Effect of anti-Mullerian hormone on Sertoli and Leydig cell functions in fetal and immature rats

Anti-Mullerian hormone (AMH) is mainly involved in the regression of Mullerian ducts in male fetuses, but it may have other functions linked to gonadal development. The present study examines the effect of AMH on steroidogenesis by Sertoli and Leydig cells in fetal and immature rats during the period where AMH is physiologically produced in the testis. The basal aromatase activity of Sertoli cells in primary culture was strongly stimulated (77-91%) by cAMP. AMH (35 nM) reduced cAMP-stimulated aromatase activity by 49-69% as early as fetal day 16 and until postnatal day 20. This effect was dose dependent and was seen after 48 h in culture. AMH also blocked the Sertoli cell aromatase activity stimulated by FSH, but LH did not stimulate this activity, confirming that the aromatase activity effectively resulted from Sertoli cells and not from contaminating Leydig cells. RT-PCR analysis showed that AMH reduced aromatase activity by decreasing the amount of aromatase messenger RNA. AMH also inhibited the LH-stimulated testosterone production by dispersed fetal Leydig cells in culture in a dose-dependent manner. The inhibitory effect of AMH did not depend on the fetal stage studied (16 or 20 days postconception) and resulted from a drop in the steroidogenic activity of each Leydig cell without affecting the number of 3beta-hydroxysteroid dehydrogenase-positive cells. These data provide the first evidence that AMH, like other members of the transforming growth factor-beta family, has an autocrine/paracrine effect on testicular steroidogenic function during the fetal and prepubertal periods.     

Expression of inhibin alpha-subunit in horse testis

Inhibin is believed to play roles in the pituitary secretion of FSH and in the paracrine regulation of testicular function. Although it has been generally accepted that inhibin is produced in Sertoli cells, there was a recent evidence for the localization of inhibin in Leydig cells of primates, rat and sheep. However, there is no report on the expression of inhibin in the adult horse testis. Therefore, using immunohistochemistry, western blotting and in situ hybridization techniques, the present study examined inhibin alpha-subunit (Ih-alpha) expression in the adult horse testis. For the detection of Ih-alpha protein, we used anti-porcine Ih-alpha antibody in immunohistochemistry and western blotting. Furthermore, digoxigenin-labeled complementary RNA probes were prepared to detect intracellular messenger RNA (mRNA) of Ih-alpha. Immunostainings for Ih-alpha were found not only in Leydig cells but also in Sertoli cells. The intensity in Leydig cells was stronger than in Sertoli cells. Immunoreactivities for Ih-alpha were found at approximately 46 kDa, 56 kDa and 90 kDa in the homogenates from testicular interstitial tissues. The bands at 56 kDa and 90 kDa agree with previous report, but not at 46 kDa. Signals for mRNA of Ih-alpha by in situ hybridization were detected in Leydig cells and in the basal region of seminiferous epithelium including Sertoli cells. These results suggest that Ih-alpha is expressed in Leydig cells and Sertoli cells of horse testis, and the expression level should be higher in Leydig cells than Sertoli cells.     

Ontogeny of estrogen receptor-beta expression in rat testis

The recently discovered estrogen receptor-beta (ERbeta) is expressed in rodent and human testes. To obtain insight in the physiological role of ERbeta we have investigated the cell type-specific expression pattern of ERbeta messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERbeta mRNA in their cytoplasm and the ERbeta protein in their nucleus. In testes of 11- and 15-day-old rats, ERbeta mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERbeta mRNA as well as ERbeta protein was found in pachytene spermatocytes from epithelial stages VII-XIV and in round spermatids from stages I-VIII. Low ERbeta expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERbeta. In adult testes, no ERbeta mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERbeta. The expression of ERbeta in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.     

Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development

BACKGROUND: Determination of the cellular distribution of the androgen receptor (AR) in testicular cells is necessary for understanding the mode of AR action in the testis. We here investigated immunohistochemically the localization of AR by use of anti-human AR polyclonal antibody NH27, with special reference to the AR in germ cells in the developing mouse testis. METHODS: ICR mouse testes taken from day 14 post coitum (p.c.) to day 56 post partum (p.p) were used for AR immunohistochemistry by the routine immunoperoxidase method at the light microscopic level and the pre-embedding method at the electron microscopic level. RESULTS: On day 14 p.c., AR immunoreactivity was present in nuclei of prospermatogonia but not in those of Sertoli cells or interstitial cells. On day 14 p.p., the AR was detected in the nuclei of spermatogonia, Sertoli cells, and myoid cells. AR immunoreactivity in nuclei of Leydig cells appeared on day 21 p.p. In the mature mouse testis, the AR was present in the nuclei of spermatogonia, Sertoli cells, myoid cells, and Leydig cells. CONCLUSIONS: AR was present both in germ cells and in somatic cells during fetal and postnatal development of the mouse testis. In the fetal testis, AR was localized exclusively in prospermatogonia and spermatogonia, suggesting that androgen may act directly on germ cells during prespermatogenesis and the early stage of spermatogenesis. Based on the fact that AR is expressed in Sertoli cells, myoid cells, and Leydig cells around the onset of spermatogenesis, the regulation of AR expression in the germ cells seems to be different from that in the somatic cells. Furthermore, our present data suggest the ultrastructural localization in nuclei of mouse testicular cells is similar to that of some other steroid receptors, both in germ cells and somatic cells.     

Expression of oestrogen receptor beta (ER beta) occurs in multiple cell types, including some germ cells, in the rat testis

The identification of a second oestrogen receptor (beta) has prompted a re-evaluation of the potential sites of action of oestrogens. The aim of the present study was to characterize immunoexpression of ER beta expression in the testis to complement earlier data which had demonstrated that expression of ER alpha is confined to testicular interstitial Leydig cells. In all testes studied, including those from both fetal (day 20.5 p.c.) and adult rats, ER beta was found to be expressed in multiple cell types. Sertoli cell nuclei were immunopositive at all ages. In adult testes expression in Sertoli cells was not stage dependent and was unaffected by ablation of Leydig cells. In fetal testes ER beta was also expressed in peritubular cells, fetal Leydig cells and gonocytes. In the pubertal and adult testis ER beta was detected in the nuclei of spermatogonia and most pachytene spermatocytes. Weak immunopositive staining was present in the cytoplasm of spermatocytes undergoing the second meiotic division. In conclusion the widespread expression of ER beta in the testis is consistent with a role for oestrogens in modulating spermatogenesis, and hence fertility, in the male.     

Macrophage migration inhibitory factor production by Leydig cells: evidence for a role in the regulation of testicular function

Macrophage migration inhibitory factor (MIF), described originally as a product of activated T lymphocytes, recently has been found to be released by monocytes/macrophages and the anterior pituitary gland. Immunohistochemical studies of the adult rat testis using an affinity-purified polyclonal antimurine MIF antibody demonstrated strong staining for MIF in Leydig cells and their putative precursors. Peritubular myoid cells and the seminiferous epithelium were negative for MIF staining; however, a weak reaction around the heads of elongated spermatids also was observed. The expression of MIF messenger RNA and protein in whole rat testis was demonstrated by Northern blot and Western blot analyses, respectively. Both MIF messenger RNA and protein immunoreactivity in Leydig cells was observed in testes obtained from long term hypophysectomized rats. Significant concentrations of intracellular MIF were detected in lysates of the TM3 Leydig cell line (7.23 +/- 2.6 pg/microgram protein), and testicular interstitial fluid contained 14.7 +/- 1.6 ng/ml MIF protein, as measured by MIF-specific enzyme-linked immunosorbent assay. To gain insight into the possible biological role of MIF in the testis, cultures of adult rat seminiferous tubules and purified Leydig cells were incubated together with recombinant murine MIF (rMIF). Neither rMIF (50 ng/ml) nor a neutralizing anti-MIF antiserum was found to affect basal or LH-stimulated Leydig cell steroidogenesis in vitro. However, a dose-dependent decrease in the secretion of inhibin by the seminiferous tubules was observed at rMIF concentrations ranging from 10-100 ng/ml. Taken together, these data indicate that Leydig cells produce MIF in vivo and suggest an important regulatory role for this newly discovered mediator of testicular function.     

Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary

Expression of the relaxin-like factor (RLF) was studied at the messenger RNA (mRNA) and protein levels in the testes and ovaries of the mouse, as well as through testicular development and differentiation in the mouse testis. In situ hybridization or RT-PCR, and immunohistochemistry using a polyclonal antibody raised against a recombinant protein, provided mutually confirmatory results for a high expression of RLF in the Leydig cells of the adult testis and at a much lower level of expression in the luteal cells of the ovary through the cycle, pregnancy, and in lactation. Analysis of protein and mRNA expression, through postnatal testicular development, indicated moderate RLF expression also in the fetal population of Leydig cells, even in the hpg mutant mouse, lacking an active pituitary-gonadal axis. Prepubertal Leydig cells, however, exhibit only very low-level RLF gene expression, this phenotype persisting in the adult hpg mouse. In summary, fetal Leydig cells express RLF in an LH/human CG-independent fashion, whereas LH/human CG is essential to induce RLF expression in the adult-type Leydig cell. In cultured adult Leydig cells or in the mouse tumor MA-10 cell line, RLF mRNA is expressed in a constitutive fashion. RLF thus seems to be a useful marker of Leydig cell differentiation status.     

Stimulatory effect of follicle-stimulating hormone on basal and luteinizing hormone-stimulated testosterone secretions by the fetal rat testis in vitro

The in vitro effect of FSH on testosterone secretion by the fetal rat testis was studied. Testes were cultured in the presence or absence of either commercial human (h) FSH (Metrodine; 200 mIU/ml) or recombinant hFSH (200 mIU/ml) for 3 days and with 100 ng/ml ovine LH during the last 4 h of culture. To avoid a stimulatory effect by the 0.4% LH that contaminates Metrodine, the cultures were performed in the presence of a monoclonal anti-hLH beta antibody and with a concentration of Metrodine that had no short term stimulatory effect on testosterone production by the fetal testes in vitro. Metrodine treatment had a positive long term effect on both basal and LH-stimulated testosterone secretion by fetal testes explanted on days 18.5, 20.5, and 22.5 postconception, which was abolished by the addition of a monoclonal anti-hFSH beta antibody. LH-free recombinant FSH also augmented basal and LH-stimulated testosterone secretion of testes explanted on days 13.5, 14.5, and 18.5 postconception. The positive effect of recombinant hFSH appeared during the second day of treatment with day 14.5 and 18.5 testes and on the third day of treatment with day 13.5 testes. As it is widely accepted that FSH receptors are exclusively localized on Sertoli cells, these results suggest that on or before day 15.5 of fetal life, 1) Sertoli cells are able to respond to FSH, 2) Sertoli cells can produce factors that are able to act on Leydig cell function, and 3) Leydig cells are sensitive to FSH-induced Sertoli cell factors. In conclusion, this study points out a potential paracrine control of fetal Leydig cell function and/or differentiation by fetal Sertoli cells as soon as fetal Leydig cells differentiate.     

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.     

Differential localization of inhibin subunit proteins in the ovine testis during fetal gonadal development

Inhibins and activins are dimeric proteins that are involved in cell proliferation, apoptosis, and differentiation in a number of systems and have previously been detected in fetal testes of many species. This study used immunohistochemistry to examine the localization of inhibin alpha-, betaA-, and betaB- subunits during ovine testicular development from days 40-135 of gestation. Localization of inhibin betaA- and betaB-subunit messenger RNAs was confirmed by in situ hybridization. The results showed that there was differential localization of inhibin alpha-, betaA-, and betaB-subunits to specific cells in the ovine fetal testis from 40 days of gestation. All three inhibin subunits were present in Sertoli cells throughout gestation, whereas the rete epithelium and gonocytes did not express inhibin alpha-subunit. These data suggest that the fetal Sertoli cells have the capacity to produce all forms of inhibins and activins, i.e. inhibin A and B, and activins A, AB, and B, whereas the rete testis epithelial cells can only synthesize activin A. In the interstitium, the fetal Leydig cells expressed all three inhibin subunits, but this was restricted to the period between 40 and 90 days of gestation. Thereafter, inhibin alpha-subunit immunoreactivity was not observed in fetal Leydig cells, which suggests that only activin ligands are produced by Leydig cells during late gestation. Collectively, the data demonstrate that fetal ovine testes have the potential to produce the full repertoire of inhibins and activins from very early in testicular differentiation. The distinct and restricted localization of the various subunits to specific cells suggests that specific dimeric proteins have particular roles in the development and function of the fetal testis.     

Vasoactive intestinal peptide is an important endocrine regulatory factor of fetal rat testicular steroidogenesis

This study elaborates our recent preliminary finding that vasoactive intestinal peptide (VIP) has a specific stimulatory effect on fetal rat Leydig cells. We examined the dose-response relationship for the effect of VIP on cAMP and testosterone production by dispersed fetal Leydig cells isolated from rat testes on embryonic day (E) 18.5. Further, we used RT-PCR to examine the expression of the VIP gene in fetal brain and testes and that of the VIP receptor genes in fetal testes and used RIA to measure VIP in testes and plasma during the fetal period. VIP stimulated fetal testicular cAMP production at a dose of 10(-9) mol/liter, whereas a dose as low as 10(-12) mol/liter stimulated testosterone production. This suggests that VIP at low doses may stimulate testosterone production using second messenger pathways other than cAMP. RT-PCR analysis could not reveal either VIP messenger RNA (mRNA) in fetal tissues or VIP1 receptor mRNA in the fetal or newborn testes, whereas VIP2 receptor mRNA was detected in fetal testes as early as E15.5. Northern hybridization analysis showed that the level of expression of VIP2 receptor mRNA is very low in fetal and neonatal testes and increases with age. The testicular VIP content was unmeasurable by our RIA method (i.e. <1 fmol/testis), whereas the circulating level of VIP was 82.9 +/- 1.1 pmol/liter on E17.5 and decreased with advancing fetal age. In conclusion, our results suggest that VIP from an extratesticular source, possibly from the maternal compartment, may regulate fetal testicular steroidogenesis through type 2 receptors as early as E15.5. These findings may be of physiological significance, because the onset of fetal testicular steroidogenesis occurs at an age (E15.5-19.5) before the onset of pituitary LH secretion.     

Differential localization of fibroblast growth factor receptor-1, -2, -3, and -4 in fetal, immature, and adult rat testes

Fibroblast growth factors (FGFs) are essential for embryonic development and have been implicated in testis development and function. The effects of FGFs are mediated through four high-affinity receptors (FGFRs), which have different binding affinities for each of the ligands. We have used indirect avidin-biotin-horseradish peroxidase-enhanced immunohistochemistry to localize FGFR-1, -2, -3, and -4 in fetal, immature, and adult rat testes. In the fetal testis, immunoreactivity for FGFR-1 was seen in gonocytes, Sertoli cells, Leydig cells, and mesenchyme, and FGFR-3 was localized in gonocytes. In the immature testis, FGFR-1 was localized to spermatogonia, and all four FGFRs were localized in pachytene spermatocytes, immature adultlike Leydig cells, and peritubular cells. In the adult testis epithelium, Sertoli cells were immunoreactive for FGFR-4, and germ cells were immunoreactive for all four FGFRs, with specific receptors localized to specific stages of germ cell development. In the adult testis interstitium, FGFR-1, -2, and -4 were localized in Leydig cells, and FGFR-1 and -4 were also localized in peritubular cells. The discrete cell- and stage-specific localization of FGFRs in the fetal, immature, and adult rat testis suggests that FGFs exert specific roles through these receptors in spermatogenesis, Leydig cell function, and testicular development.     

Induction of Ets-1 in endothelial cells during reendothelialization after denuding injury

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxra mouse embryos where the activity of the Sxra fragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxra chromosomes took place which was revealed by the mosaic expression of an X-linked lacZ transgene. The Sertoli cells, however, displayed a preferentially active XSxra chromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxra germ cells suggests that both the X and the XSxra chromosomes are active. This finding suggests that the presence of Sxra has no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxra fragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.     

Transforming growth factor beta1 and beta2 reduce the number of gonocytes by increasing apoptosis

Transforming growth factors beta1 and beta2 (TGFbetas) have recently been detected by immunohistochemistry in the fetal and neonatal rat testis, and the aim of the present study was to determine whether these factors can act as local regulators to control the number of gonocytes. Testes were kept in organ culture, and TGFbeta1 was found to have dose-dependent inhibitory effect on the number of gonocytes in testes explanted on fetal day 13.5. Either TGFbeta1 or beta2 at 10 ng/ml reduced the number of gonocytes by half after 2 days culture. TGFbetas did not decrease the BrdU labeling index of gonocytes or Sertoli cells, whereas these factors significantly increased the DNA fragmentation in gonocytes (TUNEL method). The other testicular cell types showed no positive TUNEL reaction. TGFbeta1 did not reduce the number of gonocytes in testes explanted on fetal day 17.5 (i.e. during the quiescent phase), but it did so in testes explanted on postnatal day 3 (i.e. stage of resumption of mitosis). To determine the potential cell type targets for TGFbetas, type I and type II TGFbeta receptors were immunolocalized in developing testis from fetal day 13.5 to postnatal day 3. Both receptors were present in the gonocytes throughout the whole period studied, and in the Leydig cells from fetal day 16.5 onward, but they were not detected in the Sertoli cells. Taken together, these results suggest that TGFbetas directly increase apoptosis in gonocytes without changing their mitotic activity during the developmental phases of proliferation.     

Sertoli cell-specific expression of rat androgen-binding protein in transgenic mice: effects on somatic cell lineages

The Sertoli cells of many species produce an androgen binding protein (ABP) which carries testicular androgens to the epididymis and is thought to play a role in sperm maturation. In the present report we analyzed the morphological modifications present in Leydig, Sertoli, and peritubular cells of the testis of young adult male mice transgenic for ABP gene, which overproduce ABP in testis. By in situ hybridization we demonstrated that ABP is specifically produced by Sertoli cells. Using light and electron microscopy, we detected scattered alterations of the seminiferous tubule cells which include cell degeneration and vacuolization. Leydig and Sertoli cells present morphological signs of hyperfunctioning compensatory mechanisms which include increased amounts of lipid droplets probably due to the existence of a stimulated steroid synthesis that in turn could be a consequence of the decreased unbound testosterone and/or a direct paracrine effect of ABP. Peritubular cells also present numerous signs of hyperstimulation.     

Thyroid hormone and follicle-stimulating hormone regulate Müllerian-inhibiting substance messenger ribonucleic acid expression in cultured neonatal rat Sertoli cells

Thyroid hormone is a major regulator of Sertoli cell development, and the present study sought to determine the role of T3 in Müllerian-inhibiting substance (MIS) messenger RNA (mRNA) expression. MIS, a Sertoli cell secretory protein that induces Müllerian duct regression and also may be critical for germ and Leydig cell development, is maximal perinatally, then decreases as Sertoli cells mature. The fall in MIS mRNA expression is delayed by hypothyroidism in vivo, indicating that T3 could regulate MIS mRNA. However, understanding of the hormonal regulation of MIS has been limited due partly to the lack of a primary Sertoli cell culture system in which sustained expression of MIS or its mRNA can be obtained. We have developed a Sertoli cell culture system for examining hormonal regulation of MIS mRNA. We then tested the effects of T3 and/or FSH treatment on MIS mRNA levels in this new system. Initial studies indicated that MIS mRNA production by 5-day-old rat Sertoli cells was minimal in vitro. Therefore, Sertoli cells from 2-day-old rats were cultured for 2 or 4 days. After 2 days in vitro, steady state MIS mRNA levels were decreased to 36% of the levels seen in freshly isolated Sertoli cells from 2-day-old rats. However, by day 4 of culture, steady state MIS mRNA production had recovered to 67% of that seen in freshly isolated 2-day-old Sertoli cells, which closely paralleled the decrease seen in MIS production in vivo from days 2-6. MIS mRNA levels were decreased 53%, 64%, and 86% in cultures treated with 0.01, 0.1, and 1.0 nM T3 (P < 0.05), respectively. This decrease in Sertoli cell MIS mRNA did not reflect a nonspecific effect on cell viability and/or activity, as shown by a dose-responsive increase in inhibin-alpha mRNA in these same cultures. FSH (2.5-100 ng/ml) also produced a dose-responsive decrease in MIS mRNA levels, and FSH and T3 together had an additive inhibitory effect on MIS mRNA levels, indicating that these hormones may act through distinct mechanisms. In summary, this is the first primary culture system in which sustained MIS mRNA production can be demonstrated, and it should prove useful for understanding the regulation of MIS in developing Sertoli cells. In addition, T3 and FSH are major regulators of the postnatal decrease in MIS production by the rat Sertoli cell, and these hormones may act through separate pathways.