonapeptide, is not represented, suggesting major structural differences between hypothalamic and testicular AVP gene-related mRNAs (164, 191). No function could be ascribed to these testicular AVP-like RNAs because of the lack of open reading frames and the apparent lack of association with translationally active polysomes (164). Although a developmental analysis of the transcripts showed that their detection correlated with spermatogenesis and the appearance of dividing germ cells, no other physiological manipulation was able to influence the levels of these aberrant transcripts (164). Subsequently, a testis-specific promoter for the rat AVP gene has been described, and an in vitro synthesized RNA corresponding to the longer testicular AVP gene-derived transcript was not able to act as a template for protein synthesis (192). Again, the aberrant testicular AVP gene-derived RNAs expression was closely associated with the integrity of germ cells and ongoing spermatogenesis (193). Accordingly, transgenic mice overexpressing the rat hypothalamic AVP gene were shown to have tissue-specific mRNA expression in the hypothalamus, temporal lobe, parietal cerebral cortex, cerebellum, posterior pituitary, pancreas, and lung, similar to the tissue distribution of endogenous and ectopic mouse and rat AVP expression but not in the testis (194).
Nevertheless, by applying the great sensitivity of the PCR, hypothalamic-like AVP mRNAs could be detected in rat testis beginning around late puberty (164) as well as in mouse Leydig cells and rat and mouse Leydig tumor cell lines where they are probably translated to give authentic AVP (195). Both normal and aberrant AVP gene transcripts could not be detected in human and baboon testis by PCR (168).
As a whole, these observations indicate that expression of functional AVP transcripts in the testis depends on the animal species (they are present in rat and mouse but not in primates) and are detectable only with methods more sensitive than Northern hybridization; these transcripts can be responsible for the AVP-like immunoreactive peptides found in the testis; the process of AVP gene expression differs significantly between neuronal and testicular tissues and involves differential splicing of the known AVP gene; the function of the aberrant testicular AVP gene-derived RNAs that are unable to encode the corresponding peptides is unknown.
2. Receptors.
Specific, high-affinity, low-capacity binding
sites for AVP of the V1 subtype have been identified in the Leydig
cells of adult rat testis (196, 197), in testicular interstitial cell
preparations from Brattleboro rats (188), and in the tunica albuginea
of porcine testis (169). AVP-binding sites were found in Leydig cells
from prepubertal, pubertal, and adult mice, with no marked differences
in the affinity and a 50% decrease in receptor number in the pubertal
period (180). AVP induces phospholipase C stimulation in enriched adult
rat Leydig cell preparation (198). The autoradiographic localization of
V1 receptors in the adult rat testis showed a binding to small
arteries, the seminiferous tubule epithelial surface, and in a
reticular interstitial pattern between seminiferous tubules consistent
with binding to Leydig cells (199). In rats, the testicular AVP
receptor concentration declines after hypophysectomy, and the treatment
of hypophysectomized animals with LH or GH restores the receptor levels
(200). Rat cultured PMC express a functional AVP receptor
pharmacologically and structurally identical to the V1a subtype under
developmental control, with no evidence of expression on cells prepared
before puberty and postpubertal appearance (173).
3. Local functions.
AVP produces a dose-dependent inhibition
of gonadotropin-stimulated androgen biosynthesis in cultured testicular
cells mediated by specific testicular recognition sites similar to
those mediating the pressor actions of AVP but distinct from those
involved in the antidiuretic effect (175, 176). Subsequent studies have
confirmed that long-term treatment with AVP inhibits LH-induced
testosterone production by Leydig cells in vitro (180, 182, 183, 201, 202, 203), and an in vivo experiment confirmed the
antigonadal activity of AVP (204). These effects have been accounted
for by a decreased testicular LH- binding capacity (204) and reduced
17-hydroxylase activity induced by AVP (201, 203).
In contrast, after acute treatment, AVP induces a stimulatory effect on the steroidogenic activity of mouse Leydig cells, which is more pronounced at puberty compared with prepubertal and adult periods (180). The intratesticular injection of low doses of AVP in the adult rat in vivo caused a dose-dependent decrease in total testicular blood flow, without any major effect on vasomotion, interstitial fluid volume, and testosterone production (205). The lack of an important in vivo role for AVP in the control of Leydig cell function was suggested by the reported normal plasma LH and testosterone levels as well as testicular testosterone production capacity in the AVP-deficient Brattleboro rats (206). However, the subsequent demonstration of AVP-like peptides in the testis of Brattleboro rats (188, 189) has reopened the quest for definitive proof of the influence of AVP or AVP-like immunoreactive compounds in the testis.
To summarize, further studies are needed before one can draw any conclusion about the physiological relevance of AVP in the male gonad.
G. TRH
TRH is the key regulator of the synthesis and secretion of TSH in
animals and humans and plays additional roles as a
neurotransmitter/neuromodulator in the central nervous system (CNS)
(207). Extrahypothalamic loci of function for TRH have also been
demonstrated (208). TRH acts on the target cells through a membrane
receptor which is a member of the seven-transmembrane region,
G-protein-coupled receptor family (209).
1. Expression, localization, and production.
Relatively high
levels of TRH and its immediate precursor TRH-Gly have been found in
the testis of sexually mature rats and dogs (210, 211, 212, 213), and mRNA for
pre-proTRH (ppTRH) has been identified in the rat testis (214).
Developmental studies on testicular ppTRH mRNA expression showed no expression at the earliest stages of postnatal development (day 8), while hybridization signals were found on day 20 and increased progressively up to day 70. TRH peptide concentrations measured by RIA at the same developmental periods paralleled the ppTRH mRNA expression. ppTRH mRNA and TRH peptide were colocalized to Leydig cells by Northern blot analysis and immunohistochemistry of enriched testicular cell elutriates, respectively (214). Subsequent studies have confirmed, through the use of EDS treatment of adult rats, that the Leydig cells are the only source of authentic TRH and TRH-like peptides in the rat testis (215).
Interestingly, the TRH-potentiating peptide (Ps4), which is a connecting peptide that links two copies of the TRH progenitor sequence and has the same distribution pattern of TRH in the CNS, is present in very low concentrations in peripheral tissues except in the testis where it is expressed in large amounts (216).
Methimazole-induced hypothyroidism has been found to increase the concentration of immunoreactive TRH and TRH precursor in the rat testis (212); however, others have reported that testicular mRNA concentrations are completely unaffected by experimental alterations in thyroid status (208).
In humans, the cloning and characterization of the ppTRH gene has led to the recognition of ppTRH gene expression in the testis (217).
2. Receptors.
The in vivo binding of a hybrid
protein consisting of TRH linked to a fragment of diphtheria toxin that
specifically binds to TRH receptor showed a displaceable binding in the
testis of adult rats (218). Specific binding sites for TRH and PS4, and
the mRNA for TRH receptor, have been detected in adult rat testis (216, 219, 220) and purified rat Leydig cells (219).
3. Local functions.
TRH can partially inhibit the
LH/hCG-induced testosterone secretion from rat Leydig cells in
vitro (208, 217).
Although the above data suggest a potential autocrine role for TRH in the regulation of Leydig cell function, an in vivo effect of this peptide in the testis remains to be demonstrated.
H. Somatostatin (SRIF)
SRIF is the hypothalamic 14-aa cyclic peptide that together with
GHRH regulates the GH release from the pituitary. In addition to the
pituitary, this peptide is present and plays an inhibitory role in the
normal regulation of three organ systems: the CNS and the hypothalamus,
the gastrointestinal tract, and the exocrine and endocrine pancreas
(221). A family of SRIF receptors that may mediate the distinct
biological effects of SRIF has recently been cloned. The different
cloned SRIF receptor subtypes have been designated SSTR1, 2, 3, 4, and
5 based upon the order in which they were isolated. The SRIF receptors
are membrane-bound receptors coupled to pertussis-toxin-sensitive G
proteins (222, 223).
1. Expression, localization, and production.
SRIF-like
immunoreactivity has been detected in rat (224) and human (225) testis
but not in boar testis (226). In rat testis, the levels of SRIF-like
immunoreactivity decreased after hypophysectomy (224). However, no
testicular evidence of pre-pro-SRIF gene expression could be detected
in the adult rat (227).
2. Receptors.
RT-PCR used to characterize the distribution of
mRNA encoding the SRIF receptor SSTR5 in human tissues failed to detect
mRNA expression in fetal or adult testis (228).
The paucity of data, together with the absence of studies on the local functions of SRIF, do not allow us to hazard any conjecture on the effects of this peptide within the testis.
I. Opioids
Three families of endogenous opioid peptides are recognized.
ßEND, enkephalins (ENK), and dynorphins (DYN) are the defined
peptides with morphine-like activity. The endogenous opioid peptides
are widely distributed in the brain and peripheral nervous system and
play important roles in modulating endocrine, cardiovascular,
gastrointestinal, and immune functions. Each family is derived from a
distinct precursor polypeptide. These precursors are called
pro-enkephalin (pENK), POMC, and prodynorphin (pDYN) (229). The POMC
precursor polypeptide yields the opioid ßEND, and the nonopioid
peptides, ACTH and -MSH. pENK contains six copies of Met-ENK and a
single copy of Leu-ENK. The third opioid precursor, pDYN, yields DYN-A,
DYN-B, and - and ß-neoendorphin. Three types of opioid receptors,
termed 
, , and µ, which differ in their affinity for the opioid
ligands and their distribution in the nervous system have been
characterized (230). The - and µ-receptors bind ENK and END, and
the -receptors potently bind DYN. These receptors are members of the
superfamily of the seven-transmembrane-spanning receptors and share a
high degree of amino acid sequence similarity with approximately 50%
of the residues being identical. The opioid receptors are coupled to
adenylyl cyclase and inhibit the formation of cAMP through pertussis
toxin-sensitive GTP-binding regulatory proteins (230).
1. ßEND
a. Expression, localization, and production.
The expression of
testicular POMC mRNA has initially been localized in the cytoplasm of
most Leydig cells of adult rat testes by in situ
hybridization (231). Subsequent studies showed that POMC mRNA is
expressed in purified preparations of Leydig cells and in interstitial
macrophages of the adult rat testis (232, 233, 234). Further in
situ hybridization studies in the mouse demonstrated that POMC
mRNA is most abundant in a subpopulation of somatic Leydig cells that
are found in the interstitial regions associated with discrete tubule
stages (IX-XII) of the cycling seminiferous epithelium (235),
indicating that the expression of POMC mRNA by Leydig cells is
influenced by spermatogenic cells (236). In the mouse and hamster, RNA
gel-blot experiments showed that the gene for POMC is expressed also by
germ cells and, in particular, by pachytene spermatocytes (237). The
size of POMC mRNA transcripts detected by Northern blot analysis in the
somatic and germ cells of the testis is 400 nucleotides shorter than
that of the pituitary gland, i.e., 800 vs. 1200
nucleotides (237, 238, 239). The short testicular POMC mRNA lacks exons 1
and 2 and cannot serve as a template for the POMC signal peptide
(entirely coded by exon 2) (238, 240), which is necessary in pituitary
cells for the processing of POMC during precursor migration from the
rough endoplasmic reticulum to the secretory granules via the lamellar
Golgi complex (241). Thus it is unlikely that the POMC-derived peptide
ßEND found in the testis derives from the small POMC transcript.
Subsequent studies performed with the S1 nuclease mapping technique
have shown that a very small (<1%) but definite amount of the normal
(1200 nucleotides) POMC transcript was present in rat and human testis
(238, 240, 242), and in purified Leydig cells and Leydig cell lines in
rodents (243). The low level of expression of the translatable POMC
transcript is consistent with the low production of ßEND in the
testis. This is also supported by the observation that an increased
expression of the pituitary-size POMC mRNA in human Leydig cell tumors
was associated with a dramatic increase (1,000-fold) in ßEND
concentrations compared with normal testis (244).
Immunohistochemical studies showed that ßEND is confined to the
interstitial cells of rodent (245) and human testis (246) and is
present in the interstitial cells, canaliculi of the efferent system,
spermatogonia, and spermatocytes in the frog, Rana esculenta
(247). Acetylated END forms (N-acetyl-ßEND, N-acetyl-END, and
N-acetyl-
END 1–27) were also found to be present in the rat testis
and exclusively localized to spermatogonia and primary spermatocytes
(248). END-generating endopeptidase activity has been shown to be
abundant in the rat testis and to be mainly associated with the
germinal cell fraction of the tubules (249); thus, this enzyme can be
operative in processing ßEND into - and END inside the rat
spermatogenic cells.
Studies in mice and rats showed that ßEND in the testis is developmentally regulated, with peaks at birth and after puberty (250, 251). In the mouse, at day 16 of fetal life, 50% of interstitial testicular cells stained positively for ßEND; the number dropped to 12% by day 5 of age, increased again at midpuberty, and reached a maximum in adult age (100%) (250). An analogous developmental pattern has been shown in the rat (251). Total testicular ßEND levels were very low and barely detectable from 5–20 days of age, rose sharply in parallel with testis weight from 20–60 days, and then remained unchanged through 150 days of age. In these latter studies it was shown that most of ßEND from prepubertal testes chromatographed like authentic ßEND, while with the onset of puberty and in adult life much of the total ßEND chromatographed like its precursor ß-lipotropin (251).
In fetal and adult testis, Leydig cell-derived ßEND is hormonally regulated (252). In fetal rat Leydig cells in culture, LH is a potent stimulus for ßEND production (253), while androgens, GnRH, and, to a lesser extent, dexamethasone (Dex) are inhibitory signals (252, 253). Immunohistochemical studies showed that in vivo hCG treatment increased 4-fold the number of interstitial cells positive for ßEND in prepubertal mice (250) and elevated by 100% the ßEND concentrations in interstitial fluid (254). These findings, coupled with the observation that testicular POMC gene is positively regulated by gonadotropins (255), demonstrate that LH is a stimulus for the synthesis and release of POMC-derived peptides in Leydig cells throughout rodent life. In addition to LH, CRH is another important stimulatory signal for ßEND production from adult rat Leydig cells (142, 145). In contrast to LH, which is a systemic regulator, the CRH signal generated inside Leydig cells may act as a relevant autocrine regulator of ßEND production (256). Finally, it must be noted that alcohol is a direct exogenous stimulant of ßEND secretion in adult rat testis (257, 258), and it has been suggested that alcohol may act through testicular ßEND to suppress the synthesis and release of testosterone (258).
b. Receptors.
ßEND-binding sites were initially found on
membranes obtained from adult rat testis (259). Subsequent studies
showed that Sertoli cells have specific binding sites for opiates
(260); no other testicular cell has so far been found to bind ßEND.
Opioid receptors (µ, , and ) have been cloned (261). RNA
blotting studies have shown no detectable expression of -receptor
mRNA in mouse testis (262). Since ßEND is a µ- opioid ligand, it
is possible that µ- and/or -opioid receptors are expressed in
Sertoli cells; this possibility must be verified.
c. Local functions.
The intratesticular role of ßEND has
been analyzed in various studies (239, 256, 263). In vivo
studies have proposed that ßEND in the testis inhibits
steroidogenesis (264, 265, 266, 267) and is involved in the stress-induced
attenuation of the steroidogenic testicular response to gonadotropins
(268). The lack of opioid-binding sites on Leydig cells (260) and the
absence of direct ßEND effects on Leydig cell steroidogenesis (182, 269, 270), would exclude the direct participation of intratesticular
ßEND in the regulation of testosterone secretion. Thus, it is
possible that ßEND may be an indirect modifier (via Sertoli cells) of
Leydig cell steroidogenic activity. In accordance with this hypothesis
is the observation that ßEND significantly inhibits FSH-induced INH
production by Sertoli cell cultures obtained from pubertal rats (271).
INH is one of the Sertoli cell factors known to amplify testosterone
production by Leydig cells in response to LH (272); the paracrine
inhibition of its production by ßEND would result in a reduction of
Leydig cell steroidogenic capability.
ßEND exerts other important inhibitory actions on Sertoli cell function, which include the production of ABP (260) and Sertoli cell growth (273, 274). Regarding the latter aspect, it was initially shown that naloxone treatment of fetal rat testes in culture increased basal and amplified FSH-induced Sertoli cell division (273). Subsequent studies demonstrated that ßEND inhibited FSH-induced proliferation of neonatal rat Sertoli cells by 50% and that this action involved Gi and was exerted at a point before intracellular production of cAMP (274). These studies indicate that testicularly produced ßEND may act as a local antagonizer of FSH stimulation of the Sertoli cell. This inhibitory action can be of great importance in fetal and neonatal age, which is a period of life in which there is high Leydig cell ßEND content (250) and production (252, 253) and corresponds to the proliferative period of Sertoli cells (16). Since the ultimate number of the Sertoli cells dictate the output of sperm cells at maturity both in rodents and human (16), it cannot be excluded that in the fetus, during late pregnancy, and in perinatal life an increase in testicular END might be involved in the pathogenesis of some oligozoospermias of the adult. A candidate trigger of this event could be stress, which, in adult rats, has been shown to stimulate testicular CRH gene expression and protein synthesis (134) and testicular ßEND release into the interstitial fluid (275).
Mice lacking ßEND by site-directed mutagenesis have recently been generated (276). The resulting transgenic animals display no overt developmental or behavioral alterations and have a normal functioning hypothalamic-pituitary-adrenal axis. However, they lack the opioid analgesia induced by mild swim stress and show significantly greater nonopioid analgesia. Homozygous mutant male and female mice have normal fertility based on the onset of puberty, average litter size, and the number of consecutive litters in established mating pairs. No obvious changes in endocrine function have been documented in the ßEND-deficient mice despite the extensive experimental data base concerning opiate effects in the hypothalamus (229). While it is possible that ßEND plays no role in the regulation of the hypothalamic-pituitary-adrenal axis and hypothalamic-pituitary-gonadal axis, a more likely explanation, in light of the analgesia studies, is that subtle compensating mechanisms establish themselves to maintain normal functioning of these critical neuroendocrine systems. Considering the inhibitory effects of ßEND, which include reduction of testosterone secretion and neonatal Sertoli cell growth, transgenic animals overexpressing ßEND in the gonads could help to clarify the suggested roles of this opioid peptide in testicular development and function.
2. Enkephalin (ENK)
a. Expression, localization, and production.
High levels of
pENK expression are found in somatic and germ cells, including Sertoli
and peritubular cells in immature testis and Leydig and spermatogenic
cells in adult testis (237, 277, 278, 279, 280, 281).
The analysis of postnatal development of ppENK mRNA in the rat testis showed little signal before postnatal day 20 and an increasing positivity after day 30 (282). The protein precursor for Met-ENK is encoded on transcripts of 1.4 kb in brain tissues and in those peripheral organs, such as adrenal, that are known sites of synthesis of the peptide. In the rat and mouse testis the major pENK transcript is 1.7 kb and is localized to the germ-line (283, 284). However, low levels of the conventional 1.4-kb transcript could also be detected in Sertoli cells and at lower frequency in Leydig cells (281). In humans and hamster, the germ-line appears to express only the conventional 1.4-kb mRNA (281, 283, 284). Different transcriptional initiation sites may be responsible for these differences (283, 284). These transcripts appear to be efficiently translated and associated with polysomes (285).
Extensive studies of a "cassette" sequence in the 5'-flanking region of the pENK gene have revealed that responses to several transcription factor proteins can be conferred in tissue culture cells in constructs containing <200 bases of the pENK gene 5'-flanking sequence (286, 287). Because of this distinctive feature, high testis expression and low expression in other organs have been obtained in transgenic mice containing segments of the 5'-flanking region of the rat pENK connected to a chloramphenicol acetyltransferase reporter (288) or the human 5'-flanking region connected to ß-galactosidase (289). Subsequently it has been shown that high levels of expression of the pENK gene in the testis in transgenic mice can be achieved with an even shorter "cassette" region of the pENK gene promoter, further supporting a role for pENK expression on testicular and spermatogenic development and function (290).
FSH and various compounds that activate the cAMP system transiently increase the pENK mRNA content in Sertoli cells isolated from 20-day-old rats (281, 282). Similarly, hCG or a cAMP analog treatment of cultured adult rat Leydig cells leads to a rapid increase in pENK mRNA levels (291), while the phorbol ester O-tetradecanoylphorbol 13-acetate increases the ppENK mRNA abundance in cultured peritubular cells prepared from testes of 20-day-old rats through a cAMP-independent pathway (292). A low content of pENK-derived Met-ENK sequences was found in adult testis from rat, hamster, and cattle (277). The immunohistochemical localization of ENK in adult rat testicular tissue cultures revealed positive staining in some interstitial cells, and 1 h after hCG addition specific ENK immunoreactivity was visualized in Sertoli cell cytoplasm in some tubules (293). A positive immunoreactivity for Met-ENK, which increased when the cAMP stimulator forskolin was added to the cells, was also observed in Sertoli cell cultures (294).
Interestingly, a DNA-binding factor with high affinity and specificity for the Leu-ENK-encoding sequences in the pDYN and pENK genes has been characterized (295). This factor, named Leu-ENK-encoding sequence DNA-binding factor, was present at highest levels in rat and mouse testis, cerebellum, and spleen and was generally higher in late embryonal compared with newborn or adult animals. The specific functional role of this factor is unknown. However, it could contribute to the regulation of the expression of pDYN and pENK gene transcription during development.
pENK transcripts have been detected also in human testis, and human spermatozoa show a positive immunoreactivity for Met-ENK molecules localized in the acrosome where they are stored, presumably until release at fertilization (285).
b. Receptors.
As discussed above, in the ßEND section,
opioid-binding sites are exclusively localized in Sertoli cells in
adult rats. It is still unknown which class of opioid receptor is
represented on these cells.
c. Local functions.
In immature rats the in vivo
intratesticular injection of a synthetic ENK analog suppresses in a
dose-dependent manner the basal testosterone secretion from the
injected testes excised and placed in culture (296). The effect seems
to be a consequence of the local action of the ENK analog since its
systemic injection produced no change in steroidogenesis (296).
As reported above, transgenic mice containing bases -193 to +210 of the human pENK gene and an additional 1 kb of 3'-pENK-flanking sequence driving expression of bacterial chloramphenicol acetyltransferase, and the same promoter and flanking sequences driving expression of a rat pENK cDNA, have been generated (290). The animals showed dramatic expression of the transgene in the testis, while much lower expression was observed in the brain and other ENK-producing tissues. High levels of expression in the testis can thus be achieved with a very short promoter region and do not require intron A sequences previously considered necessary. Moreover, the effect of altered ENK expression on testicular function has been analyzed. No male founder or transgenic offspring of the animals expressing the human pENK gene revealed reproductive difficulties. In contrast, one founder of the mice expressing the rat pENK gene showed no reproductive success despite normal mounting, intromission, and other motor behaviors associated with reproduction. In this animal, no sperm were seen upon dissecting the epididymis, the testes were significantly smaller compared with the testes of age-matched controls, and the seminiferous tubules were grossly abnormal, with degenerating pachytene spermatocytes and early germ cells including spermatogonia. Mature spermatids were not present in any of the tubules. Sertoli cells in the seminiferous tubules were often abnormal and incorrectly oriented. A second male founder also displayed reduced fertility, and motility measurements of epididymal sperm of this animal revealed that only 43% of the sperm cells were motile. Lack of reproductive success was also observed in two offspring from two transgenic lines. The four infertile animals displayed blood LH and testosterone levels within normal limits. In summary, overexpression of the pENK cDNA appears to have induced grossly abnormal testicular morphology in one offspring, low sperm motility in another, and reduced fertility in others (290).
These findings suggest that the role of ENK in testicular function merits closer scrutiny.
3. Dynorphin (DYN).
a. Expression, localization, and production.
Immunoreactive
DYN (297) and pDYN mRNA have been found in testicular extracts of adult
rat testis (298, 299). In the rat, pDYN-derived peptides are found in
the Leydig cells by immunohistochemistry, and immunoreactive DYN has
also been demonstrated in guinea pig and rabbit testicular homogenates
by RIA (299). Accordingly, pDYN mRNA and pDYN-derived peptides are
synthesized in the R2C rat Leydig tumor cell line and are positively
regulated by cAMP analogs (300). The chromatographic characterization
of DYN immunoreactivity in guinea pig and rat testis showed that both
authentic DYN-A and DYN-B are present in this tissue and that the
immunoreactive forms of the peptides are similar to those found in
brain and pituitary (301). Through the use of rat testicular cell
fractionation procedures and Northern blot analysis, Sertoli cells were
also found to be the site of pDYN mRNA synthesis, and in
situ hybridization to fixed adult tissue confirmed this result
(302). Moreover, the treatment of primary cultures of rat Sertoli cells
with cAMP resulted in a transient increase in steady state pDYN mRNA
and immunoreactive DYN levels (302). Immunoreactive DYN has been also
detected in the testicular perifusion effluent of perifused adult rat
testicular fragments (270).
b. Receptors.
Since DYN is the major ligand for the
-opioid-binding sites and the -receptor mRNA has not been found
in the mouse testis (262), a diffuse involvement of DYN in testicular
function, at least in this animal species, is unlikely.
c. Local functions.
The treatment of rat perifused testicular
fragments and purified Leydig cells with DYN did not alter either basal
or hCG-stimulated testosterone secretion (270).
In summary, at this point in our knowledge, despite the demonstration of the presence of DYN mRNAs and peptides in the testis, a functional involvement of DYN in the physiology of the male gonad is rather unlikely and should be considered only speculative.
J. Substance-P (SP)
The undecapeptide SP belongs to a family of related peptides, the
tachykinins, which are widely distributed in both the central and
peripheral nervous system where they function as
neurotransmitters/neuromodulators and share a common terminal amino
acid sequence (303). The receptors for SP belong to the family of
tachykinin receptors classified as NK1, NK2, and NK3. SP, neurokinin A,
and neurokinin B are the preferred endogenous ligands for the three
receptor types, respectively (304). The cloned SP receptor and the
other tachykinin receptors are membrane proteins with
seven-transmembrane helix patterns typical of G protein-coupled
receptors (305).
1. Expression, localization, and production.
A selective
immunostaining for SP has been reported in the Leydig cells of adult
human testicular tissue (306, 307), isolated human Leydig cells (308),
and in fetal and adult Leydig cells from golden hamster and guinea pig
(309, 310, 311). Using a modified PCR assay, SP mRNA could be detected in
extracts of human, mouse, and bovine testes, but not in rat or boar
testes (312). Sequencing analysis of the PCR products from human testis
showed that the transcripts expressed encode both SP and neurokinin-A
neuropeptides (312).
2. Receptors.
PCR analysis was able to demonstrate the
presence of mRNA for both SP and neurokinin-A receptors in human testis
(312).
3. Local functions.
Angelova et al. (311) reported
that, in vitro, synthetic SP can stimulate the production of
testosterone by hamster Leydig cells from neonatal and prepubertal
animals, whereas a clear inhibition of both basal and hCG-stimulated
steroid production was observed in adult Leydig cell cultures (311).
The influence of SP on the binding characteristics of the LH receptor
in purified Leydig cells from golden hamster was also studied (313). A
significant increase in the binding capacity was estimated in the
Leydig cells from young animals, while SP reduced the number of LH
binding sites in adult hamsters (313). Sertoli cells from immature rats
respond to 24 h SP treatment with an increase of transferrin and
lactate release (314).
Further studies are required before one can speculate that SP might have a regulatory role on the testicular function in vivo.
K. Neuropeptide Y (NPY)
NPY is a 36-aa peptide member of the pancreatic-polypeptide
family, isolated and extensively distributed in the brain, in
sympathetic neurons innervating cardiovascular and respiratory system,
gastrointestinal, and genitourinary tract (315). Considerable interest
has focused on the importance of NPY in the control of basic vegetative
functions. NPY stimulates food intake, modulates circadian rhythms, and
regulates the release of several hypothalamic hormones (316). NPY
appears to act upon at least four types of receptors called Y1, Y2, Y3,
and an atypical Y1 receptor that mediates the feeding response
stimulated by NPY (317).
1. Expression, localization, and production.
NPY mRNA is
expressed in immature rat Leydig cells and Sertoli cells (318). Leydig
cells treated for 12 h with LH, interleukin-1 (IL-1), or
IL-1ß exhibited an increase in NPY mRNA expression, which is also
observed after forskolin treatment. FSH treatment increased NPY mRNA
levels of Sertoli cells in a dose-dependent manner. Moreover, a germ
cell factor and a Sertoli cell factor stimulated by FSH increased NPY
gene expression in Leydig cells. A positive NPY-like immunoreactivity
in cultured immature Leydig and Sertoli cells was also shown by
immunocytochemistry (318).
To date, no evidence has been published for the presence of NPY receptors in the testis or for local in vivo or in vitro actions of the peptide .
Erickson et al. (319) have recently reported the results of knocking out NPY (319). The knockout mice appear to be normal in all respects, except for a propensity for seizures. Both male and female mutants are fertile, demonstrating that the neuroendocrine systems regulating growth and reproduction as well as the sympathetic innervation of the vas deferens are functional in the absence of NPY.
In light of these data, any hypothesis on a possible involvement of NPY in the local regulation of testicular function seems premature.
|
III. Peptides Originally Identified in the Male Gonad |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
-subunit and one of two related ß-subunits (ßA and ßB).
Both INH-A (-ßA) and INH-B (-ßB) suppress the FSH release
from the anterior pituitary. ACTs are ß/ß-dimers of which the
ßA/ßA and ßB/ßB homodimers and the ßA/ßB heterodimer have
been purified and characterized and shown to stimulate FSH secretion
from the pituitary. The mRNAs encoding INH/ACT subunits are most
abundant in the ovary and the testis but can be found in a variety of
other tissues (321). In addition to their endocrine role, these
proteins have been shown to affect the growth and differentiation of a
number of cell types, including cultured rat anterior pituitary cells,
gonadal and neuronal cell lines, and hematopoietic progenitor and
erythroleukemia cells (321).
Two distinct binding proteins for INH and ACT have been identified,
follistatin and -2 macroglobulin. Follistatin, initially discovered
for its ability to suppress pituitary FSH release (322), was
subsequently found to bind ACT directly (323) and, with lesser
affinity, INH (324, 325). It is a single-chain protein highly
cysteine-rich, product of a single gene, with different molecular
weight forms attributed to alternative mRNA splicing. It is expressed
in a variety of tissues, particularly gonads, kidney, and pituitary.
The suggested biological role of follistatin is the involvement in the
neutralization of ACT activity (326). -2 Macroglobulin is an high
molecular weight endopeptidase inhibitor present in high levels in
human serum (327, 328), known to bind proteases and a number of growth
factors, including TGFß and nerve growth factor (NGF) (329). It is
unknown whether binding of ACT or INH to -2 macroglobulin alters
their bioactivity or immunoreactivity.
Two genes coding for the ACT receptors have been cloned. One (ActRII) (330) encodes for a protein of 494 aa consisting of an ACT-binding domain, a single membrane-spanning domain, and an intracellular serine/threonine-specific protein kinase domain. The second (ActRIIB) encodes potentially four different ACT receptor isoforms belonging to the protein serine/threonine kinase receptor family, which are produced by alternative mRNA splicing (331).
To date, a high-affinity receptor for INH has not been found by molecular or biochemical techniques. However, although the identified ACT receptors exhibit a low affinity for INH (330, 331), a separate high-affinity receptor for INH is widely believed to exist, since in some systems in which ACT and INH are both active, INH exerts its activity with a much higher potency (332).
Excellent reviews have been written on the subject, to which the reader can refer for a complete description (332, 333, 334).
1. Expression, localization, and production.
All three INH
subunits have been found in the fetal, postnatal, and adult testis. In
the rat, -subunit, ßB-subunit, ßA-subunit, and follistatin mRNAs
have been found starting from day 14 post coitum (pc) by in
situ hybridization (335, 336). The ßA-subunit mRNA signal was
localized in the interstitial tissue, the ßB-subunit was localized in
the tubules, and the -subunit message was present in both the
interstitial tissue and the seminiferous tubules (335, 336). The
expression and localization of - and ßA-subunit mRNA and protein
have been evaluated by in situ hybridization and
immunocytochemistry in fetal sheep testes collected at various times of
gestation (337). As in the rat, the expression of the -subunit mRNA
was localized within the seminiferous cords and in a small number of
Leydig cells of the developing fetal testis; the expression
progressively increased with gestational age (337) as confirmed also by
immunoassay and bioassay (338). No expression of INH ßA-subunit mRNA
or protein was detected at any stage of gestation (338). Bioactive and
immunoactive INH were found to be produced and to increase throughout
gestation in bovine fetal testis (339). Roberts et al. (340)
have studied the testicular postnatal distribution of immunostaining
and mRNA signal of the INH subunits in the rat (340). In 12-day-old
rats, immunostaining and mRNA signal for the -subunit were found in
Leydig cell clusters. The ßA- and ßB-subunit staining and
ßA-subunit message were detected in isolated interstitial cells.
Positive immunostaining for each subunit was localized in a Sertoli
cell-like pattern in the seminiferous tubules. In addition, there was a
positive mRNA signal for the - and ßB-subunit over regions
containing these cell types (340). The treatment with hCG induced a
dramatic increase in the -subunit immunohistochemical staining and
mRNA signal in Leydig cell clusters (340). In adult rats, - and
ßB-subunit staining and -subunit mRNA signal were observed in the
interstitial cells. As in the immature animals, all three subunits were
localized in a Sertoli cell-like pattern in the tubule, and a positive
mRNA signal for the - and ßB-subunits was found over these cells;
contrary to what was observed in the immature animals, the hCG
treatment did not modify the intensity of the signals (340). Purified
Leydig cells from adult male rats synthesized and secreted INH in
vitro as measured by Northern blot analysis, RIA, and in
vitro bioassay; LH stimulated immunoactive INH production in a
dose-dependent manner (341). Moreover, it has been established that FSH
and to a lesser extent epidermal growth factor (EGF), but not
testosterone, are able to stimulate the production of INH by immature
Sertoli cells in culture (342).The comparative distribution of the
three INH subunits in the rat testis during prenatal and postnatal
development has been studied using an immunohistochemical approach
(343). Both - and ßB-subunit staining were present in the
seminiferous epithelium of fetal, neonatal, pubertal, and adult rat
with a distribution consistent with a localization in Sertoli cells.
The predominant localization of the ßA-subunit was in the nuclei of
pachytene and zygotene spermatocytes (343).
Interstitial cell cultures from immature rat and pig testis and
immature rat Sertoli cell cultures secrete ACT (344, 345). Cultured PMC
from pubertal animals express high levels of ßA-subunit mRNA and some
- and ßB-subunit mRNA and secrete immunoreactive and bioactive
ACT-A (346). In extracts from mature rat testis, INH -subunit RNA
can easily be detected, whereas the ßA- and ßB-subunits are
expressed at 50 and 15 times lower levels, respectively (321).
Moreover, INH -, ßA-, and ßB-subunit mRNA levels are 4- to
10-fold higher in 8- to 15-day-old animals when compared with mature
animals (321). These age-dependent changes have also been observed by
the measurement of immunoreactive testicular INH levels during
development (347), and a positive effect of FSH on the INH -subunit
expression either in vivo or in Sertoli cell cultures
in vitro has been reported (348).
The expression of INH - and ßB-subunit in the rat seminiferous
epithelium is stage dependent; Northern blot assays of seminiferous
tubule fragments extracts revealed that the expression of both subunits
is highest in stages XIII-I and lowest in stages VII-VIII (349). The
seminiferous epithelium stage-dependent expression of INH ßA- and
ßB-subunit mRNAs was also analyzed by in situ
hybridization. The ßA-subunit was expressed in Sertoli cells starting
at stage VIII of the cycle with a maximal expression during stages
IX-XI, while the ßB-subunit was maximally expressed in Sertoli cells
at stages XIII-III (350). This finding has been confirmed by the
evaluation of the INH - and ßB-subunit mRNA expression and the
levels of bioactive and immunoreactive INH in rats with
stage-synchronized spermatogenesis (351).
In human fetal testis -, ßA-, and ßB-subunits were detected at
the beginning of the second trimester of gestation by both mRNA
analysis (352) and immunocytochemistry (353, 354). The -subunit was
found in both tubular and interstitial areas in early gestation,
becoming predominant in the tubules by late gestation. The ß-subunits
were observed initially in the interstitium and subsequently also in
the tubule by the end of gestation. Moreover, it has been shown that
midgestation human fetal testicular cultures secrete detectable
immunoreactive INH -subunit in response to FSH and hCG stimulation
(353). In adult human testis, the INH -subunit has been localized in
the Sertoli cells and some Leydig cells (355, 356). In subsequent
studies, in adult nonhuman primate species and in men, INH/ACT subunits
were found in the cytoplasm of Sertoli cells and Leydig cells but not
in germ cells (357).
2. Receptors.
ActRIIB message is found in rat testis from
15–20 days pc (358). Two species of ActRII mRNA (6 and 4 kb) have been
identified in mouse and rat testis (330, 359). Both messengers are
expressed in Sertoli cells and at low levels in Leydig cell
preparations. Spermatocytes and round spermatids express only the
smaller mRNA, whereas elongating spermatids do not express ACT receptor
mRNA (359). Attisano et al. (331) observed an equal
abundance of ActRII and ActRIIB in mouse testis (331). In the rat,
Cameron et al. found a clearly present ACT receptor message
in the germ cells. ActRII is the predominant form with a strong signal
in round spermatids and a moderate signal in pachytene spermatocytes.
In contrast, ActRIIB was absent within the tubules but weakly expressed
in Leydig cells (360). The ActRII and ActRIIB2 mRNA messages were
localized in the rat seminiferous epithelium (350, 361). Maximal ActRII
expression was seen in late primary spermatocytes at stages XIII-XIV
and in early round spermatids at stages I-IV (350). ActRIIB2 mRNA was
expressed maximally in stages IX-XI in type A1 and A2 spermatogonia and
in Sertoli cells (361). In pubertal rat testis, ActRIIB2 expression was
localized in Sertoli cells around primary spermatocytes and meiotically
dividing cells (361). Woodruff et al. (362) have reported
that ACT-A binds primarily to spermatogonia, while INH-A binds all germ
cell stages (362). The changes in ACT receptor expression during sexual
maturation in rat testis were examined by Northern blot analysis. Two
ActRII mRNAs (6 kb and 3 kb) were detected. The large mRNA was low on
day 7, and gradually increased by day 35, while the small mRNA was low
on day 7, began to increase on day 21, and increased dramatically by
day 35 (363, 364). The levels of expression of ActRIIB gene did not
change during testicular development (364). In situ binding
of recombinant human 125I-labeled INH and ACT has been used
to identify binding sites and potential target cell populations in rat
testis throughout postnatal development (365). INH bound to islands of
interstitial cells in animals of all ages. In contrast, ACT binding was
seen on cells located basally within or around all seminiferous
tubules, regardless of the age of the animal, as well as on round
spermatids in stages VII-VIII of the cycle of the seminiferous
epithelium in animals older than 30 days (365). ActRII mRNA has also
been detected in pubertal PMC in culture (346). Cloning of the human
ACT receptor from a human testis cDNA library reveals high evolutionary
conservation (366).
Dynamic changes in the relative amount of the INH- and ACT-binding proteins could act to modulate ACT and INH bioavailability, and thus it is important to study the local testicular production of these substances. Both follistatin mRNA and immunoreactivity have been identified in adult testicular tissues (326, 350, 361, 367). In these studies, follistatin mRNA was localized in a similar stage-specific manner to that of the INH ßA-subunit mRNA, with maximal levels in stages IX-XI Sertoli cells (350). Sertoli cell expression of follistatin mRNA was stimulated by EGF but not by FSH or testosterone (367). In contrast to the site of follistatin mRNA expression, follistatin protein was immunohistochemically identified in spermatogenic cells (particularly spermatocytes and spermatids) and Leydig cells, but not in Sertoli cells (368). One explanation for this discrepancy is that follistatin produced by one cell type may diffuse and associate with another cell type or with components of the extracellular matrix; alternatively, the lack of protein synthesis cannot be ruled out.
Sertoli cells synthesize and secrete -2 macroglobulin in
vitro, and immunoreactive -2 macroglobulin can be measured in
tubular and rete testis fluids (369). In the testis, this protein is
believed to protect the seminiferous tubules and the rete testis from
proteases released from spermatids and other cells. The impact of -2
macroglobulin on ACT/INH activity in the testis is presently unknown.
3. Local functions.
The first evidence that INH and ACT could
serve as local modulators of testicular functions derived from the
observation of their ability to modulate the LH-stimulated biosynthesis
of androgen in rat cultured Leydig cells (272). ACT exerted an
inhibitory action on LH-stimulated androgen biosynthesis at all stages
of Leydig cell differentiation, whereas INH facilitated LH action in a
mixed testis cell preparation from neonatal and adult rats (272, 370).
In immature porcine Leydig cell cultures, ACT-A reduced the
hCG-stimulated dehydroepiandrosterone accumulation, but increased the
conversion of pregnenolone and dehydroepiandrosterone to testosterone,
suggesting that ACT may alter the activity of either the cholesterol
side chain cleavage enzyme or the 3ß-hydroxysteroid dehydrogenase
(3ß-HSD)/isomerase (371). In cells obtained from pubertal and adult
rats, ACT stimulated the proliferation of spermatogonial cells (372, 373) and caused a rapid reaggregation of Sertoli cells and germ cells
in the absence of basement membrane or peritubular cells (372). This
effect is accompanied by an ACT-stimulated increase in thymidine
incorporation into germ-Sertoli cell cocultures and, interestingly,
these effects can be differentially regulated by follistatin (374).
However, ACT has a different function during rat gonadal development in
that it inhibits thymidine incorporation in rat testes on day 14 pc and
has no effect in testes on day 15 or 18 pc (358). INH decreases
spermatogonial proliferation when injected locally into adult hamster
testis without affecting the controlateral testis treated with control
fluid (375). ACT-A has a slight inhibitory effect on proliferation of a
rat Leydig cell testicular tumor line (376). ACT inhibits
FSH-stimulated aromatase activity, androgen receptor mRNA expression,
and androgen binding without significantly affecting FSH receptor mRNA
expression in cultured Sertoli cell isolated from 21-day-old rats
(345).
Targeted disruption studies have provided some interesting clues
about local functions of INH/ACT in the testis in vivo. Male
mice homozygous for a deletion of INH -subunit develop mixed or
incompletely differentiated gonadal stromal tumors as early as 4 weeks
of age (377), and show a 200-fold increase in testicular expression of
ACT ßA-subunit mRNA accompanied by a tissue- specific reduction in
the expression of its type II receptor (378). The predisposition of
INH-deficient mice to gonadal tumors identified INH as the first tumor
suppressor protein with a gonadal specificity. The gonads and external
genitalia of male and female INH-deficient mice were normal before
tumor development by gross examination and detailed histology,
suggesting that INH is not essential for normal sexual differentiation
and development. Moreover, as mature spermatozoa were initially found
in the homozygote INH-deficient animals, INH did not seem necessary
for the formation of mature germ cells. Only when the tumor becomes
much more destructive was an arrest of spermatogenesis observed. Even
in the contralateral tumor-free gonads of some male mice, germ cell
maturation was arrested. This suggests that the tumors secrete a
substance(s) that may be toxic for spermatogenesis. The exact mechanism
of tumor formation in INH-deficient mice is still unclear. The deletion
of INH -subunit results in 2- to 3-fold elevation of serum FSH
levels. The prolonged elevation of FSH could contribute to, but is not
directly responsible for, the development of gonadal tumors,
considering that humans who have FSH-secreting adenomas with FSH levels
more than 20-fold higher than normal do not develop gonadal stromal
tumors (379). The finding that ACT, but not INH, can stimulate the
proliferation of gonadal tumor cell lines derived from the
INH-deficient mice (380) suggests that the unopposed action of ACT,
together with prolonged FSH action, might take part in tumorigenesis.
Also ACT-, follistatin-, and ActRII-deficient mice have been produced (381, 382, 383). Jaenisch and colleagues (381) created mouse strains deficient in the ßB- subunit expected to be deficient in ACT-B, ACT-AB, and INH-B. Homozygous mutant embryos had defects in eyelid development. Homozygous mutant females were unable to rear offspring normally while homozygous males bred normally, and histological examination of their testes failed to detect overt abnormalities. Mice lacking the ßA-subunit, thus unable to express ACT-A and INH-A, die within 24 h of birth. They have no whiskers or lower incisors and have a cleft palate (382). Surprisingly, defects in ActRII-deficient mice show little resemblance to those of other ACT-deficient animals (383). Most individuals developed into adults whose only significant problem was the suppression of FSH and the consequent defective reproductive performance that progressed to infertility in the females but only to a delay in fertility in males (383). The testes of the homozygous animals were small, but spermatogenesis could occur despite reduced FSH and without signal transduction through ActRII. Finally, follistatin-deficient mice are small and have a number of serious defects leading to death within a few hours of birth (384).
In conclusion, ACT and INH have local regulatory roles in the testis both in the control of androgen secretion and germ cell proliferation. Although the gene deletion studies indicate that many aspects of gonadal development proceed in the absence of INH and ACT, the identification of INH as a tumor suppressor gene with gonadal specificity is remarkable.
B. PModS
PModS is a nonmitogenic paracrine factor isolated from the
conditioned medium of peritubular cells that modulates Sertoli cell
function and whose secretion is increased by testosterone (385, 386).
Further characterization of PModS showed that there are two forms of
the substance, named PModS A and B, with respective mol wts of 54,000
and 56,000 (387). PModS stimulates transferrin, ABP, and INH secretion
from Sertoli cells in culture (385, 388, 389), whereas it exerts no
effect on Leydig cell basal and hCG-stimulated testosterone and
immunoreactive INH production (390). PModS was found to dramatically
increase mRNA levels for c-fos, but had no effect on
c-jun mRNA. The treatment of Sertoli cells with an antisense
c-fos oligonucleotide was found to inhibit the actions of
PModS on transferrin expression, suggesting that PModS acts indirectly
through transcription factors to induce Sertoli cell-differentiated
functions (391). PModS stimulates cGMP levels in Sertoli cells, without
influencing cAMP levels or calcium mobilization or the metabolism of
PI, and increases phosphorylation of specific proteins (392). Since it
has been observed that cGMP does not directly mediate the actions of
PModS, it is postulated that tyrosine phosphorylation and alteration in
the expression of transcription factors may mediate the Sertoli cell
differentiation induced by PModS.
In spite of the reported in vitro effects and hormonal regulation of PModS, the lack of biochemical characterization of the substance, identification of receptors, and in vivo function do not allow the expression of a critical judgment on its role in the male gonad.
|
IV. Growth Factors |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
The biological actions of the IGFs are modulated by a family of at least six IGF-binding proteins (IGFBPs) that are found in the circulation and in extracellular compartments. The IGFBPs can inhibit or enhance IGF effects and may also have ligand-independent effects.
The reader can refer to recent reviews on IGFs, IGF receptors, and IGFBPs for detailed information (393, 394, 395).
1. Expression, localization, and production.
Testicular
immunoreactive IGF-I was first discovered in media conditioned by
seminiferous tubule cultures from adult rats, Sertoli cells cultured
from immature rats (396), and in acid extracts of rat testes (397, 398). Sertoli cells and PMC prepared from sexually immature rats
accumulate IGF-like activity in the medium, and the Sertoli cell
accumulation is inhibited by cycloheximide and stimulated by cyclic
nucleotide analogs (399). The partial characterization of this IGF
immunoreactive material demonstrated that immature rat Sertoli cells in
culture secrete a peptide that is the equivalent of human IGF-I (400).
A high homology between the IGF-I-reactive material in culture media
conditioned by porcine Sertoli cells and human IGF-I, whose secretion
could be increased by FGF and EGF, was also observed by other groups
(401, 402, 403). Evidence has been presented that apart from Sertoli cells
and PMC, cultured Leydig cells from immature rats also produce IGF-I
(404, 405). Peritubular cells display the highest production rate
followed by Sertoli cells and Leydig cells.
A marked age-related change of IGF-I-like immunoreactive material has been reported in the rat testis by means of immunohistochemistry (406, 407). Up to 2 weeks after birth, all cells in the growing testis show IGF-I-like immunoreactivity; thereafter, the frequency declines and the Sertoli cells become negative. During puberty, there is a rapid increase in the number of spermatogenic cells showing IGF-I-like immunoreactivity. In the adult rat, IGF-I immunoreactivity is exclusively localized in the cytoplasm of spermatocytes. No significant stage-dependent changes have been observed in testicular IGF-I concentrations in stage-synchronized rat testes (408).
The expression of the mRNA for IGF-I was originally demonstrated in adult and immature rat testis (409, 410, 411). Subsequently, Leydig cells have been shown to express this gene; the expression was enhanced by GH treatment (412). The analysis of the ontogeny of IGF-I and IGF-II mRNA in the rat has revealed that both mRNAs are maximally expressed in the testes of day 2 animals and decrease with age (25). The quantification of mRNA for IGF-I during pig testicular development showed a steady increase from fetal 100–102 days up to 25 weeks postnatally (413). The pattern of expression of IGF-I gene expression in the mouse testis has been studied by in situ hybridization analysis (414). In the testis of prepubertal mice (postnatal day 14) IGF-I transcripts were localized in the interstitial compartment. In older animals (postnatal day 35) IGF-I transcripts were detected in the seminiferous epithelium, but not in the interstitial cells, with a strong signal evident only in spermatids. In immature hypophysectomized rats, the administration of LH, FSH, and GH all resulted in a significant increase in testicular IGF-I mRNA expression (411). It has been shown that IGF-I production by cultured immature pig Leydig cells and Sertoli cells is stimulated in a dose-dependent fashion by LH and FGF or FSH and FGF, respectively (415). Both FSH and GH trigger a modest increase of IGF-I levels in Sertoli cell cultures prepared from pubertal rats (399). FSH treatment partially prevents the marked decrease in intratesticular IGF-I in hypophysectomized or GnRH antagonist-treated rats but not in GnRH antagonist plus EDS-treated animals, providing in vivo evidence for the importance of Leydig cell-Sertoli cell interactions in regulating testicular IGF-I content (416, 417). This effect of FSH is potentiated by GH and testosterone (418).
In the human testis the immunohistochemical staining of IGF-I is preferentially localized in Sertoli cells; less evident staining is found in primary spermatocytes and in some Leydig cells (419, 420). The slight positive reactions at the levels of spermatocytes and Leydig cells have been interpreted as being due to IGF-I-receptor complexes. In situ hybridization studies in human testis failed to reveal mRNA for IGF-I (421). This disagreement with the immunohistochemical findings has been explained by an accumulation of circulating IGF-I bound to IGFBP-2 or to IGF-I receptors localized in the germinal cells or alternatively to a cross-reactivity in the immunological detection of IGF-I with IGF-II derived from local testicular synthesis within the seminiferous tubule.
No mRNA for IGF-II was detected in the adult rat testis (410); low mRNA expression, unmodified by hormonal treatments, was found in the immature hypophysectomized rat (411). In the pig, IGF-II mRNA was expressed in the fetal and immature testis, and its expression decreased with age (413). Human fetal testis contains abundant IGF-II mRNA, which significantly decreases from 13 to 25.8 weeks of gestation (422). IGF-II mRNA was abundantly localized in vascular cells and peritubular cells of adult human testis by in situ hybridization (421).
2. Receptors.
Sertoli cells and pachytene spermatocytes from
immature rats (399, 423, 424) and Sertoli cells from immature pigs
(425) were found to possess IGF-I receptors. IGF-I receptors have been
identified in both whole testicular homogenates and isolated Leydig
cells from adult rats (398, 424, 426, 427, 428). IGF-I binds to total testis
membrane fractions from immature and adult rat testes, the maximal
specific binding being reduced between 21 days of age and adult life
(395). It has been suggested that Leydig cell type I IGF receptors can
be up-regulated by LH, FSH, and GH, with hCG/LH being the most
important factor (428). In both pig (429) and rat (428, 430), Leydig
cell LH/hCG up-regulates the IGF receptors.
Strong IGF-I receptor gene expression, which does not appear to change with developmental age, is evident in Leydig cells, PMC, and occasionally in spermatogonia of 14- and 35 day-old mice testis by in situ hybridization (414).
In human testis the major positivity for the IGF-I receptor was found in secondary spermatocytes, early spermatids, and in some Leydig cells, whereas Sertoli cells were less positive (419). In situ hybridization to localize mRNA for IGF-I receptors in human testis showed positive expression in the germinal epithelium (421).
Both Sertoli and germinal cells from adult rat and mice contain IGF-II receptors (423, 431). IGF-II receptor mRNA has been localized by in situ hybridization in the germinal epithelium of both human (421) and rat (432) adult testis. IGF-II receptor immunoreactivity was present in human testis from 23 weeks of gestation to 2 yr of age (433).
The actions of IGFs are modified by IGFBPs that may enhance or inhibit the effects of IGFs. As reported above, at least six different IGFBPs have been identified in the rat. Northern analysis of the IGFBP3 and IGFBP2 mRNAs in rat tissues showed an abundant expression in the testis (434, 435). Cultured immature rat Sertoli, Leydig, and peritubular cells produce IGF-I BPs (404). Both ligand blot analysis and RNA blot hybridization indicated that, in the rat, cultured peritubular cells synthesize primarily IGFBP-2, while IGFBP-3 is predominantly synthesized by prepubertal Sertoli cells (436, 437). The expression and regulation of IGFBP-1, -2, -3, and -4 in purified adult rat Leydig cells have been evaluated (438). None of the testicular crude interstitial cells, purified Leydig cells, or seminiferous tubules expressed IGFBP-1 mRNA. Large amounts of IGFBP-2 were expressed in purified Leydig cells and small amounts were expressed in seminiferous tubules. IGFBP-3 mRNA was predominantly expressed in purified Leydig cells but not in seminiferous tubules, while small amounts of IGFBP-4 were expressed by the Leydig cells.
In the adult human testis IGFBP-1 mRNA was not detected; IGFBP-2 was expressed in both Leydig cells and Sertoli cells; IGFBP-3 was detected only in the endothelium of testicular blood vessels; IGFBP-4 was localized in the endothelium, Leydig cells, and interstitial connective tissue; IGFBP-5 was abundant in connective tissue and was detected at low levels in the Leydig cells; IGFBP-6 was present in some peritubular cells and in some cells of the interstitial compartment (421).
3. Local functions.
IGF-I and IGF-II stimulate the
proliferation of rat and pig prepubertal Sertoli cells (423, 439), and
IGF-I has a small mitogenic effect on immature Leydig cells (401, 440, 441). Both IGFs stimulate spermatogonial DNA synthesis and have a
maintaining effect on premeiotic DNA synthesis in the rat under
in vitro conditions (442), and IGF-I induces the
differentiation of mouse type A spermatogonia (443).
IGF-I stimulates testosterone production and potentiates hCG-induced testosterone formation by cultured Leydig cells of rodent (427, 444, 445, 446, 447, 448), and porcine origin (401, 429, 440); this effect is more pronounced in immature than in adult cells (448, 449).
The IGF-I potentiating effect on hCG-induced testosterone production by Leydig cells in vitro has been confirmed in human (450).
An intraperitoneal injection of recombinant IGF-I to immature Snell dwarf mice (dw/dw) for 7 days caused a significant increase of testicular LH receptors and an acute steroidogenic response to hCG similar to that induced by recombinant hGH treatment, suggesting that the effects of GH on the testis are probably mediated by IGF-I (451). These effects could not be observed in phenotypically normal control (dw/-) animals. The GH-deficient rat has been used as animal model in which to analyze the effects of GH on reproduction. These dwarf rats, although fertile, have a decreased testicular size (452). The administration of GH in the GH-deficient rats from days 21 to 49 of life does not induce significant differences in testicular IGF-I concentrations or in the number of germ cells per cross-section of the seminiferous tubules (418), although it has been suggested that testicular development in the first 30 days of life is pituitary hormone- and probably GH-dependent (453).
Neither GH nor IGF-I pretreatment enhanced acute gonadal responses to gonadotropin stimulation of prepubertal non-human primate testis in vivo (454).
Transgenic mouse models have provided invaluable information to our understanding of IGFs physiology. Wolf et al. (455) characterized the effects of elevated IGF-II on body and organ growth at 4 and 12 weeks of age in transgenic mice expressing human IGF-II under the transcriptional control of the rat phosphoenolpyruvate carboxykinase promoter (455). Body growth was not significantly influenced by elevated IGF-II, but transgenic mice displayed increased kidney and testis weight at the age of 4 weeks, suggesting that elevated IGF-II in postnatal life may have subtle time-specific effects on testis growth. Disruption of the IGF-II gene showed that IGF-II is essential for normal placental and fetal growth, but not for postnatal growth, since the viable and fertile IGF-II null mutants exhibit dwarfism at birth but grow postnatally at a normal rate (456). In contrast, the postnatal growth of IGF-I null mutants, which survive variably depending on genetic background, is reduced (457). The IGF-I null mutation also has a dramatic impact on the development and physiology of the reproductive systems of both sexes (414). Homozygous mice are infertile dwarfs. The testes are reduced in size and sustain spermatogenesis only at 18% of the normal level. The most likely explanation for the infertility of IGF-I null males is predominantly the absence of sex drive, which is apparently due to inadequate serum testosterone levels probably insufficient for perinatal androgenization. Interestingly, androgen deficiency in the mutants can be correlated with an apparent retarded differentiation of Leydig cells. This assumption is supported by the following considerations. The Leydig cells lack the membranous whorls and produce low levels of testosterone both in basal conditions and after LH stimulation, and basal and LH-stimulated androstenedione production is much higher in the IGF-I null mutants than in wild type controls. All these are features of immature Leydig cells. Does testicular IGF-I have some degree of autonomy from other hormonal stimuli? The answer seems to be yes. GH receptor genes do not appear to be expressed in the testis (458, 459, 460). In contrast to the IGF-I null mutants, male little mice carrying a missense mutation of the gene encoding the receptor for GHRH (38, 39, 41), dr/dr rats, which have a base substitution in the GH gene (461, 462), and dw/dw rats, which have a deficit in GHRH signal transduction pathway (463), are fertile. All these natural mutants have low serum levels of IGF-I. Thus, in addition to being apparently GH-independent, the testicular functions of IGF-I seem to be served by its local production without an endocrine contribution by its circulating form. As a whole, these results strongly suggest that testicular IGF-I has a determinant role in Leydig cell development and differentiation and that the lack of this substance in the testis induces infertility. More direct proof of this action will derive from selective testicular targeted deletion studies.
B. Transforming growth factor-ß (TGFß)
TGFß is a polypeptide composed of two disulfide-linked monomers,
synthesized as large precursors. It belongs to a large family of
polypeptides including ACT/INH, Müllerian inhibiting substance,
the decapeptaplegic (dpp)/Vg-related factors, and the BMPs (464). At
least five different TGFß molecular species are recognized (213). The
TGFß receptors comprise multiple components. The main TGFß-binding
components are membrane proteins called receptor types I, II, and III,
respectively. TGFß receptor types I and II are involved in signaling,
whereas the type III receptor, otherwise known as betaglycan, regulates
access of the TGFß to the signaling receptors. A model in which the
type I receptor requires the type II receptor for ligand binding and
both receptors upon heterodimeric receptor complex formation are
required to activate serine/threonine kinase domains has been suggested
(465, 466). TGFß has a wide range of effects on cell proliferation,
differentiation, and organization. Depending on the conditions, TGFß
can either inhibit or stimulate proliferation; the ability of TGFß to
elicit multiple cellular responses of opposite sign is the subject of
great debate (467).
1. Expression, localization, and production.
Both Sertoli
cells and peritubular cells isolated from 20-day-old rats synthesize
and secrete TGFß in culture (468). Reverse phase chromatography of
secreted proteins indicates that Sertoli cells primarily produce
TGFß1, and peritubular cells produce both TGFß1 and TGFß2, while
a mixed population of germinal cells at various stages of development
was not found to contain the TGFß message. TGFß3 mRNA has been
detected in substantial amounts in murine adult testis (469).
The developmental expression of multiple forms of TGFß (TGFß1, TGFß2, and TGFß3) in whole testis and isolated peritubular cells and Sertoli cells from prepubertal, midpubertal, and late pubertal rat testes has been determined using nuclease protection analysis (470). TGFß1 and TGFß2 mRNA expression was predominant in the immature testis and decreased at the onset of puberty. TGFß3 mRNA expression peaked at an early pubertal stage. Peritubular cell mRNA expression of TGFß1, TGFß2, and TGFß3 decreased during pubertal development upon differentiation of this cell type. Sertoli cell expression of TGFß1 increased slightly and plateaued during pubertal development, TGFß2 mRNA was evident only in immature prepubertal cells, and TGFß3 mRNA increased transiently at the onset of puberty. Immunoblot analysis indicated that both cultured peritubular cells and Sertoli cells can produce the proteins for TGFß1, TGFß2, and TGFß3 (470). It has been shown that, in the mouse, the germinal cells express a unique transcript for TGFß1 of 1.8 kb (471). The precise in vivo localization of TGFß1 and TGFß2 during rat testicular development has been determined by means of immunohistochemistry (472). TGFß1 immunoreactivity was detected in Sertoli cells throughout testicular development. TGFß2 was found in fetal Sertoli cells but became undetectable rapidly after birth. In fetal animals the Leydig cells contained TGFß1 and TGFß2; after birth TGFß1 persisted whereas TGFß2 became undetectable. Before puberty, TGFß1 and TGFß2 were absent in a portion of the Leydig cells; when the adult stage was reached, TGFß1 was no longer detectable and TGFß2 staining was faint to absent. During spermatogenesis, TGFß1 predominated in spermatocytes and early round spermatids, but as the spermatids elongated around stages VIII-IX of the cycle, the TGFß1 levels declined. TGFß2 was undetectable in spermatocytes and early round spermatids, but as spermatogenesis progressed, around stages V-VI, the spermatids rapidly acquired a TGFß2-positive reaction (472).
TGFß-like activity has been identified in conditioned medium obtained from cultures enriched in immature porcine Sertoli cells by both bioassay and RRA (473), and the release of TGFß-like material is modulated by hormonal treatment. FSH decreases the release of TGFß-like material to undetectable levels in a dose-dependent way, while the TGFß secretion is enhanced by estradiol, Dex, or T4 treatment; the inhibitory action of FSH seems to be predominant since the stimulatory action of steroid and thyroid hormones is not observed in the presence of FSH (473). An analogous FSH-induced reduction of TGFß secretion by cultured rat Sertoli cells has been reported by others (470).
2. Receptors.
TGFß specifically binds to immature porcine
Leydig cells and Sertoli cells (474, 475). The testicular cell
expression and developmental appearance of mRNAs for the three
high-affinity TGFß receptors has been examined in the rat (476).
Transcripts for receptors I and II were essentially detected in the
immature 10-day-old testis, whereas receptor III mRNAs were present
throughout testicular development. Somatic cells contained mRNAs for
the three receptor types extending also to the PMC the previous
observation of the presence of TGFß receptors on Leydig cells and
Sertoli cells. In germ cells, transcripts for types I and II were in
low abundance compared with type III mRNA, which was expressed in three
different transcript forms (476).
3. Local functions.
TGFß1 appears to play a role in
reproductive function both during embryogenesis and in the adult.
TGFß1 inhibits proliferation of cultured primordial germ cells (PGC)
isolated from 8.5 pc mouse embryos (477). Cultured genital ridges exert
a chemotropic effect on PGC, which is blocked by antibodies to TGFß1,
and TGFß1 mimics the chemotropic effect of genital ridges. These
observations suggest that TGFß1 may be one of the factors that
modulate the migration of PGC to the genital ridges in vivo
(477). TGFß has a potent inhibitory effect on Leydig cell
steroidogenesis. In immature pig and rat Leydig cells, TGFß reduces
the number of hCG receptors, and the cAMP and testosterone response to
this hormone, without affecting Leydig cell proliferation (478, 479, 480). A
potent TGFß inhibition of both growth and steroidogenesis was also
observed in a rat Leydig cell testicular tumor line (376). Morera
et al. (474) reported that TGFß exerts a biphasic effect
on hCG-stimulated testosterone production: stimulatory at low
concentrations and inhibitory at high concentrations. TGFß1 at a
concentration of 1 ng/ml was found to enhance the hCG-stimulated
testosterone formation in primary cultures of purified immature porcine
Leydig cells, and this effect was additive with EGF, as a result of a
complex interaction occurring at the levels of cholesterol substrate
availability in the mitochondria and of 3ß-HSD/isomerase activity
(481).
TGFß has been found to influence peritubular cell function and
migration, increasing the production of extracellular matrix and
promoting colony formation (468). TGFß elicits an increased
contractility and shape changes of purified pubertal rat PMC embedded
in collagen (482), and a synergistic effect of TGFß and
platelet-derived growth factor (PDGF) on collagen gel contraction by
PMC has been demonstrated (483). TGFß1 stimulates lactate production
and glucose uptake in Sertoli cells isolated from immature porcine
testes (475), but has no effect on transferrin production by
differentiated Sertoli cells (468). TGFß1 inhibits the TGF-induced
PMC DNA synthesis at each stage of pubertal development but neither
stimulates nor inhibits Sertoli cell DNA synthesis or growth (470) and
enhances the expression of a 50 kDa protein in human spermatozoa (484).
A role for TGFß in testicular immunosuppression has been suggested on the basis of three observations: antibodies neutralizing TGFß reverse the suppression of rat peripheral blood lymphocyte proliferation induced by rat abdominal testis extract, recombinant TGFß1 dose-dependently inhibits testicular IL1-like factor-induced proliferation of murine thymocytes, and extracts of seminiferous tubules contain a TGFß-like growth inhibitor of a mink lung epithelial cell line (485).
Despite the above data, preliminary results obtained with the generation of mice homozygotes for the disrupted TGFß1 allele showed no gross developmental abnormalities until about 20 days after birth when the animals succumbed to a multifocal inflammatory disease (486, 487). The only reported testicular abnormality in these animals was a mild to moderate inflammation of the serosa of the testis; however, since all the animals die before puberty, the potential effect of the gene knockout on adult testicular function is unknown. Nevertheless, it has been later reported that one homozygous mutant male survived to reproductive age and was able to impregnate two superovulated females (488). The pregnancy progressed to term, but the outcome of the pregnancy, in terms of number and viability of offspring, was uncertain because the offspring were cannibalized.
A transgenic mouse model, in which mature TGFß1 is overexpressed in the liver and causes hepatic fibrosis, has been generated (489). The transgenic line expressing the highest level of the transgene also had high (>10-fold over control) plasma levels of TGF-ß1. Several extrahepatic lesions developed in these animals, including glomerulonephritis and renal failure, arteritis, and myocarditis, as well as atrophic changes in the pancreas and testis. The atrophic changes in the testis consisted of thickened tubular basement membranes, small and more widely spaced seminiferous tubules, and more prominent Leydig cells. This thickening of the basal membranes is consistent with the reported in vitro stimulatory effect exerted by TGFß on the extracellular matrix production by PMC (468).
TGFß3 null mutant mice have also been generated by gene targeting (490). Within 20 h of birth, homozygous TGF-ß3 -/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis.
In conclusion, despite the established in vitro suppression on Leydig cell differentiated functions, the stimulatory effects on PMC, and the developmentally regulated expression of TGFßs and TGF receptors within the testis, the potential local role of TGFß in vivo is unknown. The reported normal fertility in one homozygous mutant TGFß1-deficient mouse seems to suggest that TGFß1 action is not an absolute requirement for testis development and function. However, breeding studies involving intercrosses of heterozygous mice carrying one disrupted TGFß1 allele have shown a reduction in both heterozygous and homozygous mutant offspring compared with the wild type (488). The decrease in the number of homozygous mutants is consistent with the role exerted by TGFß1 in embryological development and implantation. Nevertheless, the reduced number of the heterozygotes also seen has raised the possibility that TGFß1 may be important in haploid germ cell function. It could be that in the absence of a functional TGFß1 gene, germ cell function or survival is impaired leading to a reduction in the frequency of conceptions involving the mutant germ cells.
C. TGF/EGF
EGF and TGF together with amphiregulin belong to the EGF family
of growth factors. A variety of basement membrane and extracellular
matrix proteins, as well as plasminogen activator and mammalian
clotting factors, also contain EGF-like domains. EGF is a 53-aa
polypeptide first isolated from the submandibular gland of the male
rat; TGF contains 50 aa and has approximately 40% sequence homology
to EGF. Both EGF and TGF prohormone molecules have hydrophobic
transmembrane domains near the COOH termini, and either prohormone can
be produced as an integral membrane protein. The mature EGF is a
proteolytic processed polypeptide of the EGF precursor that exhibits
eight EGF-like repeats in addition to the mature EGF (491). EGF and
TGF appear to be the major EGF family peptides involved in mammalian
development, and both have been shown to bind to a single EGF receptor
(EGFR). EGFR has intrinsic tyrosine kinase activity (492, 493).
1. Expression, localization, and production.
Several lines of
evidence indicate that TGF and EGF are produced within the testis.
Peritubular cells and Sertoli cells isolated from 20-day-old rats
contain a mRNA species that hybridizes in a Northern blot analysis with
a human TGF cDNA probe, and an immunoblot with a TGF antiserum
confirmed the production of TGF by both Sertoli cells and
peritubular cells (494). The analysis of the developmental expression
of TGF in whole testis and isolated cell types showed that the
TGF gene was predominant early in testis development and decreased
during puberty and that TGF mRNA content was greatest in prepubertal
peritubular cells but remained relatively constant in Sertoli cells,
with a slight decline at the later pubertal stages (495).
The testicular immunohistochemical localization of TGF at various
ages has also been described (496). In neonatal rats intense staining
was seen in Leydig cells. In the 21-day-old rat, TGF was visualized
exclusively in most but not all the Leydig cells, as well as in
interstitial cell cultures, but not in Sertoli cells, germ cells, or in
Sertoli cell cultures. In contrast to the observations in tissue
sections, PMC, which contaminated the Sertoli cell preparations, showed
intense staining for TGF. In adult testis, all the Leydig cells were
positive and no staining was found in the seminiferous tubules. After
EDS treatment, no significant staining for TGF could be detected in
the interstitium, confirming the Leydig cell origin of the
immunoreactivity.
Immunoreactive EGF has been also observed in mouse and human testis (497, 498). In mouse, mature EGF-positive immunostaining was localized in Sertoli cells, pachytene spermatocytes, and round spermatids, while EGF precursor immunostaining was limited to pachytene spermatocytes and round spermatids (499). In vitamin A-synchronized rat testis, testicular EGF levels significantly higher at stages IX-XIV of the cycle of the seminiferous epithelium have been demonstrated (408). In contrast, Northern blot analysis did not detect EGF expression in isolated rat peritubular, Sertoli, and germ cells (494).
2. Receptors.
The presence of EGFR in testicular cells of
several species has been demonstrated by binding, Northern blot, and
immunocytochemistry. Specific binding sites have been shown in a clonal
strain of Leydig tumor cells (500), in enriched interstitial cell
preparations from adult rats (501), in immature porcine Leydig cells
(502), as well as in murine Leydig cells (503). Specific EGF binding
was also observed in isolated interstitial cell preparations from both
intact and Leydig cell-depleted adult rat testis, suggesting that EGF
binds to Leydig cells and to an interstitial cell fraction, possibly
the precursor of the mature Leydig cell (504). Immunocytochemical data
established that EGFR is present in Sertoli cells of adult and immature
rat testes (505). In immature rats, the receptor was also found in
presumed peritubular cells and in the cell surface of Sertoli cells
where it exhibited autophosphorylation upon stimulation with EGF (494, 506). The EGFR gene expression during the rat testis pubertal
development showed that EGFR mRNA levels were higher in the early
pubertal stages, with a predominant localization in peritubular cells
and a low level of expression in Sertoli cells, pachytene
spermatocytes, and round spermatids (495). Scatchard analysis confirmed
the presence of high-affinity receptors on peritubular cells; however,
no functional receptors were detected on Sertoli cells from any stage
of development examined (495). In adult mice, Western blotting of
testis membrane fractions revealed a specific band corresponding to
EGFR (506).
In adult nonhuman primates, a positive immunostaining for EGFR was shown in Leydig cells, Sertoli cells, and PMC, and immunoblotting of testicular membrane preparations revealed a specific band corresponding to EGFR (505, 507). Moreover, changes in the concentration of EGFR mRNA in the testes of monkeys approaching puberty have been reported (508). In humans, specific EGF binding activity was detected in testicular membrane preparations (498). Subsequent studies have confirmed the presence of EGFR in a particulate fraction of human testicular tissue (509). Cross-linking experiments revealed major binding species in a region that is thought to represent a proteolysed form of the receptor, and immunohistochemical localization of EGFRs demonstrated their presence in the interstitial tissue (509). EGFR has been observed in PMC and Sertoli cells of adult human testis by means of immunofluorescence (510).
3. Local functions.
TGF and EGF have been shown to
influence Leydig cells, Sertoli cells, and PMC functions. The effects
vary depending on time of exposure and animal species. In the MA-10
tumor cell line, EGF induces a substantial decline in the number of LH
receptors with a corresponding reduced ability of hCG to stimulate
steroidogenesis (500, 511). Similarly, EGF inhibits the gonadotropin
stimulation of testosterone production by primary cultures of adult rat
Leydig cells (512). EGF capability to decrease the hCG-stimulated
testosterone production can also be exerted via inhibition of
17-hydroxylase and 17,20-lyase activities (501).
Contrasting results have been obtained by other authors. Verhoeven and Cailleau (513) reported a stimulatory effect of EGF on basal and LH-stimulated androgen production, while an inhibitory effect was only observed in cells that, after a prolonged culture in the absence of LH, were acutely challenged with LH and EGF (513).
In porcine immature Leydig cells, EGF enhances the gonadotropin action
on testosterone formation through an increase in the availability of
cholesterol substrate and in the activity of 3ß-HSD (504). Acute
treatment of pig Leydig cells with EGF stimulated testosterone
secretion (2-fold) without affecting cAMP production; long-term
treatment produced a dose-dependent decrease of hCG receptor number but
negligible effects on hCG-induced testosterone production (514).
Similar acute EGF effects have been observed with a clonal strain of
cultured murine Leydig tumor cells (515). In humans, EGF can stimulate
steroidogenesis of isolated Leydig cells, without further enhancing the
steroidogenesis induced by maximal concentrations of hCG (450). TGF
interacts synergistically with LH to promote DNA synthesis of immature
rat Leydig cells in culture (441) and stimulates peritubular cell
proliferation, migration, and colony formation (494) but has no effect
on Sertoli cell growth and transferrin production (495). Very recently,
it was reported that TGF increases the mRNA levels and the secretion
of plasminogen activator inhibitor I in peritubular cells from
20-day-old rat testes (516). These data suggest that TGF may be
involved in the control of net protease activity of the seminiferous
tubule influencing the restructuring events occurring at specific
stages of spermatogenesis and the extracellular matrix turnover.
Intraperitoneal injection of EGF stimulates DNA synthesis in adult mice testes, the maximal stimulation occurring 8 h after injection (517).
The addition of EGF to the culture medium of seminiferous tubule segments from adult rats (518) and rat primary Sertoli cell-enriched cultures (320) results in the stimulation of INH production.
Moreover, nanomolar concentrations of EGF induce a 2-fold increase of
lactate production and inhibit by more than 50% FSH-stimulated
estradiol synthesis of prepubertal rat Sertoli cells (519). It has been
reported that EGF contains three sequence homologies with human
FSH-ß, the hormone-specific subunit of FSH, and this homology seems
to be of some significance since EGF can inhibit the in
vitro binding of [125I]human FSH to calf testis
membranes (520). Prepubertal rat Sertoli cells respond to EGF with a
small but significant rise in the secretion of both transferrin and ABP
(521, 522, 523) whereas cells from older animals are less responsive (524, 525). EGF stimulates DNA synthesis and proliferation of immature pig
Sertoli cells (439) and, alone or in combination with PDGF and TGFß,
stimulates ornithine decarboxylase (ODC) activity in Sertoli
cell-spermatogenic cell cocultures and cultured peritubular cells
(526). Accordingly, TGF or EGF single subcutaneous injections to
8-day-old mice result in a 22-fold increase of testicular ODC activity
(527). The treatment of porcine cultured Sertoli cells with EGF
increases lactate production, glucose transport, and lactate
dehydrogenase activity (528). EGF inhibits the FSH-stimulated germ cell
differentiation of adult mice testicular fragments by blocking the
proliferation of type A spermatogonia (529).
The original in vivo observation by Tsutsumi et al. (530) suggesting that submandibular gland EGF may control testicular function in an endocrine fashion gave rise to an array of studies regarding the possible role of circulating and intratesticular EGF in testicular physiology (530). Tsutsumi et al. reported that removal of the salivary glands of the mouse results in a decrease of circulating EGF to an undetectable level with a concomitant impairment of spermatogenesis, not accompanied by a modification of the circulating levels of testosterone or FSH. These alterations in spermatogenesis can be reversed by the administration of EGF. In line with this evidence, it has been reported that streptozotocin-induced diabetes in mice results in a decrease in EGF content in the submandibular glands, reduction in plasma EGF levels, and oligozoospermia, which can all be normalized by EGF administration (531). More recently, it has been shown that sialoadenectomy of sexually mature male mice results in a decline in sperm count, motility, and fertility that can be reversed by EGF administration (532). However, the submandibular gland and EGF involvement in the control of spermatogenesis has been questioned. Tokida et al. (533) reported that sialoadenectomy did not decrease plasma EGF or cause infertility in male mice, and Russell et al. (534) suggested that although sialoadenectomy decreased certain functional testicular parameters, the effects were negligible.
Mice homozygous for either disrupted TGF or EGFR gene have been
generated (535, 536, 537, 538, 539, 540). The mice defective for the TGF gene show
dramatic derangement of hair follicles and curly whiskers but are
healthy and fertile (535). The recruitment of other factors might
account for the absence of predicted pathology in TGF -/- mice. In
the case of TGF, there is a distinct possibility of functional
substitution, since TGF is known to compete with EGF binding to
EGFR, and therefore EGF (or a member of the EGF family) might be
recruited to compensate for the TGF deficiency. EGFR -/- mice show
periimplantation, midgestational, and postnatal mortality dependent on
genetic background (537, 538, 539). The homozygous mutants that lived up to
3 weeks showed abnormalities in skin, kidney, brain, liver, and
gastrointestinal tract. Organ systems with no gross histological
differences between homozygous and wild type littermates included the
pancreas, heart, skeletal muscle, skeleton, teeth, ovary at postnatal
day 12, and testis at postnatal day 8 (538).
On the other hand, mice transgenic for TGF under the control of the
MT-1 promoter (495, 541, 542) overexpress the gene in several
reproductive organs, including the seminal vesicles and the testis. The
transgenic mice have no abnormal testicular morphology or alterations
of the spermatogenesis. These observations suggest that TGF
overexpression may not perturb adult and prepubertal testis function.
On the contrary, whether or not the absence of EGFR may impair
testicular development or function is still unclear. Although the above
results demonstrate that the testis is a site of EGF and TGF
production and that testicular germ cells and somatic cells are targets
for EGF/TGF, their potential roles in the testis is uncertain
because conclusive evidence in vivo is lacking.
D. Fibroblast growth factor (FGF)
FGF belongs to a family of at least seven polypeptides that
include acidic FGF (aFGF or FGF-1), basic FGF (bFGF or FGF-2), int-2
(FGF-3), kaposi sarcoma FGF (K-FGF or FGF-4), FGF-5, FGF-6, and
keratinocyte growth factor (KGF or FGF-7) (543). FGFs are widely
distributed in many tissues and exhibit a variety of actions, including
stimulation of cell division and differentiation of cells derived from
embryonic mesoderm and neuroectoderm, neuronal growth, and angiogenesis
(544, 545).
The FGF receptors are single-chain transmembrane glycosylated proteins characterized as a subgroup of the tyrosine kinase superfamily with two or three extracellular immunoglobulin-like domains. Four distinct FGF receptor genes have been cloned. They are classified into FGFR-1/flg, FGFR-2/bek, FGFR-3, and FGFR-4 (546, 547, 548). The FGF receptor genes have a distinct pattern of expression during early embryonic development and during organogenesis.
The most widely studied FGFs are aFGF and bFGF, which exhibit 55% sequence homology (543, 545). Since both aFGF and bFGF lack a signal sequence to facilitate their secretion, they are not secreted in a soluble form, but in association with some compounds of the extracellular matrix from which they are released to exert their paracrine/autocrine role. Interestingly, FGF, apart from the high-affinity signaling receptors, also binds with lower affinity to cell surface proteoglycans that cannot transmit signals alone, but somehow modulate the ability of the growth factor or the signaling receptor to generate a biological response (549). Furthermore, it has been suggested that bFGF can act also in an intracrine fashion (550).
1. Expression, localization, and production.
bFGF gene
expression and protein production have been studied during rat prenatal
and postnatal development. The immunohistochemical distribution of bFGF
in the 18-day-old rat fetus revealed that the sex cords are completely
negative but are underlined by a strongly positive basement membrane
(551). The fibroblast-like cells of the interstitium show moderate
immunoreactivity whereas the basement membrane and the peritubular
cells are more strongly stained and in the Leydig cells the staining is
both intracellular and cell surface associated (551). In whole testis,
bFGF gene expression is predominant early in prepubertal testicular
development and decreases with sexual maturity (552). Both freshly
isolated peritubular cells and Sertoli cells express bFGF mRNA and
protein at relatively constant levels during pubertal development (552, 553), with a slight suppression at the late pubertal stages (552).
Freshly isolated mature Leydig cells also express low levels of bFGF,
and FSH increases the Sertoli cell production of bFGF-like proteins
(552). Western blots of rat and mice testicular homogenates identified
a 30-kDa bFGF-like protein (554). Immunohistochemical staining at
various postnatal ages was also studied. In 5-day-old rat testis,
prespermatogonia were immunoreactive (554). In the adult rat testis,
the cytoplasm of pachytene spermatocytes and Leydig cells was positive
while Sertoli cells were negative (554, 555). In mice a similar
distribution of bFGF mRNA and protein is observed (555, 556). bFGF has
also been isolated from bovine and human testis (557, 558).
FGF-6 mRNA can be detected in the adult but not in the pubertal mice testes (559).
2. Receptors.
The presence of high-affinity, low-capacity
receptors for FGF has been demonstrated in cultured testicular cells
from neonatal rats (560) and in immature rat (561) and porcine (562)
Leydig cells. The bFGF receptor was localized by immunohistochemistry
in Leydig cells, round and elongated spermatids, and Sertoli cells. The
presence of the receptors appeared more pronounced in stages I-VIII of
the seminiferous epithelium (555).
3. Local functions.
FGF inhibits LH-stimulated testosterone
production by cultured neonatal rat testicular cells and cultured
immature rat Leydig cells through a reduction of 17-hydroxylase and
5–3ß-HSD activity (560, 563) but enhances hCG-stimulated
testosterone formation (481), and aromatase activity (564) in cultured
immature porcine Leydig cells. Other studies have shown that bFGF
inhibits hCG-stimulated 5-reductase activity in cultured immature
rat Leydig cells (565). Subsequently, it has been shown that treatment
of immature rat Leydig cells with increasing bFGF concentrations had a
biphasic effect on 3ß-HSD activity and LH/hCG receptor number, with
lower concentrations being progressively inhibitory and higher
concentrations progressively stimulatory (566, 567). Interestingly, it
has been demonstrated that the secondary increase in 3ß-HSD activity
after addition of higher bFGF concentrations can be blocked by heparin,
while the inhibition of 3ß-HSD by low bFGF concentration can be
partially reversed by either insulin or IGF-I (566). The treatment with
FGF alone or together with hCG of mixed testicular cells from adult rat
did not affect testosterone production (501).
bFGF stimulates the secretion of IGF-I by pig Leydig cells, Sertoli cells, and Leydig cell-Sertoli cell coculture (514).
Unlike bFGF, aFGF inhibits 3ß-HSD, hCG-stimulated 5-reductase
activity, and hCG binding in cultured immature rat Leydig cells both at
low and high concentrations (567).
bFGF increases c-jun mRNA levels in immature cultured pig Leydig cells, but its effect on c-fos and jun-B mRNA is lower than that produced by hCG; furthermore, bFGF potentiates the stimulatory action of hCG on c-fos and jun-B, whereas, hCG potentiates the effect of bFGF on c-jun (568). bFGF has been shown to influence Sertoli cells because it stimulates the proliferation of immature pig Sertoli cells (410), increases c-fos and transferrin mRNAs in cultured rat Sertoli cells (553, 555, 569), and augments the production of extracellular sulfated glycoprotein-1 by a Sertoli cell line (TM4) (570). Finally, it has been reported that bFGF treatment of rat PMC induces a dose-dependent increase in the production of plasminogen activator inhibitor I, suggesting an involvement of this growth factor in the regulation of protease activity of the seminiferous tubule (516).
Although the above data demonstrate that testicular bFGF expression is developmentally regulated, and that bFGF can modulate Leydig cell, Sertoli cell, and PMC functions in vitro, the physiological role of this peptide remains unknown.
E. Platelet-derived growth factor (PDGF)
PDGF, a major mitogen for cells of mesenchymal origin, is widely
expressed in normal and transformed cells (571, 572, 573). PDGF is composed
of two polypeptide chains, named A chain and B chain, respectively,
which can combine in three disulfide-linked dimers, AA, AB, and BB,
with a mol wt of approximately 30,000 (574). The PDGF isoforms exert
their biological actions via binding to cell surface receptors that
belong to the protein tyrosine kinase family of receptors (575). Two
receptor subunits have been identified, the -subunit, which can bind
both the PDGF A chain and PDGF B chain, and the ß-subunit, which can
bind only the PDGF B chain. These subunits, upon ligand binding,
dimerize to form three high-affinity binding sites for the dimeric PDGF
ligand: an -receptor, an ß-receptor, and a ßß-receptor
(576). Due to the binding specificities of the - and ß-subunit,
PDGF-AA can bind only to -receptors, PDGF-AB can bind to -
and ß-receptors, and PDGF-BB can bind to all three (577). Apart
from being a mitogen, PDGF is a potent chemoattractant for a number of
cell types both in vitro and in vivo and
activates the early transcription of otherwise quiescent genes, several
of which encode potent cytokines and others of which are protooncogenes
(578). The widespread expression of the PDGF system in the developing
embryo has been proposed to indicate multiple functions for PDGF in the
prenatal development of mesenchymal structures (579, 580, 581, 582, 583, 584, 585, 586, 587). Mice
deficient for PDGF-B (588), PDGFR-ß (589), and PDGF-A (590) develop
cardiovascular, hematological, renal, and pulmonary abnormalities in
late gestation.
1. Expression, localization, and production.
The expression of
PDGF A chain and B chain genes was observed in rat testicular RNA 2
days before birth; it increased through postnatal day 5 and fell to low
levels in adults (591). The predominant cell population expressing
transcripts of the PDGF genes during prenatal and early postnatal
periods were Sertoli cells, while in adult animals PDGFs were confined
to Leydig cells. Accordingly, early postnatal Sertoli cells, but not
cells from older animals, produced PDGF-like substances, and this
production was inhibited dose-dependently by FSH, whereas cultured
adult Leydig cells produced PDGF-like molecules under positive hCG
control (591). This kind of developmental expression for mRNAs and
proteins of the two PDGF subunits was also confirmed by
immunohistochemistry and immunocytochemistry (591). Loveland et
al. (592) examined the expression of mRNAs encoding the PDGF
subunits in Leydig cells, primary spermatocytes, round spermatids
isolated from adult animals, and enriched preparations of Sertoli cells
obtained from 20-day-old animals. Although Leydig cells and Sertoli
cells contained mRNAs encoding both PDGF subunits, neither PDGF subunit
mRNA was detected in the germ cells (592).
2. Receptors.
In situ hybridization to localize the
expression of the PDGFR ß-subunit mRNA during organogenesis in the
mouse embryo revealed the expression of the receptor throughout the
mesenchyme of the testis starting from 12.5 day pc (593).
Immunohistochemical studies conducted in the rat fetus have shown that
little or no PDGFR-ß immunoreactivity could be detected in day 13 rat
fetus; by day 16, a strong immunoreactivity was seen among other
tissues in the gonads, and the intensity of staining peaked at day 18
and decreased by day 21 (594). Strong PDGFR-ß immunostaining has been
reported in the sex cords of the developing gonad in a 12-week-old
human fetus (594). Purified adult rat Leydig cells and PMC from
neonatal rats possess specific high-affinity, low- capacity receptors
for PDGF as revealed by ligand binding, immunohistochemistry, and
Northern analysis (591, 595). Immunohistochemical analysis in the rat
showed PDGFR-expressing cells scattered in the intertubular compartment
in the fetal testis that become organized in a cellular layer
corresponding to PMC during postnatal development (591). Purified PMC
from prepubertal rats show PDGF receptors after 24 h in culture,
and PDGF treatment increases the cytosolic Ca++
concentration in these cells (596).
3. Local functions.
The combination of PDGF and EGF induces a
significant activation of ODC in rat Sertoli cell-spermatogenic cell
cocultures after 6 h of incubation (526). After 24 h of
incubation, PDGF does not appear to affect activity if either EGF or
TGF-ß is present, while PDGF alone inhibits ODC activity in Sertoli
cell-spermatogenic cell cocultures. Moreover, PDGF significantly
stimulates ODC activity of PMC after 6 h of incubation both alone
or in combination with EGF and TGF-ß, while after exposure for
24 h no effect can be observed. These findings raise the
possibility that PDGF has the ability to down-regulate ODC activity in
Sertoli cell-spermatogenic cell cocultures but not in nonepithelial
peritubular cells. This contrasting effect led to the suggestion that
PDGF might play an important role in spermatogenic cell differentiation
and a more mitogenic function in the peritubular cell population (526).
The predicted activity on PMC was later confirmed since the PDGF
treatment of cultured rat PMC stimulated cellular proliferation and
increased the release of type IV collagen, fibronectin, type V
collagen, and laminin (596) and synergistically with TGFß stimulated
the contraction of PMC in culture (483). In contrast, PDGF did not show
any stimulation of spermatogonial differentiation of type A
spermatogonia in organ culture of adult mouse cryptorchid testis (443).
Moreover, PDGF shows a strong chemotactic activity for purified
immature rat PMC (591). In cultured immature Leydig cells, PDGF
inhibits hCG-stimulated 5-reductase activity, basal
5-3ß-HSD activities, and hCG-stimulated testosterone
formation (597). Also in cultured purified adult rat Leydig cells,
exposed to a minimally stimulating dose and maximally stimulating dose
of LH, PDGF-BB stimulates testosterone production in a dose-dependent
manner (598, 599). PDGF markedly inhibits aromatase activity stimulated
by cAMP of human adipose stromal cells (600).
Based on the above data, a tentative model of the developmental regulation that could be exerted by the PDGF system in the rat testis has been presented (591). In the prenatal and early postnatal period the PDGF molecules produced by the Sertoli cells could chemotactically attract the intertubular precursors of the PMC to the peritubulum and induce them to divide. In adults, PDGF could modulate Leydig cell production of testosterone in an autocrine way. However, in vivo evidence is needed to verify this assumption. The generation of PDGF-B-, PDGF-A-, and PDGF-ß-receptor-deficient mice has not helped to address this question for the following reasons. Both PDGF-B and PDGF ß-receptor knock-out mice die perinatally and show kidney glomerular defects due to the absence of mesangial cells, hemorrhages, anemia, and thrombocytopenia (588, 589). PDGF-A null allele is also lethal with two restriction points, one prenatally before embryonic day 10 (50% loss of embryos) and one postnatally (30% perinatal death, 20% postnatal day 20 death) (590). Postnatally surviving PDGF-A-deficient mice develop lung emphysema secondary to the failure of alveolar septation. This is apparently caused by the loss of alveolar myofibroblasts. The fact that these animals do not survive to reproductive age and the absence of description of the testis phenotype do not provide information on the developmental and regulatory effects of the PDGF system in the testis. However, it is interesting to note that in all cases of PDGF and PDGF receptor gene disruption, a myofibroblast-type cell is affected (mesangial cells and alveolar myofibroblasts), and during testicular development the PMC, which are also a myofibroblast-type cell, are the principal target of the PDGF system action.
In conclusion, although circumstantial evidence indicates an involvement of PDGF in the control of testicular development, further studies are needed to confirm this hypothesis.
F. Nerve growth factor (NGF)
NGF is a protein essential for the development and maintenance of
sensory and sympathetic neurons of the peripheral nervous system (601).
It interacts with two distinct receptor entities that are distributed
among both responsive and nonresponsive cells. One, which has been
identified as the protooncogene product of trk (generally
designated TrkA), is a prototypical growth factor receptor containing a
tyrosine kinase in its internal domain. The second receptor, called
LNGFR (identifying it as the low-affinity NGF receptor), lacks this
type of entity. It does possess a G protein consensus interaction site
at the C terminus, but an activity involving such a molecule linked to
this receptor has yet to be demonstrated (602).
1. Expression, localization, and production.
A positive
immunoreactivity for NGF has been reported in the adult mouse testis
(603). This immunoreactivity appeared to be localized to the cells of
the germ line. Most stages, from primary spermatocytes to mature sperm,
were positive. The presence of NGF mRNA and protein in adult rodent
testis has also been demonstrated by in situ hybridization,
Northern analysis, immunohistochemistry, enzyme-linked immunoassay, and
a test of biological activity (604). NGF mRNA and NGF-like
immunoreactivity was detected in spermatocytes and early spermatids of
adult mouse and rat testis. The presence of NGF-like substance in a
testicular extract was also confirmed by a bioassay. An analysis of the
stage-specific expression of NGF during the cycle of the seminiferous
epithelium in the rat revealed NGF mRNA and protein at all stages of
the cycle (605).
A quantitative determination by immunoassay of NGF in human testis revealed the presence of 5.44 ng of ß-NGF per g wet weight (606).
2. Receptors.
A high level of LNGF receptor mRNA was detected
in the mouse testis (604). Subsequently, the LNGFR mRNA was found in
Sertoli cells of adult rat testis in tubules at stages VI to VIII of
the seminiferous epithelial cycle, and its expression was
down-regulated by testosterone (607). This characteristic distribution
of NGF and NGF receptor has been confirmed in adult human testis (608).
Further studies conducted in human testicular tissue have shown that
NGF receptors are localized on cells of the lamina propria (606).
TrKA mRNA, encoding an essential component of the high-affinity NGF receptor, is present at all stages of the seminiferous epithelium (605, 609); in contrast, the expression of LNGFR mRNA was found only in stages VII and VIII of the seminiferous cycle (605). The LNGFR protein was present in the plasma membrane of stages VII to XI on the Sertoli cells apical part facing the lumen of the tubules and in the basal compartment (605). Further studies on LNGFR mRNA expression during testicular development have shown that LNGFR is expressed in the peritubular cells of the embryonic mouse testis (610). Immunohistochemical analysis in the rat showed LNGFR-expressing cells scattered in the intertubular compartment in the embryonic testis that become organized in a cellular layer that surrounds PMC during postnatal development (610). Furthermore, in peripubertal and adult mouse and rat testis, the expression of a shorter mRNA of 3.2 kb that cross-hybridizes to the LNGFR transcript (3.7 kb) has been identified. This shorter mRNA has been localized by in situ hybridization particularly in spermatids at stages VII-IX of the mouse seminiferous epithelium cycle (610).
3. Local functions.
Addition of NGF to the culture medium of
mechanically isolated seminiferous tubules from human testes stabilizes
specific functions of the seminiferous tubules such as the maintenance
of the myoid phenotype of the lamina propria, the prevention of
thickening of the tubular wall, and the stabilization of Sertoli cell
morphology and function (611). In the adult rat, NGF dose-dependently
increases DNA synthesis of seminiferous tubule segments with
preleptotene spermatocytes at the onset of meiosis while other segments
remain nonresponsive (605). The infusion of NGF into adult rat testis
in vivo causes an increase in the level of testicular ABP
mRNA probably caused by prolongation of stages VII-VIII of the
seminiferous epithelium that display the maximal ABP mRNA expression
(612).
NGF- and TrkA-targeted mice have been generated (613, 614). Both mutants show reduced growth and survival rate and suffer complete loss of sympathetic neurons and sensory neurons responsive to temperature and pain. NGF (-/-) mice have a maximal life span of 4 weeks, which is somewhat shorter than the life span observed for TrkA (-/-) mice (8 weeks). The fertility potential of NGF mutant could not be analyzed, and a detailed histological analysis of nonneuronal tissue has not been conducted. Mice that are heterozygotes for NGF gene disruption display a mild phenotype but grow and breed normally. As reported above, the majority of TrkA (-/-) mice die after weaning stage; however, it has been reported that those that reach adulthood remain infertile (615). Also in this case the lack of histological analysis and the profound systemic effect of knocking out do not allow conclusions on the suggested role of NGF in the male reproductive system.
Mice carrying a mutation of the gene encoding the LNGFR have also been produced (616). The homozygous animals displayed a complex phenotype with deficit in cutaneous innervation and heat sensitivity but were viable and fertile. The observed phenotype is much less severe than might have been expected considering the expression patterns of the LNGFR gene. This indicates either that LNGFR has no essential role in the development of the many tissues that express it or that its function is redundant and its absence can be compensated for by another protein.
In conclusion, the data reported above suggest that NGF may be involved in the regulation of testicular morphogenesis and function. However, definitive in vivo experimental proofs are lacking.
G. Steel factor (SLF)
SLF, also called mast cell growth factor, stem-cell factor, or
hematopoietic cell growth factor, is a protein whose gene maps to
chromosome 10 in the vicinity of the steel (Sl) locus. The
SLF protein can be expressed in a biologically active form as either a
membrane-bound protein or as a soluble factor. SLF is the ligand for
the c-kit protooncogene receptor encoded by the murine
dominant white spotting (W) locus. The product of the
c-kit protooncogene is a member of the PDGF family of
tyrosine kinase receptors. The SLF/c-kit system is essential
in melanogenesis, gametogenesis, and hematopoiesis during embryonic
development and postnatal life (617, 618).
1. Expression, localization, and production.
SLF gene
transcripts have been detected in the mouse embryo along the migratory
pathways of the PGC and in the gonads (619), and its postnatal
testicular expression is relatively high at all ages (620). The forms
of SLF mRNA present in the testis suggest that from postnatal day 5
onward the membrane-bound form of SLF predominates (620). As observed
by in situ hybridization and immunohistochemistry, the SLF
expression is distinct in Sertoli cells but not in germ cells from
postnatal day 1 to postnatal day 9; thereafter the intensity of
expression declines. SLF in Sertoli cells appears to be concentrated
basally at the stage of the cycle of the seminiferous epithelium where
it is known to interact with differentiating type A spermatogonia
(620). Treatment of Sertoli cell cultures with cAMP analogs led to a
significant increase in the SLF mRNA levels (621). The production of
the membrane-bound form of SLF by mouse Sertoli cells has been
demonstrated by means of a bioassay, based on the ability of Sertoli
cell cultures from Wv/Wv but not
Sld/Sld mice, to support the growth of
cocultured mast cells (622). Further studies on the regulation of SLF
production by mouse Sertoli cells have produced contradictory results.
Rossi et al. (623) reported that FSH and cAMP were able to
increase the mRNA levels for both the soluble form and the
transmembrane form of SLF in cultured primary mouse Sertoli cells. The
inductive effect of cAMP was more pronounced in cultures from
13-day-old animals than in cultures from 18-day-old animals (623).
Tajima et al. (624) showed that the treatment of mast
cell-Sertoli cell coculture with cAMP increased SLF production whereas
FSH had no significant effect.
In human testicular tissue, a positive staining for SLF was demonstrated within the Leydig cells and less intensely in the area of Sertoli cells (625).
2. Receptors.
In situ hybridization studies showed
that c-kit is expressed during mice embryogenesis in the PGC
before and after migration into gonadal ridges (626). The
c-kit mRNA was found to be expressed at high levels in
spermatogonia, and at lower levels in meiotic pachytene spermatocytes
purified from 18-day-old mice (627). Rossi et al. (628)
cloned a spermatid-specific 3.2-kb cDNA in mouse that encodes a
truncated c-kit protein lacking the extracellular and
transmembrane domains and part of the tyrosine kinase domain. In
situ hybridization revealed c-kit transcripts in mouse
spermatogonia beginning at 6 days after birth and labeling of Leydig
cells at all ages examined (629). The synthesis of the c-kit
receptor by isolated type A spermatogonia from 9-day-old rat testis was
established by Northern blot hybridization, immunocytochemistry, and
Western blot analysis and was found to be constitutively
autophosphorylated on tyrosine with a significant increase in
phosphorylated protein upon stimulation with the kit ligand
SLF (630).
Normal human testis stained for c-kit in Leydig cells, in the cytoplasm of early spermatogenic cells, as well as in the acrosomal granules of the round spermatids and in the acrosome of testicular spermatozoa (625).
3. Local functions.
After the demonstration that
c-kit is allelic with W locus in mice and SLF is
mapped to Sl locus, the relationship between W
and Sl mutations has been clarified (631). The phenotype
analysis of W or Sl mice proved that
c-kit and its ligand are indispensable for gametogenesis,
melanogenesis, and hematopoiesis (632). During gametogenesis, receptor
activation by SLF acts primarily as a homing mechanism for migration or
as a viability factor critical for clonal survival of PGC. Expression
of the mRNA for c-kit and SLF at the destination site
continues after migration is complete, suggesting that receptor
signaling may also have a late role in local proliferation and
differentiation. The transmembrane form of SLF supports the survival
but not the proliferation of mouse PGC (633, 634). Soluble recombinant
SLF stimulates, in a dose-dependent fashion, thymidine incorporation in
cultures of isolated germ cell populations enriched in spermatogonia.
Autoradiographic analysis shows that SLF selectively stimulates DNA
synthesis in type A spermatogonia (623). Despite the evidence for
postnatal expression of c-kit and SLF, whether
c-kit and its ligand are required for the maintenance of the
germ cells in postnatal life is difficult to address by phenotype
analysis because virtually no germ cells are present in the gonads of
W and Sl mice.
In a very interesting paper, Yoshinaga and colleagues reported that intraperitoneal injection of an anti-c-kit monoclonal antibody to prepubertal mice completely blocks the mitosis of type A spermatogonia but not the mitosis of gonocytes, spermatogonial cell precursors, or Sertoli cells, and intravenous injection of the same antibody into adult mice causes a depletion in the differentiating type A spermatogonia (635). These data demonstrate that the maintenance and/or mitosis of the differentiating type A spermatogonia requires c-kit and its ligand (635). In summary, the c-kit receptor system is essential in several stages in gametogenesis. During development it provides a proliferative, migratory and/or cell survival signal for PGC. Postnatally, during spermatogenesis c-kit/SLF is thought to facilitate proliferation and/or survival of spermatogonia.
H. Gastrin-releasing peptide (GRP)
GRP is a 27-aa peptide that belongs to the family of the bombesin
(BN)-like peptides. The BN-like peptides are a diverse family of
peptides originally characterized in frog skin and later found to have
a wide distribution and range of actions in mammals including secretion
of gastrointestinal peptide hormones, regulation of smooth muscle
contraction, modulation of neuronal firing rate, and growth (636). The
BN-like peptides have classically been divided into three subfamilies,
the BN subfamily, of which GRP was considered the mammalian form; the
ranatensin subfamily, of which neuromedin-B was considered the
mammalian form; and the phyllolitorin subfamily, which to date has only
been characterized in amphibians. After the identification and
molecular cloning of a true amphibian gut GRP distinct from amphibian
BN (637) and phylogenetic analysis of BN-like peptide prohormone
sequences (638), the BN-like peptides were reclassified into GRP
subfamily, neuromedin-B subfamily, and skin peptide subfamily.
Three receptors for BN-like peptides have been cloned to date, a GRP-preferring subtype (639), a neuromedin B-preferring subtype (640), and a third subtype, designated BN receptor subtype 3 (BRS-3), with an as yet unknown ligand (641). All the receptors belong to the seven membrane-spanning domain G protein-coupled receptor superfamily. A fourth BN receptor subtype (BB4), with high affinity for the BN-related peptides distinct from GRP, has been cloned very recently from frog brain and suggested to be present also in mammals (642).
1. Expression, localization, and production.
Purified adult
rat Leydig cells were found to produce immunoreactive GRP by RIA; the
GRP production was not stimulated by hCG treatment of the cell cultures
(643, 644). HPLC analysis of the Leydig cell-conditioned medium extract
revealed the presence of two biologically active C-terminal fragments
of the GRP, GRP 18–27 and GRP 14–27. Immunohistochemical studies in
the adult testis showed specific staining for GRP localized in the
Leydig cells in both rat and human tissue (643, 644).
2. Receptors.
Recently, human genomic and cDNA clones encoding
a new BN-like peptide receptor subtype, called BN receptor subtype 3
(BRS-3), have been isolated and characterized (641). Chromosome mapping
studies indicate that the BRS-3 gene is located in human chromosome X.
Interestingly, mRNA expression in rat tissues is limited to the testis
on secondary spermatocytes and has also been found to be widely
expressed in a panel of human cell lines from all histological types of
lung carcinoma (641). Expression of BRS-3 cDNA in Xenopus
oocytes encodes a functional receptor that is activated by BN-like
peptides (BN, GRP, Phe8-phyllolitorin, and ranatensin)
applied at 10-5 M concentration. Whether BRS-3
may be a low-affinity receptor for BN agonists examined thus far or for
a high-affinity BN-like ligand yet to be defined is unknown. It is
noteworthy that since BRS-3 maps on chromosome X and its mRNA
expression is limited to secondary spermatocytes, which are haploid and
possess either an X or Y chromosome, only half of these cells could
potentially express BRS-3. Moreover, specific high-affinity,
low-capacity binding sites have been found on cultured PMC isolated
from prepubertal rat testis by using [125I]GRP as ligand
(L. Gnessi, unpublished results), and it has been reported that GRP can
transmodulate the binding of PDGF to PMC by reducing the number, but
not the affinity, of its receptors (645).
3. Local functions.
To date no evidence for local testicular
functions of GRP have been reported.
Although the above data demonstrate the presence of GRP-like substances and BN receptor subtypes in the testis, the potential physiological significance of testicular GRP remains to be established since in vitro and in vivo evidence supporting a local action of the peptide is lacking.
|
V. Immune Derived Cytokines |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
IL-1 is the term for two polypeptides (IL-1 and IL-1ß). Although
both forms of IL-1 are distinct gene products, they recognize the same
cell surface receptors and share the various biological activities.
IL-1 is the prototype of the proinflammatory cytokines in that it
induces the expression of a variety of genes and synthesis of several
proteins that, in turn, induce acute and chronic inflammatory changes.
An endogenous antagonist for the IL-1 receptor (IL-1ra) has recently
been isolated and cloned and may play a role in modulating the effects
of IL-1. IL-1, IL-1ß, and IL-1ra all bind to the two IL-1 receptor
subtypes isolated and cloned to date. The IL-1 receptor type I
(IL-1RtI) and IL-1RtII are both members of the Ig superfamily of
receptors. The major difference between the type I and type II receptor
is the truncated cytoplasmatic portion of the type II receptor. No
clear picture has emerged from the studies on the signal transduction
events after the binding of IL-1 to either one of its cell surface
receptors (646).
Il-2 is a glycoprotein with an apparent molecular mass of 15.5 kDa,
which plays a pivotal role in T cell activation (647). There are three
forms of IL-2 receptors that can be distinguished on the basis of their
affinity for the ligand. The receptor is an heterodimeric complex
composed of three receptor subunits, IL-2R, IL-2Rß, and IL-2R.
Independent IL-2R or IL-2Rß subunits generate low and intermediate
affinity receptors, whereas high-affinity receptors are formed when all
receptor subunits are noncovalently associated also with IL-2R,
which plays a pivotal role in facilitating IL-2 binding by IL-2Rß and
in receptor signaling. The ß- and -subunits have been shown to
bind to cytoplasm protein-tyrosine kinases of the jak family
(648).
IL-4 is a glycoprotein with an approximate molecular mass of 15–19
kDa. It is made in response to immunological recognition, principally,
although not exclusively, by CD4+ T lymphocytes. IL-4 has a
wide range of functions on B cells, T cells, macrophages, hematopoietic
precursor cells, and stromal cells (649). IL-4 mediates its functions
by binding to a high-affinity receptor complex, composed of the
IL-4-binding receptor chain and a common -chain. The proposed
mechanism for signaling through the IL-4 receptor complex involves the
activation of protein tyrosine kinases associated with the cytoplasmic
domains of the -chain and the IL-4R chain (650).
IL-6 is a multifunctional cytokine that acts as a differentiating and proliferative factor on B cells, T cells, hepatocytes, and hematopoietic progenitor cells (651). Binding of IL-6 to the IL-6 receptor results in the homodimerization of a non-ligand-binding 130-kDa signal-transducing molecule, gp130, activation of the tyrosine kinase jak2, and the consequent initiation of the Ras-dependent mitogen-activated protein kinase cascade (651, 652).
1. Expression, localization and production.
In 1987 Khan
et al. (653) reported the isolation of an IL-1-like factor
from intact testis of adult rat. IL-1-like activity was found in the
extracts and medium conditioned by cultures of normal and cryptorchid
seminiferous tubules but not in interstitial cells or in culture media
conditioned by 10-day-old rat Sertoli cells. This indicates that the
testicular IL-1-like factor was produced by cells in the seminiferous
tubules, most probably by Sertoli cells, in adult but not in
prepubertal rats (653). The appearance of the IL-1-like factor during
postnatal development and its cellular origin have been further
investigated by a murine thymocyte proliferation assay (654). Very low
IL-1 activity was seen in culture medium conditioned by seminiferous
tubules from rats aged 10 or 20 days. From 30 days of age, increasing
amounts were detected, with maximum levels being attained in adult
animals. No IL-1 activity was found in medium conditioned by
peritubular cells. Sertoli cell-enriched seminiferous tubules obtained
from experimentally cryptorchid or from prenatally irradiated rats
produced much higher levels of IL-1 activity than did those obtained
from intact testes, suggesting that the testicular IL-1-like factor is
produced by Sertoli cells and that its appearance during development
coincides with the initiation of active spermatogenesis (654). An
IL-1-like factor is produced by adult rat seminiferous tubules segments
containing all stages of the spermatogenic cycle except for stage VII
(655).
Human testicular cytosol preparations exhibited significant IL-1-like
activity that could be neutralized by specific IL-1 antibodies in a
bioassay (656). An IL-1-like factor has been isolated from adult rat
testis interstitial fluid (657). In vitro experiments have
confirmed that both human and rat seminiferous tubules secrete IL-1,
and cultured medium from rat Sertoli cells contains IL-1 bioactivity
that could be neutralized by an anti-IL-1 antibody (658). However,
further reports revealed that other cell types contribute to the
production of IL-1 in the testis. Human recombinant IL-1ß and
bacterial lipopolysaccharide (LPS) cause a dose-dependent increase in
IL-1 mRNA expression in purified mature rat Leydig cells, indicating
Leydig cells as a potential source of IL-1 (659). Lin et al.(660) have demonstrated that both IL-1 and IL-1ß mRNAs are
expressed in IL-1ß-stimulated adult rat Leydig cells, with IL-1ß
being the predominant species, and that in Leydig cells IL-1 mRNA can
be induced by a single injection of hCG in vivo.
Interestingly, the phagocytosis of residual bodies or cytoplasts from
elongated spermatids, or even the phagocytosis of latex beads, induces
the secretion of IL-1 by rat Sertoli cells in vitro
(661). Freshly isolated macrophages from adult rat testis secrete a
lymphocyte-activating activity that is responsive to bacterial LPS
stimulation and is blocked by IL-1ra, a naturally occurring antagonist
that specifically inhibits both IL-1 and IL-1ß (662). However, the
levels of IL-1 secreted by the testicular macrophages are considerably
reduced compared with a population of resident macrophages from
peritoneum cultured under identical conditions.
Sertoli cells prepared from rats of increasing ages were found to
secrete IL-6 in vitro; LPS, latex beads, residual bodies,
and cytoplasts from elongated spermatids markedly stimulated IL-6
secretion at all ages investigated (663). The levels of IL-6 varied
throughout different stages of the seminiferous epithelium cycle; the
highest levels were observed in stages II-VI and the lowest were seen
in stages VII-VIII. Furthermore, FSH differentially stimulated IL-6
secretion during the seminiferous epithelial cycle (663). IL-6 is also
secreted from enriched preparations of adult rat Leydig cells, and its
release is increased in a dose-dependent manner by hCG and IL-1ß
(664). Cultured mouse Sertoli cells produce IL-6 and its production is
enhanced by IL-1, IL-1ß, TNF, and LPS and inhibited by
interferon- (INF) (665).
2. Receptors.
Specific IL-1 receptors have been identified in
crude membrane preparations of mouse testis (666). IL-1 showed
significantly higher specific binding than IL-1ß. However, binding of
IL-1 was species-dependent, with a barely detectable signal in rat
and guinea pig testis (667). In autoradiographic studies, IL-1
receptors were heterogeneously distributed, with highest densities
present in the interstitial areas of the mouse testis (668). Type 1
IL-1 receptor mRNA, localized in the mouse testis with in
situ hybridization, confirmed the autoradiographic data with an
intense signal observed over the interstitial cells (669). The paucity
of IL-1 binding in the rat (667) is somewhat puzzling, since
recombinant human IL-1 produces a variety of actions in the rat testis
(see below), suggesting the possibility of the presence of a novel IL-1
receptor in the rat that may be important in mediating the effects of
IL-1 in this species.
3. Local functions.
Verhoeven et al. (670) showed
that IL-1ß alone stimulates steroid production in short-term cultures
of enriched rat Leydig cells from 19-day-old animals, whereas
LH-induced androgen formation is inhibited under longer incubation
conditions (670). IL-1 is a potent inhibitor of adult Leydig cell
function in that it diminishes hCG binding and blocks hCG-stimulated
testosterone and cAMP formation in primary cultures of mature rat
Leydig cells (671, 672, 673). The observed inhibitory effects of IL-1 on
Leydig cell testosterone production are predominantly due to a block of
the expression of P450c17 and P450 scc (674, 675, 676). A dose-dependent
inhibitory action of IL-1ß on hCG-stimulated testosterone production
was found in a primary culture of neonatal rat testicular cells (677).
In immature porcine Leydig cells, IL-1 and, to a lesser extent,
IL-1ß are potent inhibitors of hCG-stimulated testosterone production
(678).
Only two studies have reported a stimulatory effect or a lack of effect
of IL-1 on Leydig cell steroidogenesis. In one, an IL-1ß mediated
stimulation was observed in both basal and LH-induced testosterone
production by adult rat Leydig cells in culture (679), while in another
the lack of effect of both IL-1 and IL-1ß on LH-stimulated
testosterone production was reported (680).
These data suggest that the actions of IL-1 on Leydig cell steroidogenesis in vitro may be stimulatory, inhibitory, or ineffective under different culture regimens.
In the tubule, IL-1ß inhibits FSH-induced aromatase activity in immature rat Sertoli cells (681) and stimulates Sertoli cell transferrin secretion (682).
IL-2 is also a potent inhibitor of adult rat Leydig cell
steroidogenesis in primary culture. IL-2 inhibits hCG-stimulated
testosterone and cAMP formation but has no effects on the binding of
hCG to Leydig cells. It also blocks cAMP and forskolin-induced
testosterone formation (683). Meikle et al. (684) reported
that although IL-2 inhibits hCG-stimulated testosterone synthesis in
isolated murine Leydig cells, it stimulates testosterone production in
minced testes, suggesting an IL-2-mediated release of regulatory
factors from other cells that are able to overcome the direct
inhibitory effect of IL-2. Locally injected IL-1ß, but not IL-1,
induces acute inflammatory-like changes in the testicular
microcirculation of adult rats (685). IL-1 is able to enter the
testis in intact form and to cross the blood-testis barrier, which is
consistent with the finding of a direct action of IL-1 on gonadal
function (686).
Intraperitoneal injection of the bacterial endotoxin LPS induces a significant increase of IL-1ß concentration and a decrease in the density of IL-1 receptors in the testis (687).
IL-1 may act as a growth factor for spermatogonia and thus participate in the regulation of spermatogenesis (655, 688). A similar mitogenic effect on Leydig cells isolated from immature rats has been reported for IL-1ß (689), and Lin et al. (690) demonstrated that IL-1ß can inhibit IGF-I mRNA expression in adult rat Leydig cells, both in vivo and in vitro. Interestingly, the ontogeny of testicular IL-1 activity during normal sexual maturation and photoperiod-induced regression in the seasonal breeding of bank voles is in accordance with a proposed role for IL-1 as a mitogen for germ cells (691). Testicular IL-1 activity can first be detected at puberty as spermatogenesis is activated and increases until full sexual maturity is reached, whereas it fails to appear when pubertal development and the initiation of spermatogenesis is arrested by light deprivation (691).
IL-2 and IL-6 increase the release of transferrin from rat Sertoli cells obtained by microdissection of IX-XI- and XIII-staged seminiferous tubule segments; moreover, IL-6 increases basal and FSH-induced transferrin secretion (682, 692).
It has recently been demonstrated that IL-4 is a positive regulatory factor for mouse PGC in vitro in that the IL-4 protein is present in the vicinity of proliferating PGCs in vivo. Both subunits of the high affinity IL-4 receptor are expressed in PGC-containing tissues, suggesting a developmental role for IL-4 as a survival factor for mouse PGCs in vivo (693).
Because of their pronounced effects on the immune system and the hematopoietic system, IL- and IL receptor-deficient mice have been generated (see Refs. 694 and 695 for review). The results from these studies indicate that ILs do not play unique roles in the development of the immune system and steady state hematopoiesis. However, impairment or modulation of the immune and/or inflammatory responses has been shown to occur in each of the IL/IL receptor-deficient mice, demonstrating that no IL is dispensable or completely redundant in vivo. Regarding the reproductive function, IL-1 (696), IL-2 (697), IL-2 receptor (698, 699), IL-4 (700, 701), and IL-6 (702, 703) deficient mice are healthy and fertile. Thus it appears that in normal conditions the testicular effects of these cytokines are either redundant or probably subtle.
Mice overexpressing IL-2, IL-4, and IL-6 have been generated. Mice with an IL-6 transgene driven by the Ig enhancer (Eµ-IL-6), analyzed at ages varying from 7 to 18 weeks, develop oligoclonal plasmacytoses; there is no information on testicular appearance or fertility in these animals (704).
IL-4 transgenic mice, despite their normal gross appearance at birth, develop severe runting and die within the first 2 weeks of life (705). They show severe hypoplasia of the thymus and marked lymphocyte depletion in the spleen. When transgene expression was attenuated, a marked increase in serum IgE levels and the appearance of an inflammatory ocular lesion resembling the inflammatory reaction observed in human allergic disease was observed. The few attenuated transgenic founders generated were capable of breeding.
Interestingly, male transgenic mice with the murine MT-1 promoter-human IL-2 fusion gene have atrophic testes and motor ataxia due to infiltration of lymphocytes into the cerebellar tissue (706). In these mice, the testes were depleted of spermatogenic cells without any apparent signs of an immunological response. Whether this dramatic effect could be ascribed to the reported potent inhibitory action of IL-2 on testosterone production in Leydig cells (619, 620) is unclear.
In summary, the effects of ILs in the testis are complex. The ILs secreted by various cellular testicular components may be involved in discrete local paracrine/autocrine modulatory actions. Nevertheless, it is important to notice that IL production can be induced within the testis under appropriate conditions, such as after LPS administration (659, 661, 662, 663, 665), and this could be a potential mechanism for the reduction of fertility and androgen secretion that accompanies testicular inflammation (707).
B. Tumor necrosis factor- (TNF)
TNF is a potent cytokine, produced mainly by activated
macrophages, that has numerous biological functions, including
hemorrhagic necrosis of transplanted tumors, cytotoxicity, and an
important role in endotoxic shock and in inflammatory,
immunoregulatory, proliferative, and antiviral responses (708).
Receptors for TNF are expressed in the majority of mouse and human
cell lines. Two distinct TNF receptor subtypes (type I and type II)
have been identified (709).
1. Expression, localization, and production.
Recent studies
have demonstrated that mouse spermatids secrete TNF and that both
pachytene spermatocytes and round spermatids contain mRNA for TNF as
detected by both Northern blot analysis and in situ
hybridization (710). There was much less overall hybridization with
TNF antisense probes on sections of testis from a 16-day-old mouse
compared with that in adult testis (710). Presumptive interstitial
macrophages were also labeled (710). Testicular macrophages from adult
rats release TNF in vitro, while medium from cultured
Sertoli cells, Leydig cells, and peritubular cells did not contain
TNF activity (711). Further studies revealed that testicular
macrophages do not constitutively release TNF both in
vivo and in vitro and that the reported release of
TNF in vitro was due to the activating effect on
macrophages of the collagenase used to isolate the cells (712).
However, testicular macrophages release TNF when exposed to
bacterial endotoxins (712).
2. Receptors.
Purified cultured porcine Leydig cells from
immature animals appear to posses specific TNF receptors (713), and
adult mice Sertoli and Leydig cells contain mRNA for the TNF
receptor (710).
3. Local functions.
TNF augments testosterone secretion in
both whole testis cell cultures, as well as purified Leydig cells from
adult rat, and produces maximal increases of hCG-induced testosterone
secretion (679). The effect of TNF administered by continuous
intravenous infusion on testicular function in rats has been also
studied (714). Testicular weight decreases within 24 h, and germ
cells damage is observed together with a fall in plasma testosterone
and an increase in LH and FSH levels. The decrease in testosterone
concentration and increase in gonadotropin levels suggest that TNF
interferes with Leydig cell function; germ cell damage may be either
direct or secondary through Leydig and/or Sertoli cell dysfunction.
Moreover, TNF enhances the inhibitory effect of IL-1ß on Leydig
cell steroidogenesis (715).
TNF reduces hCG-stimulated, but not basal, testosterone secretion by
cultured purified Leydig cells isolated from immature porcine testes in
a dose- and time-dependent manner, antagonizing the LH/hCG action on
testosterone formation predominantly through a decrease in the
availability of cholesterol substrate in the mitochondria (713).
Subsequently, Xiong and Hales (716) reported that TNF inhibits both
basal and cAMP-stimulated testosterone secretion from isolated adult
mouse Leydig cells, and this inhibition was parallel to the
TNF-mediated repression of the cholesterol side-chain cleavage
enzyme P450scc and P450c17 mRNA and protein levels.
Interestingly, the experimental autoimmune orchitis induced in adult
male mice can be attenuated when the affected animals are injected with
neutralizing antibody to TNF, but not neutralizing antibody to
INF- (717).
A recent study has shown that TNF stimulates proliferation of PGCs
in culture, suggesting possible involvement of TNF in the
proliferative regulation of the PGCs during embryonic development
(718).
However, homozygous TNF receptor-1 (719, 720) and TNF receptor-2 (721)
gene-targeted mice show higher susceptibility to infections (719) and
fail to develop germinal centers in peripheral lymphoid organs (720)
but are viable, breed normally, and show no apparent phenotypic
anomalies. The absence of defects in the germ cell compartment of the
TNF receptor mutants suggests that other signals in vivo
(SLF, IL-4) may be a sufficient supply for PGCs during normal embryonic
development. In light of the above results, TNF may be considered
one of the factors involved in the detrimental effects of inflammation
on testicular function.
|
VI. Vasoactive Peptides |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
ET-1 is produced mainly in endothelial cells and vascular smooth muscle cells, but it is also produced in neurons and astrocytes, endometrial cells, hepatocytes, kidney mesangial cells, and breast epithelial cells. The majority of ET-1 secretion from cultured endothelial cells is toward the vascular smooth muscle side of the cells, where it can bind to specific receptors and cause vasoconstriction.
ET-2 is produced predominantly within the kidney and intestine, with smaller amounts produced in the myocardium, placenta, and uterus.
ET-3 has been found in high concentrations in the brain (726) and may regulate important functions in neurons and astrocytes, such as proliferation and development. It is also found throughout the gastrointestinal tract, in the lung and kidney.
Two distinct ET receptor subtypes, A and B, have been cloned (727, 728). The two ET receptors differ in their ligand selectivities: ET receptor B binds to the three ET isopeptides with similar affinity, whereas ET receptor A does not bind ET-3. The receptors are members of the superfamily of receptors linked with guanine nucleotide-binding proteins.
1. Expression, localization, and production.
Adult rat testis
contains ET-1 mRNA (729, 730) and peptide (731) and ET-3 mRNA (732) in
relative abundance. Further studies have demonstrated that cultured rat
Sertoli cells, but not Leydig cells, from 20-day-old rats secrete a
peptide with the immunological and chromatographic properties of ET-1
and that this secretion is greatly inhibited by FSH (733).
Immunocytochemical studies also confirmed that ET-1 is exclusively
localized in Sertoli cells in immature rats, but in adult testis some
staining is also observed in the interstitium (733). Subsequent
immunohistochemical studies in adult rat testis showed immunoreactive
ET-1 in Sertoli cells, endothelial cells, and most, but not all,
interstitial cells. The intratesticular levels of immunoreactive ET-1
are significantly increased after hCG treatment (734). The recent
demonstration that the testis contains exceptionally high levels of
ET-converting enzyme (725) and that this enzyme is localized in Leydig
cells and not in the seminiferous tubules (735) suggests that Leydig
cells could be the principal site of testicular ET-1 synthesis.
The expression of a specific transcript for ET-1, and ET-1-like immunoreactivity, has been found in human testis (736). Positive staining was confined to the Sertoli cells and a small number of peritubular and interstitial cells (736).
2. Receptors.
Specific high-affinity ET receptors have been
demonstrated in mouse Leydig tumor cells (737), in normal immature rat
Leydig cells (733), and PMC (738, 739), while the receptors were
expressed at very low concentrations in Sertoli cells and spermatogenic
cells. The immunochemical characterization of the ET receptors in the
bovine testis revealed that the ET receptor A subtype is the major form
in this tissue (740), in agreement with the report by Sakurai et
al. (728), who showed that no mRNA for ET receptor B occurs in rat
testis (728). Northern analysis of mRNA extracts from marmoset testis
revealed the presence of both ET receptors with the prevalence of the
ET receptor A mRNA; interestingly, the animals maintained on a
high-fat/high-cholesterol diet exhibited a significant increase of both
receptor mRNAs in all tissues and in the testis (741). In the rat
testicular PMC, ET induces a rapid production of inositol triphosphate
and mobilization of intracellular calcium in a concentration-dependent
fashion (739, 742). Autoradiographic studies have confirmed the
presence of abundant ET-binding sites both in the interstitial space,
containing Leydig cells, and in the peritubular myoid layer of the
adult rat testis (743).
Specific ET receptor A and B transcripts have been identified in human testis, with a more abundant expression of ET A subtype (736). The binding sites were mostly concentrated into the seminiferous tubules, although interstitial cells and PMC were also positive. Within the seminiferous tubules ET-1 binding was confined to spermatocytes and spermatids, whereas the Sertoli cells were negative (736).
3. Local functions.
ET-1 stimulates progesterone production
and transient expression of the protooncogenes c-jun and
c-myc in mouse Leydig tumor cells (737). In purified adult
rat Leydig cells, ET positively regulates basal and hCG-induced
steroidogenesis (743, 744). This effect on testosterone production in
Leydig cells could be suppressed by a selective ETA receptor
antagonist, but not by ETB receptor antagonists, confirming that rat
Leydig cells are mainly provided with the ETA receptor subtype (743).
PMC treatment with ET induces a phenotypic response involving the
rearrangement of the cytoskeleton with a switch from a round to a flat
and elongated morphology (739). Moreover, ET-1 stimulates DNA synthesis
and contraction of rat PMC in culture (742). Although ET receptors have
not been found in rat Sertoli cells isolated from 20-day-old rats (739)
and in adult human Sertoli cells (736), Sharma et al. (745)
have reported that in Sertoli cells isolated from 9-day-old animals,
ET-1 activates an intracellular signaling pathway involving
[Ca2+] and attenuates FSH-stimulated cAMP and estradiol
accumulation (745).
Local intratesticular injections of physiological and pharmacological doses of ET-1 cause a dose-related decrease in testicular blood flow that can be blocked by concomitant injection of an ET receptor A antagonist (734). Since hCG treatment in high doses causes a major decrease in intratesticular blood flow at 4–6 h after treatment and the intratesticular levels of ET-1 are increased after hCG treatment, the possibility that ET-1 could be a mediator of the vascular effects of hCG has been suggested.
Studies of ET-1 gene-targeting mice have provided direct evidence for an unsuspected developmental role in neural crest-derived tissues for ET-1 in addition to the previously recognized regulatory effect on vascular tone in the adult context (746, 747). Also ET-3 and ET receptor B gene-targeted neonates display a phenotype that suggests an important role of ET-3/ET receptor B in neural crest formation (748, 749). The phenotype resulting from the homozygous ET-1 mutations is lethal within 15–30 min from birth while the heterozygotes are phenotypically normal and fertile, but develop elevated blood pressure. The viability of the ET-3 and ETB receptor varied greatly with a mean life span of 21 days. Also in this case the heterozygous mice were phenotypically normal and fertile. The limited viability of the homozygous mutants does not allow the study of the reproductive effects of these genotypes.
In conclusion, despite the evidence for production, receptor expression, and in vitro action of ET, the physiological role of this peptide in the testis remains unknown.
B. Angiotensin-II (AT-II)
The renin-AT system is one of the major control systems of the
blood pressure and water and electrolyte balance. The octapeptide AT-II
is the biologically active product generated by a cascade of
proteolytic cleavages. Renin from the juxtaglomerular cells of the
kidney cleaves angiotensinogen, a glycoprotein precursor from the
liver, to form the decapeptide AT-I, and AT-converting enzyme (ACE)
removes a dipeptide from the C-terminal end of AT-I to form AT-II
(750). AT-II cell surface receptors mediate the action of the hormone
(751). Other evidence suggests the existence of two AT receptor
subtypes. The structure of the AT1 receptor subtype has
seven-membrane-spanning domains and is coupled through G-binding
proteins to pathways involving PI hydrolysis, PKC, and
Ca2+. The AT1 receptor is widely distributed and is the
receptor that mediates signal transduction in the cardiovascular
system, adrenal, and kidney. The signaling pathway and function of the
AT2 receptor are not clear, but tissue distribution of the receptor
suggests a role in brain function (751).
Here the evidence of the testicular expression of the various components of the renin-AT system is described.
1. Expression and localization.
Renin immunoreactivity has
been found by means of immunohistochemistry in adult rat Leydig cells;
some heterogeneous staining was observed in the interstitial cells of
animals between 30 and 40 days of age, and no immunoreactivity was
detected in animals younger than 30 days (752). Staining was suppressed
or abolished by hypophysectomy and estrogen treatment and was reduced
by gonadotropin stimulation, leading to the speculation that in
gonadotropin-treated rats the increased secretion of the renin-like
substance, not compensated for by a parallel increase in synthesis,
might lead to decreased storage, explaining the weaker specific
staining of stimulated Leydig cells (752). Subsequently, the
renin-specific immunohistochemical staining exclusively localized in
the Leydig cell was confirmed, and the biochemical determination of
renin activity in the rat testis was also reported together with a
decrease after hypophysectomy and a remarkable increase after
gonadotropin treatment (753). The existence of renin enzyme activity in
Leydig cells purified from adult rat testis has been demonstrated
together with an inactive form of latent renin that is activated by
sulfhydryl reagents (754). A renin mRNA has been detected in adult
mouse (755) and rat testis (756) and in the testis of a transgenic mice
carrying the human renin gene (757). The immunocytochemistry and
in situ hybridization of renin and its mRNA in adult rat
testis confirmed the Leydig cell localization of the enzyme (758).
LH and cAMP elicit a dose- and time-dependent induction of renin activity in a cultured mouse Leydig tumor cell line, which parallels the increase in steroid biosynthesis (759). Further studies have demonstrated that the LH-induced renin activity in these cells is retained within the cells while about 15% of AT-I and the majority of AT-II are secreted (760).
ACE has been implicated in at least two other areas, both of major physiological and pathophysiological interest, which are not related to the control of blood pressure and fluid electrolyte homeostasis: reproduction and immunology (761). Leydig cells isolated from adult rat testes were found to contain renin, ACE, and AT-I, AT-II, and AT-III as determined by HPLC and RIA, while in germinal cells only AT-II and AT-III were found at significant levels (762). Subsequent studies showed that testicular ACE activity is associated primarily with germinal cells of the mature rat and rabbit (763, 764). The testicular transcripts encode for the ancestral, nonduplicated form of the enzyme in both rodents and men (765, 766, 767). This form appears in the rat testis at puberty and is testosterone dependent (763, 768, 769). The transcription of germinal ACE occurs via a testis-specific promoter located within the intron 12 of the ACE gene (770, 771, 772, 773, 774), which is completely extinguished in somatic tissues (775). Characteristically, testicular ACE is smaller than its pulmonary counterpart, has different N- and C-terminal sequences and mRNA, yet arises from a common gene (776, 777). It probably represents an internal portion of pulmonary ACE and has similar enzymatic properties (778, 779). By using immunoperoxidase detection and in situ hybridization, the temporal expression and cell distribution of the germinal isoform of ACE have been studied in the testis of normal mice and rats (780). In both species, specific testicular ACE mRNA and its gene product are present only after completion of meiosis. The study of the pattern of expression of germinal ACE during spermatogenesis in mouse and rat showed that ACE mRNA and its corresponding protein are first synthesized during stages IV-VII, the maximum expression occurs at stages VIII-XII, and ACE mRNA is no longer detectable in spermatids beyond stage XIV, whereas its gene product is expressed until the end of spermatid maturation (780, 781). By using specific isoenzyme antisera, immunofluorescent staining of sections of adult, but not immature, pig and rabbit testes revealed specific staining to testicular ACE within the lumen of seminiferous tubules in the cytoplasm of spermatocytes, whereas the antiserum specific for endothelial ACE showed the presence of this isoenzyme only in blood vessels (782, 783).
Although no angiotensinogen mRNA has been detected in the adult rat testis (784), AT-II has been found in adult rat Leydig cells and germinal cells (762) and is produced by mouse Leydig tumor cells treated with LH (760).
In human testis, renin immunoreactivity was found in the Leydig cells (785). Studies conducted assaying prorenin, renin, and angiotensinogen in internal spermatic venous blood from young men suggested that the only form of renin secreted by the human testis is prorenin (786). In other studies it has been reported that under basal conditions very low levels of renin and AT-II are released into internal spermatic vein blood, while after treatment with hCG the secretion of both renin and AT-II are significantly higher (787, 788).
In man the results of genomic DNA analysis are consistent with the presence of a single gene for ACE in the aploid human genome. According to the results obtained in different animal species, the ACE gene is transcribed as a 4.3-kb mRNA in vascular endothelial cells whereas a 3.0-kb transcript was detected in the testis, where a shorter form of ACE is synthesized (777). Immunolocalization of ACE in the human male genital tract has shown an intracellular positivity in spermatids on the acrosomal cap (789).
2. Receptors.
High-affinity receptors for AT-II have been
reported in the Leydig cells in rat and primate testis, including human
(790). Studies on the AT-II receptors in rat testis revealed the
presence of high receptor density in newborn rats, which decreased
toward the adult age parallel to the fall in the ratio of interstitial
to tubular tissue (790, 791, 792).
3. Local functions.
AT-II inhibits adenylate cyclase activity
in Leydig cell membranes and reduces basal and hCG-stimulated cAMP
pools and testosterone production in intact cells from adult rats
(791). Male but no female mice homozygous for a disrupted ACE gene have
impaired fertility despite being potent and having sperm of normal
appearance, suggesting that sperm produced by homozygous male mutants
have a reduced fertilizing ability (793). The mechanisms responsible
for the reduced fertility in these male mutants is currently unknown. A
direct involvement of AT-II seems unlikely. Thus far, three
laboratories have studied the phenotype of homozygous mice carrying
null mutation of the angiotensinogen gene primarily focusing on the
blood pressure control of the mutant. While one study does not document
structural abnormalities in major organs (794), the others report
kidney abnormalities similar to those observed in ACE-deficient mice
(795, 796). The absence of a functional angiotensinogen gene is
compatible with survival to birth, but postnatal survival is severely
compromised. However, an occasional survival to adulthood of
angiotensinogen -/- animals, tested for fertility, produced three
litters of normal size, demonstrating that AT is not essential for male
fertility in mice. Accordingly, although gene targeting of the AT1
receptor (797) and AT2 receptor (798, 799) in mice has marked effects
on blood pressure and central nervous system and cardiovascular
function, it does not impair the animals’ fertility. Moreover, in
transgenic mice carrying both human renin and human angiotensinogen
genes leading to overproduction of AT-II, no change in reproductive
performance was reported (800).
In conclusion, several components of renin-AT system are expressed within the testis, but the complete system has not yet been identified. For example, in the rat testis, renin, ACE, AT-I, and AT-II have been found but angiotensinogen could not be detected. The evidence for AT-II production, receptor expression, and in vitro action on Leydig cell testosterone production are convincing. However, the physiological role of AT-II in testicular functional control is unclear. The reported impaired fertility in male mice ACE knock-out is interesting but probably involves pathophysiological mechanisms that do not relate to AT-II generation.
C. Atrial natriuretic peptide (ANP)
ANP is a peptide hormone released from the cardiac atrial myocytes
that regulates blood pressure and fluid volume (801). In addition to
its natriuretic, diuretic, and vasorelaxant activities, ANP inhibits
the release of aldosterone, renin, and AVP while stimulating the
release of androgen, progesterone, and GH. ANP selectively activates
particulate guanylate cyclase and inhibits adenylate cyclase, thereby
increasing the intracellular accumulation of cGMP and decreasing cAMP
(802). The biological actions of ANP are mediated by specific cell
surface receptors. The diverse cellular responsiveness to ANP action is
thought to be due to the heterogeneity in the ANP-binding sites. Two
types of ANP-specific receptors have been identified in target tissues:
two different guanylate cyclase-linked receptors that appear to mediate
most of the ANP biological effects, and a clearance receptor (803).
1. Expression and localization.
Low levels of an
immunologically recognized ANP prohormone together with ANP mRNA
transcripts have been found in the adult rat testis (804, 805, 806), and
immunoreactive ANP has been localized in mouse and rat spermatids and
elongating spermatozoa (807).
2. Receptors.
Pandey et al. (808, 809, 810) have
characterized ANP receptors in cultured mouse Leydig tumor cells that
contain predominantly the guanylate cyclase-coupled form of the ANP
receptors, and ANP receptors have been detected also in purified mouse
Leydig cells (811). The radioautographic localization of ANP-binding
sites in the adult rat testis demonstrated specific binding only in
interstitial cells (812). An ANP-dependent membrane guanylate cyclase
has been demonstrated in mouse and rat testis (813), as well as three
immunologically and biochemically similar but distinct ANP receptors
(809, 814). Kinetic analysis of the fate of the ANP receptors on Leydig
cells demonstrates internalization, recycling, and redistribution of
the receptors (815). Recently, evidence that both Gs and Gi subunits of
G proteins seem to be involved in the regulation of Gs catalytic
activity of the ANP receptors in the plasma membrane of a murine Leydig
tumor cells line (MA-10) has been provided (816).
3. Local functions.
ANP has been demonstrated to stimulate
testosterone and cGMP production in isolated mouse Leydig cells
(817, 818, 819, 820) and a murine Leydig tumor cell line (810), while in rat
Leydig cells a lower stimulation of cGMP formation and a lack of
testosterone production enhancement, probably due to an insufficient
formation of cGMP in these cells, was observed (818). However,
subsequent studies showed that ANP exerts stimulatory effects on
steroidogenesis and calcium flux in rat Leydig cells (821). ANP
increases the levels of cGMP, inhibits cAMP accumulation and
progesterone secretion in response to hCG, and negatively regulates the
phosphorylation of PKC in cultured Leydig tumor cells (822, 823). More
recently it has been reported that a specific antagonist inhibits the
testosterone production and cGMP accumulation stimulated by ANP in
purified mouse Leydig cells (824). Moreover ANP enhances basal and
LH-stimulated testosterone production of purified mouse Leydig cells
involving both the 4 and 5 pathways of
steroidogenesis (811). Although ANP receptors have not been found in
Sertoli cells, treatment of cultured Sertoli cells with ANP resulted in
both a dose-dependent and time-dependent elevation of cGMP levels
(392).
In humans the intravenous administration of 100 µg of ANP induced a rapid significant increase of spermatic vein testosterone levels without affecting peripheral testosterone and LH concentrations, suggesting that ANP may directly influence the Leydig cell function in man (825).
Mice with either proANP or ANP receptor-disrupted gene have been generated (826, 827). Genetically reduced production of ANP leads to salt-sensitive hypertension (826) while mice lacking the guanylyl cyclase-A receptor for ANP develop salt-resistant hypertension (827). Both homozygous mutants, except for the blood pressure, appear normal and fertile.
A transgenic mouse line expressing ANP fusion genes has been also produced (828). These mice have large amounts of pro-ANP in the liver, and their plasma ANP levels are significantly elevated. The chronic endogenous hypersecretion of ANP results in sustained perturbations in cardiovascular homeostasis not accompained by significant changes in several other physiological parameters including fertility.
|
VII. Conclusions and Perspectives |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
Similar to other endocrine-regulated tissues, the testis is subject to
a hierarchy of controls. In this light, the systemic hormones represent
the first-step regulators. In addition, the local factors synthesized
by the cellular components of the testis may act as paracrine or
autocrine/intracrine hormones, or, as a further variation of this
theme, as juxtacrine factors, regulatory molecules that remain exposed
on the cell surface and modulate adjacent cells by contact. Polypeptide
factors are among the most widely studied of the second-step local
modulators of testicular function, and other molecular classes may
serve such functions as well. The second-step substances act through
cell surface or intracellular receptors (third step) that trigger the
cascade of intracellular events (fourth step) from the early signal
transduction to the final cell response. The interactions of all these
elements within the structural context of the testis regulate its
function. This hierarchical vision, while acknowledging FSH, LH, and
testosterone as prime regulators, recognizes that an imbalance of each
of the substances involved in the subsequent steps may potentially
impair organ function. Moreover, the actors of the intratesticular
control mechanisms may not be the same during testicular ontogenesis.
Also in the mature organ, the environment to which each cellular
component is exposed varies cyclically, due to the marked morphological
and functional changes of Sertoli cells and germ cells throughout the
seminiferous epithelium cycle. This is clear from the data summarized
in Tables 6
and 7.
Why, one might wonder, should such a complex network of regulators be
required for the organization of testicular function? Why wouldn’t a
simple feedback relationship suffice? The answer to this questions
seems to be that a classic feedback mechanism might be enough if the
testis were only devoted to the secretion of testosterone, but in any
case such a vision would be too simplistic since all the endocrine
glands possess a complex network of intraglandular regulators.
Furthermore, the testis is the organ in which spermatogenesis takes
place, and this is a dynamic process organized so that, at a given time
point, adjacent tubules are at different stages of the cycle of the
seminiferous epithelium (Fig. 1). This structural complexity implies
that to furnish the optimal focal intratesticular environment, local
regulators should be produced to generate the unique microchemical
context required for germinal cell development.
Thus, the next question is, to what extent do local regulatory peptides
play a role in the control of testicular function? Coming back to the
classic definition, the basic requisites for a regulatory peptide to be
considered a potential local modulator are: 1) the substance should be
produced in the target tissue; 2) the cell/tissue should contain
specific functional receptors; 3) the substance should affect at least
one single function of the target cell. Evidence for the presence of
numerous regulatory peptides in the testis that fulfil these criteria
has been provided. However, despite the enormous amount of data, they
can hardly fit into a physiological picture. As noted in the
introduction, this is mainly due to the difficulty in distinguishing
between effects that are restricted to the artificial conditions of the
in vitro systems and effects that are relevant to testicular
control in vivo. Notwithstanding, evidence from in
vivo experiments suffers from some intrinsic limitations as well.
For example, the pharmacological doses employed and the ubiquity of the
regulatory substances make it difficult to distinguish between direct
effects and effects mediated by interference on other systems. Another
limit is the redundancy of the actions of the regulatory peptides. This
concept is readily apparent if one looks at the in vitro
effects on the cellular components of the testis as listed in Table 4.
This redundancy is probably less active in vivo because of
three conditions: 1) it is presumably the interaction between several
factors that ultimately determines the net response of the cell to the
stimulus and, given the complexity of such interactions, it is
conceivable that even if two different stimuli induce the same response
in vitro, small environmental variations might result in
qualitatively different patterns of subsequent effects in
vivo; 2) the expression of some of these substances and of their
receptors is developmentally regulated; thus there are time windows in
their potential actions (Table 6); 3) the expression of substances and
receptors may also vary depending on the stages of the seminiferous
epithelium (Table 7). Again, these variables can be hardly evaluated
in vitro. Another important element that should be
considered is cross-regulation among the regulatory factors. For
example, the production of IGF-I by Sertoli cells is stimulated by FGF
and EGF (401, 402, 403, 415), and IL-1 and IL-1ß increase Sertoli cell
production of IL-6 (665) and Leydig cell secretion of NPY (318).
Once released, other mechanisms are involved in the control of availability and domain of influence of the testicular regulatory peptides, such as proteolytic processes and interactions with extracellular matrix and binding proteins. These events can function in two directions either to increase or to decrease the availability of polypeptide regulators. For example, studies of TGFß1 processing show that the growth factor is secreted as an inactive molecule and that extracellular proteolytic cleavage is required to release the biologically active dimer from the precursor molecule (829). Complexes formed by matrix proteins with regulatory factors might have either higher affinity for specific receptors or remain sequestered in the extracellular matrix before release by enzymes such as heparinase and plasmin to function as diffusible regulators. In this way the extracellular matrix can provide a further mechanism for regulating the cytokine’s sphere of influence.
Thus, at present, there is a great need for more information about how combinations of signaling molecules are used to organize the control of the testis in vivo.
Despite the experimental evidence reported in this chapter, for many of the locally produced regulatory peptides, their relevance in the control of testicular function is still speculative. On the other hand, the need to provide answers to the open questions on the control of the gonadal function is becoming increasingly important in light of the prevalence of male idiopathic infertility. In this context, the suspicion that regulatory peptide pathways are involved in the control of testicular function and in the pathogenesis of some cases of male infertility deserves much further investigation.
Promising new information has been obtained from studies that employ molecular genetic approaches, using targeted gene inactivation or overexpression in transgenic animals. However, in interpreting the results from these models one must be conscious of the limits of the technique. A major consideration concerns the ability of the biological system to adapt or respond to the alterations. Such physiological adaptations or compensations may obscure other important functions of the gene under investigation. For example, this is particularly evident if one considers the defects generated by single knockouts of receptors or substances belonging to a family, which can be milder than expected because of functional redundancy. As a matter of fact, the deficiency of any one of the family members may be compensated for by the action of other members of the same group. Furthermore, many of the presently available mutations cannot reveal all of the functions of a given gene within a given organ because they may result in a lethal phenotype. Mutant mice represent in vivo models that involve the entire organism, and thus the effects produced within an organ cannot be easily ascribed to a direct effect of the gene product on that organ. The gene expression in vivo is spatially and temporally regulated while transgenic animals are chronically exposed to the alteration of the gene.
Nevertheless, given all these major premises, significant advances have been made over the last few years in defining the local requirements for normal testicular development and function. Among the substances described in this review, particular mention should be made of INH, IGF-I, TGFß, SLF/c-kit, and ENK. The studies on IGF-I and SLF/c-kit have defined the important role of IGF-I in Leydig cell differentiation and of SLF/c-kit in PGC and spermatogonia migration and survival. INH has been shown to be a tumor suppressor gene with gonadal specificity. The testicular overexpression of pENK has been shown to lead to histological abnormalities of the seminiferous tubule. Persistent overexpression of TGFß1 elicits a cellular overresponse that may lead to serious fibrotic or proliferative disease in various organs and in the testis. In the future, for many of the substances described here, their testicular actions will be more clearly revealed, limiting gene overexpression or gene deletion spatially and temporally and allowing their circulating or endocrine functions to remain intact. New technologies that provide a mechanism to limit gene disruption to specific tissues have recently emerged (830).
There are two more lines of evidence that deserve mention here because of their potential relevance in revealing the influence of locally produced regulatory substances in the control of testicular function.
Very recently, mutant mice lacking the cAMP-responsive element
modulator (CREM) gene have been generated and shown to have
spermatogenic arrest at the spermatidic stage (831, 832). These animals
may constitute a tool for studying some cases of altered physiological
processes leading to infertility because spermatogenic arrest is also a
feature of many cases of infertility in human males. The CREM gene
encodes multiple regulators of the cAMP-transcriptional response by
alternative splicing (833). A developmental switch in CREM expression
occurs during spermatogenesis, whereby CREM function is converted from
an antagonist to an activator, CREM, which accumulates from the
premeiotic spermatocyte stage onward (834) and is particularly abundant
in round spermatids, predominantly at stage VII-VIII of spermatogenesis
(835). Mutant mice lacking the CREM gene are sterile, as their
developing spermatids fail to differentiate into sperm (831, 832). In
these animals the inhibition of sperm development is not accompanied by
decreases in the levels of FSH or circulating and intratesticular
levels of testosterone (831, 832). This provides an interesting
experimental model that resembles the frequent condition found in
infertile men in which the defect in the production of mature
spermatozoa is not accompanied by a concomitant modification of the
gonadotrophic hormones. It has been established that FSH is responsible
for the CREM switch (836). The effect of FSH is not direct, however,
because germ cells do not have FSH receptors. Thus, it has been
suggested that another hormonal message, originating from the Sertoli
cells upon FSH stimulation, mediates CREM activation in germ cells
(837).
Another example that seems to indicate a role for locally produced regulatory peptides in controlling testicular function comes from a study conducted to investigate the role of the bone morphogenetic protein 8B (BMP8B) during spermatogenesis (838). BMPs are members of the TGFß superfamily synthesized as large precursors and processed and proteolytically cleaved to yield carboxy-terminal mature protein dimers (839). BMPs are multifunctional regulators of embryonic development required for the organogenesis of kidney, lung, heart, teeth, gut, and skin. In particular, BMPs may help to mediate inductive interactions between mesenchymal and epithelial cells in these tissues. Zhao et al. (838) have generated mice with a targeted mutation in BMP8B. The homozygous BMP8B mutant males exhibit variable degrees of germ cell deficiency and infertility. Interestingly, two different defects can be observed in the mutant testes. During early puberty, the germ cells of the homozygous mutants either fail to proliferate or show a marked reduction in proliferation and delayed differentiation. In adults there is a significant increase in apoptosis of spermatocytes that leads to germ cell depletion and sterility. Although an endocrine pathway influencing gonadotropin production in the pituitary gland by BMP8B cannot be completely ruled out, the lack of any significant changes in morphology, proliferation, or apoptosis in Leydig cells and Sertoli cells does not support a gonadotropin-mediated BMP8B function. Also, androgen production by Leydig cells appears to be normal since the morphology of the androgen-dependent tissues in all homozygous mutant males is unaffected. Thus the BMP8B proteins function locally within the seminiferous tubules, either through an autocrine effect on germ cells and/or through a short-range paracrine effect on Sertoli cells. This hypothetical model of action is further reinforced by the notion that BMPs bind ACT receptors (840), which are expressed in germ cells and Sertoli cells at specific stages of the seminiferous cycle (350, 361).
Finally, a promising new technique has been developed that in the near future may allow the investigation of the interactions of germ cells with Sertoli cells and other cellular components of the testis. The procedure consists in the transfer of spermatogonial stem cells from a donor mouse (841, 842) or rat (843) testis into individual seminiferous tubules of an infertile recipient mouse. The donor cells are able to establish complete spermatogenesis in the seminiferous tubules of the host and to produce normal spermatozoa. Moreover, cryopreserved mouse spermatogonia can be successfully transferred to recipient host testis where they produce viable and morphologically normal sperm (844). The transfer of spermatogonia between species will represent a radical contribution to reproductive biology and promises benefits in areas such as animal conservation and cryopreservation of spermatogonia of patients before radical chemotherapy, since stem cells from males with azoospermia may be capable of generating spermatogenesis in a surrogate. The ability to manipulate the donor or the host could represent a further opportunity to study the local control mechanisms of the testis. For example, the use of genetically sterile mice as recipients should help in determining the role played by locally produced regulatory substances in the engrafting and development of donor spermatogonia.
In conclusion, the testis is a totally integrated cellular system. Regulation at a local, intragonadal level is an integral part of the overall regulation of development and functional control of the male gonad. Interaction between cells within the testis extends beyond the same cell type to include germ cell-somatic cell interactions as well. Gonadal peptides are among the signals that are used to communicate within the testis, and it is conceivable that an alteration in their function either during development or in adult life may be responsible for some cases of idiopathic male infertility.
Hopefully, the knowledge and experimental tools generated during more than 20 yr of research and summarized in this review will help to elucidate the role of gonadal peptides in the pathophysiology of the testis.
|
Footnotes |
|---|
1 This work has been supported in part by the Italian National Research
Council (CNR), Contract 94.04666.26, and Italian Ministero
dell’Università e della Ricerca Scientifica e Tecnologica quota
60% (article 63, 3° co. DPR 382/80). 
|
References |
|---|
|
TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
|---|
and opioid receptors from mouse brain. Proc Natl Acad Sci USA 90:6736–6740, ßA and ßB subunits in
various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251-2 macroglobulin. Immunol Today 11:163–166[CrossRef][Medline]
- and inhibin
ßA-subunits in the fetal sheep testis. J Endocrinol 145:35–42-subunit expression in rat Leydig cell cultures.
Mol Cell Endocrinol 66:119–122[CrossRef][Medline]
-subunit gene
in vivo and in vitro. Mol Endocrinol 3:29–35 and ß-B subunits during the cycle
of the rat seminiferous epithelium. Endocrinology 124:987–991-subunit
in the human testis. Cell Tissue Res 269:221–227[CrossRef][Medline]
in the testes of normal men and in men with
testicular disorders. Int J Androl 13:463–469[Medline]
2-macroglobulin.
Biochemistry 29:1063–1068[CrossRef][Medline]
-inhibin is a tumour-suppressor gene with gonadal
specificity in mice. Nature 360:313–319[CrossRef][Medline]
-inhibin deficient mice. Biochem Biophys Res Commun 203:105–112[CrossRef][Medline]
and p53-deficient mice:
the role of activin as an auto
