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Clusterin, also known as SGP-2 or TRPM-2, is expressed in the male reproductive tissues at different levels. The genomic structure of the rat clusterin gene was recently reported by our laboratory and others. In this study, we have determined the promoter responsible for the basal expression of the rat clusterin gene in testicular cells by analyzing the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in MA-10 cells driven by different segments of the 5′-flanking region and the first intron of the clusterin gene. The region required for maximal basal expression was identified at − 266 to + 54. Addition of DNA fragments of the rat clusterin gene from − 1298 to − 266 bp, or from + 54 to + 1153 to ( − 266/+54)CAT resulted in a 87% decrease in CAT activity, suggesting the presence of inhibitory DNA elements in both the 5′-flanking region and the first intron. When DNA fragment in the first intron, + 1153 to + 2874, was included, CAT activity in the ( − 266/+2874)CAT construct increased to 70% of the clusterin promoter ( − 266/+54)CAT, indicating that stimulatory DNA elements may be present in this region of the first intron. Treatment of MA-10 cells with cyclic AMP (cAMP) neither decreased CAT activity driven by any of the clusterin/CAT chimeric plasmids examined in transient transfection studies, nor reduced the synthesis of nuclear clusterin RNA in nuclear run-on assays, indicating that the reduction of clusterin mRNA levels by cAMP previously reported in our laboratory is not exerted at the transcriptional level. Furthermore, addition of transcriptional or translational inhibitors (actinomycin D and cycloheximide respectively) abolished the cAMP effect observed in MA-10 cells. In summary, we have demonstrated that the basal transcription of the rat clusterin gene in testicular cells is under the control of both positive and negative regulatory sequences at the 5′-flanking region as well as in the first intron. The reduction of clusterin mRNA after exposure of MA-10 cells to cAMP is not due to a decrease in its transcriptional activity, but rather to an increase in the degradation of this mRNA through synthesis of a destabilizing protein(s) and its mRNA.

We and others have shown that the inhibin/activin beta B-subunit gene is expressed differently in the gonads. Two species of 4.8- and 3.7-kilobase (kb) beta B-subunit messenger RNA (mRNA) with equal concentrations were identified in the testis, whereas 1 predominant 4.8-kb and a minor 3.7-kb mRNA were observed in the ovary. In this study, we analyzed the structures of these 2 mRNAs in rat testis and showed that both 4.8- and 3.7-kb beta B-subunit mRNAs were terminated at the region proximal to 2.2 kb down-stream from the translation stop codon. However, only 4.8-kb mRNA could be detected when RNA probes prepared from the 5'-region 1 kb up-stream from the translation start site were used for Northern blot analysis. Our observations suggested that the 2 heterogeneously sized beta B-subunit mRNAs are transcribed from different initiation sites. Transcription of the 4.8-kb mRNA was initiated at 3 adjacent nucleotides, GGA, 1.1 kb up-stream from the translation start codon ATG, whereas multiple transcription initiation sites spreading over 150 nucleotides upstream from the ATG codon were previously identified for 3.7-kb mRNA. Neither of the 2 transcripts contained TATA and CAAT boxes in their promoters. The 5'-flanking DNAs required for transcription of the 4.8- and 3.7-kb mRNA were examined by their ability to induce transient expression of the chloramphenicol acetyltransferase (CAT) gene in MA-10 Leydig tumor cells. A marked increase in CAT activity was detected when the 5'-flanking DNA for the 4.8- or 3.7-kb transcript was progressively shortened from its 5'-end. Maximal CAT activity was observed when -409 and -139 basepair beta B-subunit DNA up-stream from the 4.8- and 3.7-kb transcription initiation site, respectively, were fused to the CAT gene, suggesting the presence of a negative regulatory element(s) at the up-stream regions of these promoters. Although putative AP-2 sites were identified, treatment of the transfected cells with cAMP and/or phorbol 12-myristate 13-acetate did not apparently change CAT activity driven by either the 4.8- or 3.7-kb promoter. Our results concluded that 1) the two inhibin/activin beta B-subunit mRNAs were transcribed from different initiation sites; 2) both promoters may be controlled by up-stream negative regulatory elements; and 3) neither of these promoters is responsive to cAMP and/or phorbol esters under the conditions employed.

The promoter/regulatory sequences responsible for the transcription of the rat inhibin alpha subunit gene in the testis were identified by the transient expression in an MA-10 Leydig tumour cell line of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), which was driven by different regions of the 5' flanking sequence of the inhibin alpha subunit gene. The CAT activity was elevated when the 2.0 kb 5' flanking alpha subunit gene fragment was progressively shortened from its 5' end, and a maximal increase was reached when the CAT gene was driven by an alpha subunit gene promoter extending to -163 bp. This construct was termed A alpha BstCAT. Furthermore, when either the -2.0 to -1.6 kb or the -2.0 to -1.0 kb alpha subunit DNA fragment was fused to A alpha BstCAT, and CAT activity was markedly suppressed, indicating the presence of negative regulatory DNA elements (NREs) in the upstream region of the gene. The cyclic AMP (cAMP) responsiveness of the alpha subunit gene, which was dependent upon the putative cAMP response element within the 67 bp alpha subunit promoter, was not affected by the upstream NREs. The inhibitory effect was also demonstrated when the -2.0 to -1.0 kb fragment was placed in either orientation with respect to the alpha subunit promoter or to a thymidine kinase promoter, suggesting that the NRE(s) can act as a silencer. Based on our observations we conclude that the basal expression of the rat inhibin alpha subunit gene in testicular MA-10 cells may, at least in part, be controlled by the upstream silencer(s) and NRE(s).

In addition to the feedback regulation of pituitary FSH secretion, gonadal activins have been shown to modulate physiological functions in the reproductive tissues. These observations suggested that activins and the receptors for these peptides may be coexpressed in gonadal tissues. In this study, we have cloned cDNAs encoding two species of type II activin receptors (ActRII and ActRIIB) from rat testicular mRNA and have shown that the two rat activin receptors share 97% similarity in the nucleotide sequence with those reported in the mouse. Two species [6 and 3 kilobases (kb)] ActRII mRNA were identified in all reproductive tissues of adult male and female rats. The 6-kb ActRII mRNA was the abundant form in most of the reproductive tissues. In placenta, the 6- and 3-kb mRNA were present in equal intensity. Interestingly, the ratio of the expression of two species of ActRII mRNA in rat testis changed with age. The 6-kb mRNA was the predominant form in immature 15- and 20-day-old testis, while the 3-kb mRNA increased with age and became the major form in mature testis. In female rats, however, the 6-kb ActRII mRNA was the abundant species in all of the ovaries examined, including immature, normal cycling, and pregnant rats. One major 2.25-kb species of ActRIIB mRNA was identified in all of the reproductive organs examined. Nucleotide sequence analysis of the isolated ActRIIB cDNA clones revealed that ActRIIB2 was the major isoform found in rat testis. The levels of expression of ActRIIB gene in rat testis or ovary were not changed during development. We conclude that 1) both type II and IIB activin receptor genes are widely expressed in the male and female reproductive tissues; and 2) the expression of 6- and 3-kb ActRII mRNA is tissue dependent as well as age dependent in rat testis.

Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene FecB, when compared with that from non-carrier rams. In order to determine whether the FecB gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (+ +) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2·3 kb and a minor transcript in some tissues at 0·8 kb. Densitometry of the autoradiographs revealed no FecB-specific differences in the densities of the hybridization signals from + + and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the FecB gene.

The aim of this study was to determine the bidirectional release of Immunoreactive inhibin-a (irINH.u) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-a was measured by RIA in fluid samples collected concurrently from the three testicular compartments-the seminiferous tubules, the interstitium, and the vascular system-through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively, Overall, the concentration of iriNHix in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-ot concentration in rete testis fluid was inverselyrelatedto that in testicular lymph, but i,v, administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibln levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-u concentration so that, overall, the total output of Inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-a In the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an F5H-induced release of a series of proteins in the M, range of 30 000-32 000. These results point to an FSH-induced increase in vascular permeability as a major mechanism of inhibIn release by the outer compartment of the seminiferous epitheium.

The efficacy and specificity of human recombinant inhibin alpha-subunit as an immunogen were determined by studying its effect on the ovulation rate and serum gonadotropin levels in ewes. Beginning on July 12 (day 1 of the experiment), 2-yr-old Rambouillet ewes were immunized four times at intervals of 20, 30, and 30 days, respectively, by im injections of the alpha-subunit each time with 100 micrograms. Blood was collected from the jugular vein as required before and after ovulation. Ovulation rates were determined, beginning on day 87, by laparotomy before estrous synchronization by means of vaginal progestagen sponges. After synchronization, ewes were examined again. The ovulation rate before estrous synchronization was over 4 times higher in immunized than control ewes [8.7 +/- (+/- SE) 1.9 vs. 2.0 +/- 0.0]. After estrous synchronization, ovulation rates in the immunized and control ewes were 6.3 +/- 2.4 and 1.3 +/- 0.3, respectively; this increase was accompanied by higher pre- and postovulatory FSH levels, but there was no change in the pattern of circulating LH. Furthermore, the postovulatory FSH surge persisted for longer periods of time in the immunized than control animals. These results demonstrate the potency of the human recombinant alpha-subunit as an immunogen and illustrate its specificity in suppressing the effects of endogenous inhibin. The results also point to inhibin as an important regulatory factor in controlling the ovulation rate by modulating both pre- and postovulatory FSH levels.

Adult Suffolk rams were immunized four times against the human recombinant inhibin alpha-subunit over a period of 80 days. Blood samples were collected at weekly intervals and serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were determined by radioimmunoassay procedures. The results show that season-related elevations of gonadotropin levels in immunized rams was delayed by 1-2 wk and, in these animals, it was more pronounced and extended than in vehicle-treated controls. Peaks of circulating testosterone were higher in control rams than in immunized animals. The capacity of the antisera to bind 125I-labeled inhibin alpha-subunit increased significantly in each immunized animal within 30 days of treatment, even though neutralizing antibodies were detected with a rat pituitary cell culture bioassay in only one of the four immunized rams. Epididymal sperm reserves tended to be greater in immunized than in control animals. These results show that inhibin controls the release of FSH during the breeding season, thereby regulating spermatogenic activity; it may also exert its effect on testicular function by a local effect on Leydig cells, as evidenced by changes in serum testosterone profiles and increased serum LH levels in rams immunized against the inhibin alpha-subunit.

Clusterin, also known as sulphated glycoprotein-2 or testosterone-repressed prostate message-2, is a ubiquitous protein found in a variety of tissues and species. In the reproductive tract of the male rat, clusterin is regulated in a complex age-dependent and cell-specific manner. It is expressed at high levels in the epididymis and testis and at very low levels in the prostate under basal conditions. The expression of this gene in the prostate and seminal vesicles is associated with androgen withdrawal, while in the testis clusterin mRNA is repressed by cyclic AMP (cAMP). To understand the mechanisms that control the expression of the clusterin gene better, we isolated and characterized the gene encoding rat clusterin, and analysed its cytosine methylation pattern in various tissues. Several putative regulatory DNA elements were identified, including a consensus AP-1 site in the 5′ flanking region. Two AP-1 sites and two transforming growth factor-β inhibitory elements, one AP-2 site and eight half-sites for glucocorticoid/androgen response elements were found within the first intron, and one cAMP response element was found in the first exon. The cytosine methylation pattern indicated that testicular or epididymal DNA in the rat is hypomethylated in the region between positions −534 and −99 of the clusterin gene, when compared with tissues with lower levels of expression such as prostate as well as liver, lung, kidney and spleen.

IGF-I mRNA has been demonstrated in testicular tissue and, more recently, localized specifically to Leydig cells. This study investigated the expression of IGF-I and side-chain cleavage enzyme (SCC) mRNA in two preparations of rat interstitial testicular cells which were separated by buoyant density into Leydig cell-enriched and -depleted fractions. RNA was prepared from interstitial cells obtained from the testes of untreated adult and immature rats and adult rats treated with human chorionic gonadotrophin (hCG) or ethane dimethanesulphonate (EDS; to destroy Leydig cells). IGF-I mRNA was detected in all samples, with five major transcripts ranging from 7·5 to 0·6 kb. Leydig cells (3β-hydroxysteroid dehydrogenase-positive and sensitive to EDS) expressed abundant IGF-I and SCC mRNAs, and levels of both were increased following hCG treatment. However, in addition, IGF-I mRNA which was derived from non-Leydig interstitial cells was detected, in the complete absence of SCC message, either in the more buoyant interstitial cells or in both interstitial cell fractions following the destruction of Leydig cells by EDS treatment. IGF-I expression in the Leydig cell-depleted cell fraction was also increased by hCG treatment, and it is therefore suggested that at least part of this non-Leydig interstitial cell IGF-I mRNA originates in Leydig cell precursors. In conclusion, Leydig cells are not the sole origin of IGF-I mRNA in the testis, and the non-Leydig cell expression may be an important component of testicular IGF-I production.

The clusterin protein and its messenger RNA were identified in many tissues including testis. In this report, we demonstrate the expression of clusterin gene in four Leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540. When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed. A 60-70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in I-10 cells, and no apparent change in LC-540 cells. The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines. The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration. Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA. The suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels. Our observations suggest: 1) cAMP negatively regulates clusterin gene expression in two Leydig tumor cell lines, MA-10 and R2C; 2) The inhibitory effect of cAMP on clusterin gene expression is probably acting through the protein kinase A pathway; and 3) The four Leydig tumor cell lines respond differently to cAMP in the expression of clusterin and side-chain cleavage genes.

Publisher Summary Proopiomelanocortin (POMC)-derived peptides act as paracrine and autocrine regulators of Sertoli and Leydig cells, respectively. Proopiomelanocortin is a precursor molecule that gives rise to multiple peptides that appear to have been used throughout phylogeny for various purposes. In prokaryotes, these peptides may act as chemotactic agents whereas in eukaryotes the same peptides may act by multiple actions. For example, in the pituitary gland, these peptides are secreted as classic hormones; in the central nervous system, they act as neurotransmitters; and in the testis as autocrine and paracrine regulators. The fact that immune-stainable β -endorphin and other POMC-derived peptides in Leydig cells appear to increase during periods of testosterone synthesis in fetal life and again at puberty suggests that the expression of these peptides might be dependent upon gonadotropin secretion. The chapter also explains the effect of human chorionic gonadotropin (hCG) treatment on testicular POMC-like mRNA in hypo-physectomized animals. It has been found that treatment with hCG resulted in an increase in total content of POMC-like mRNA in the testis.

Using antibodies against peptides derived from different portions of the POMC molecule, immunocytochemical evidence suggests that this precursor and/or the peptides present within it are localized in testicular Leydig cells of at least five species. There is no evidence for the localization of these peptides or their precursor in any other cell type in this organ. Examination of testicular extracts by gel filtration, SDS-PAGE, and RP-HPLC indicate that the testis contains low concentrations of POMC-derived peptides relative to brain. Further analysis indicates that POMC is processed to alpha-MSH and beta-endorphin similar to its processing in intermediate pituitary lobe and brain. The relative mobilities of immunoreactive alpha-MSH and beta-endorphin on RP-HPLC columns indicate that they are in the unacetylated state as in brain and in contrast to the acetylated forms in the intermediate pituitary lobe. The potential for Leydig cells to synthesize POMC and its peptides was suggested by the demonstration of POMC-like mRNA in total testis and Leydig cell cultures. The size of the POMC-like mRNA is approximately 150 base pairs shorter than anterior or intermediate pituitary POMC mRNA. POMC-like mRNA activity has also been localized to Leydig cells in sections of testes using in situ hybridization. Immunostainable beta-endorphin and other POMC-derived peptides are present in testicular Leydig cells during fetal life and following puberty at times when testosterone secretion is maximal. The accumulation of immunostainable POMC-derived peptides in Leydig cells is dramatically increased by LH and hCG. A variety of observations suggests that testicular cells can respond to POMC-derived peptides. ACTH and the MSHs stimulate growth and cAMP accumulation in Sertoli cells. By contrast, studies using antagonists suggested that beta-endorphin and/or another testicular opioid inhibit Sertoli cell proliferation and ABP secretion. These observations are consistent with the postulate that different portions of the POMC molecule may have opposite effects on Sertoli cell function and suggest a mechanism by which Leydig cells could modulate Sertoli cell activity. Intratesticular administration of opiate antagonists inhibits testosterone secretion both in vivo and in vitro. These observations suggest that Leydig cell-derived beta-endorphin may facilitate testosterone secretion either directly or indirectly. The finding of POMC and its derivative peptides in testis, ovary, adrenal, and placenta suggests that all steroid hormone-secreting organs in mammals may utilize this peptidergic system.

It has been postulated that some endocrine responses to stressful stimuli are mediated through the activation of hypothalamic pro-opiomelanocortin (POMC)-derived peptides. The aim of the present study was to analyse the effect of chronic stress on expression of the POMC gene in the medial basal hypothalamus and pituitary, and on serum concentrations of LH, β-endorphin and corticosterone. Adult male rats were killed after being subjected to restraint stress for 6 h/day over 2, 3 or 4 days. Chronic restraint induced an increase in serum concentrations of β-endorphin and corticosterone and a decrease in serum LH levels. To determine whether chronic stress induced any change in POMC synthesis, a dot-blot method was used to measure POMC mRNA levels. No significant changes were detected either in the β-endorphin content or in POMC mRNA levels in the medial basal hypothalamus after 2, 3 or 4 days of chronic restraint. This observation contrasts with the stimulation of POMC mRNA levels in both lobes of the pituitary. The data suggest that although chronic restraint induces an increase in POMC synthesis and secretion in the pituitary and a decrease in LH secretion, it has no effect on hypothalamic POMC neurones.