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Dipartimento di Fisiopatologia Medica, Università di Roma "La Sapienza," Rome, Italy
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Abstract |
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 Top Abstract I. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
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/EGF (TNF) |
I. Introduction |
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TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
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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
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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.
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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.
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II. Neurohormones/Neuropeptides |
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TopAbstractI. IntroductionII. Neurohormones/NeuropeptidesIII. Peptides Originally...IV. Growth FactorsV. Immune Derived CytokinesVI. Vasoactive PeptidesVII. Conclusions and...References |
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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,
