Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

Leptin secreted from testicular microenvironment modulates hedgehog signaling to augment the endogenous function of Leydig cells

Although testosterone deficiency (TD) may be present in one out of five men 40 years or older, the factors responsible for TD remain largely unknown. Leydig stem cells (LSCs) differentiate into adult Leydig cells (ALC) and produce testosterone in the testes under the pulsatile control of luteinizing hormone (LH) from the pituitary gland. However, recent studies have suggested that the testicular microenvironment (TME), which is comprised of Sertoli and peritubular myoid cells (PMC), plays an instrumental role in LSC differentiation and testosterone production under the regulation of the desert hedgehog signaling pathway (DHH). It was hypothesized that the TME releases paracrine factors to modulate LSC differentiation. For this purpose, cells (Sertoli, PMCs, LSCs, and ALCs) were extracted from men undergoing testis biopsies for sperm retrieval and were evaluated for the paracrine factors in the presence or absence of the TME (Sertoli and PMC). The results demonstrated that TME secretes leptin, which induces LSC differentiation and increases testosterone production. Leptin’s effects on LSC differentiation and testosterone production, however, are inversely concentration-dependent: positive at low doses and negative at higher doses. Mechanistically, leptin binds to the leptin receptor on LSCs and induces DHH signaling to modulate LSC differentiation. Leptin-DHH regulation functions unidirectionally insofar as DHH gain or loss of function has no effect on leptin levels. Taken together, these findings identify leptin as a key paracrine factor released by cells within the TME that modulates LSC differentiation and testosterone release from mature Leydig cells, a finding with important clinical implications for TD.Subject terms: Stem-cell differentiation, Translational research     

Detection of anti-Mullerian hormone receptor II protein in the postnatal rat testis from birth to sexual maturity

Anti-Mullerian hormone (AMH) produced by the immature Sertoli cells negatively regulates the postnatal Leydig cell (i.e. adult Leydig cells/ALC) differentiation, however, the mechanism is sparsely understood. AMH negatively regulates the steroidogenic function of fetal Leydig cells (FLC) and ALC. However, when this function is established in the ALC lineage and whether AMH has a function in FLC in the postnatal testis are not known. Therefore, the objectives of this study were to examine the presence of AMH receptor type II (AMHR-II) in FLC and cells in the ALC lineage in the postnatal mammalian testis using the rat model Male Sprague Dawley rats of days 1, 5, 7-21, 28, 40, 60 and 90 were used. AMHR-II in testicular interstitial cells was detected in testis tissue using immunocytochemistry. Findings showed that the mesenchymal and the progenitor cells of the ALC lineage, were negative for AMHR-II. The newly formed ALC were the first cell type of the ALC lineage to show positive labeling for AMHR-II, and the first detection was on postnatal day 13, although they were present in the testis from day 10. From days 13-28, labeling intensity for AMHR-II in the ALC was much weaker than those at days 40-90. FLC were also positive. The time lag between the first detection of the newly formed ALC in the testis and the first detection of AMHR-II in them suggests that the establishment of the negative regulatory role of AMH on ALC steroidogenesis does not take place immediately upon their differentiation; no change in cell size occurs during this period. The absence of AMHR-II in mesenchymal cells suggests that it is unlikely that the negative regulatory effect of AMH on ALC differentiation in the postnatal testis is achieved via a direct action of AMH on mesenchymal cells. The presence of AMHR-II in postnatal FLC suggests a possible role by AMH on FLC, which warrants future investigations.     

Role of prolactin on Leydig, Sertoli and germ cellular neutral lipids in bonnet monkeys, Macaca radiata.

To elucidate the specific influence of prolactin on neutral lipids in Leydig, Sertoli and germ cell compartments of the testis in immature and mature monkeys, the present study was carried out by injecting ovine prolactin (oPRL) (1 mg/kg body weight/twice daily for 10 days ip), to both age groups. Similarly, bromocryptine (an ergot alkaloid which inhibits prolactin secretion) was given to other sets of immature and mature monkeys (1 mg/kg body weight/twice daily for 10 days ip) to induce hypoprolactinemia. It was observed that after oPRL administration the total lipid accumulated in the germ cells of immature and mature monkeys. Total lipid was markedly decreased in the Leydig cells of mature monkeys only. But no such influence of PRL was evident in the Leydig cells of immature monkeys, suggesting an age-dependent effect of PRL on the Leydig cells. The increase in total lipid in the germ cells following PRL treatment was contributed by mono, di- and triacyl glycerols and free cholesterol. However, an opposite effect of PRL was evident in the Leydig cells of mature monkeys, where the cholesterols and glyceride fractions registered a decrease. The reduced cholesterol fractions in the Leydig cells following PRL treatment suggests the utilization of cholesterol for steroidogenesis. Sertoli cells were found to be comparatively resistant to change in PRL status. Bromocryptine treatment brought about the opposite effect of PRL in almost all parameters studied in both immature and mature monkeys. In general, these findings with prolactin suggests that PRL has a specific and definite influence on testicular neutral lipids and the response of different cellular compartments was found to vary.     

Stem Leydig cells: from fetal to aged animals

Leydig cells are the testosterone-producing cells of the testis. The adult Leydig cell (ALC) population ultimately develops from undifferentiated mesenchymal-like stem cells present in the interstitial compartment of the neonatal testis. Distinct stages of ALC development have been identified and characterized. These include stem Leydig cells (SLCs), progenitor Leydig cells, immature Leydig cells, and ALCs. This review describes our current understanding of the SLCs in the fetal, prenatal, peripubertal, adult, and aged rat testis, as well as recent studies of the differentiation of steroidogenic cells from the stem cells of other organs.     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.     

Studies on H-Y antigen in different cell fractions of the testis during pubescence

Summary Various cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.     

Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in vitro

We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Paracrine control of Leydig cell activity by FSH dependent proteins from Sertoli cells: an in vitro study

The regulating effect of follicle-stimulating hormone (FSH) on Leydig cell function was studied using a model of immature porcine Leydig and Sertoli cells cultured in a hormone supplemented defined medium. FSH pretreatment for 2 days of Leydig cells cultured alone was with no effect. FSH pretreatment of Leydig cells cocultured with Sertoli cells increases Leydig cell activity in an FSH dose-dependent manner with a maximal effect observed at 50 ng/ml porcine FSH (pFSH). Leydig cells cultured for 2 days in conditioned medium (CM) by FSH stimulated (FSH-CM) Sertoli cells, as compared to CM by unstimulated (control) (C-CM) Sertoli cells show an increase of their activity with a maximal effect observed at 50 ng/ml pFSH. Leydig cells cultured in CM as compared to non CM, show a marked development of organelles (smooth endoplasmic reticulum and mitochondria) involved in the steroidogenic activity. The activity of FSH-CM as compared to C-CM on Leydig cell function was non dialyzable and trypsin sensitive. These data suggest that Sertoli cells exert a regulatory action on Leydig cell steroidogenic activity via FSH dependent secreted proteins.     

Regulation of pig Leydig cell aromatase activity by gonadotropins and Sertoli cells

The present work was done to investigate the cell localization of testicular aromatase activity and its regulation in immature pig testis using an in vitro model. Leydig cells and Sertoli cells were isolated from immature pig testes and cultured alone or together in the absence or presence of human chorionic gonadotropin (hCG) or porcine follicle-stimulating hormone (pFSH) for 2 days. At the end of incubation, the amounts of testosterone (T), estrone sulfate (E1S) and estradiol (E2) were measured. Then the cells were incubated for 4 h in the presence of saturating concentrations of delta 4-androstenedione (3 microM) and the amounts of E1S and E2 were measured again (aromatase activity). The ability of Sertoli cells to produce estrogens was very low and neither hCG nor pFSH had any significant effect. hCG stimulated, in a dose-dependent manner, the secretion of T and E1S by Leydig cells cultured alone as well as the aromatase activity of these cells. The main estrogen produced by Leydig cells was E1S. pFSH also stimulated the above parameters of Leydig cell function; this may have been due to the contamination of this hormone with luteinizing hormone (LH). Coculture of Leydig cells with Sertoli cells without gonadotropins had very small effects on T and E1S production and on aromatase activity. However, treatment of coculture with increasing concentrations of hCG had a dramatic effect on Leydig cell functions. For each hCG concentration, the amounts of T and E1S secreted, as well as the aromatase activity of the coculture, were 2- to 3-fold higher than those of Leydig cells cultured alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

WNT/CTNNB1 signaling is involved in the regulation of multiple embryonic developmental processes, adult tissue homeostasis, abd cell fate determination and differentiation. Many WNTs and components of the WNT/CTNNB1 signaling pathway are expressed in the testis, but their physiological roles in this organ are largely unknown. To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in both Leydig and Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Although Cre activity was expected in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Leydig cells, no evidence of Cre-mediated recombination of the floxed allele or of WNT/CTNNB1 pathway activation could be obtained, and testosterone levels were comparable to age-matched controls, suggesting that genetic recombination was inefficient in Leydig cells. Conversely, sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. The latter often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, and this was accompanied by an increase in the expression of the immature Sertoli cell marker AMH. In addition, a poorly differentiated, WT1-positive somatic cell population accumulated in multilayered foci near the basement membrane of many seminiferous tubules. Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis.     

Expression in vitro du gène de l'aromatase dans les cellules testiculaires du rat

The ability of the male gonad to convert androgens into estrogens is well known ; the microsomal enzymatic complex involved in this transformation is named aromatase and is composed of a specific cytochrome P450 aromatase (P450arom) and a ubiquitous reductase. Using a highly specific RT-PCR method we have measured the amount of P450arom mRNA in purified Leydig and Sertoli cells prepared from 20, 40 and 70–80 day-old rats. The amount of P450arom mRNA in the Leydig cells is independent of age (40 × 10⁻³ attomoles/μg of total RNA); in contrast, in the immature rat Sertoli cells, after 5 days of culture the amount of P450arom mRNA is 20-fold lower when compared to that of 20-day-old rat Sertoli cells (71 × 10⁻³ attomoles/μg of total RNA). Nevertheless, irrespective of the age, the addition of either FSH or dbcAMP for 6 h increases the level of P450arom mRNA in the rat Sertoli cell preparations. Therefore, we evidenced that during testicular maturation not only the Leydig cells but also the Sertoli cells of the rat have the capacity to express the gene for cytochrome P450 aromatase.     

Variations in testicular androgen receptors and histology of the lamb testis from birth to puberty

Changes in testicular androgen receptor numbers were studied in lambs from 25 to 100 days of age. During this period, cytoplasmic receptors increased from 5 to 80 pmol/testis and nuclear receptors from 1 to 12 pmol/testis, while the total volume of Leydig cells increased 7-fold. The total number of Sertoli cells doubled between 25 and 40 days of age. From 40 days onward their number remained constant while their cellular and nuclear sizes increased by a factor of 3 and 1.5 respectively. Cytoplasmic receptor concentration was positively correlated with the number of Sertoli cells per section of seminiferous tubule, and negatively correlated with the number of germinal cells per cross section. One explanation for these results could be that Sertoli cells are the main androgen target cells in lamb seminiferous tubules.     

Interaction between Leydig and Sertoli cells in vitro

The interaction between Leydig and Sertoli cells grown in co-culture was studied. After 3 to 4 days in culture, Leydig and Sertoli cells formed monolayers. To distinguish functional Leydig cells from Sertoli cells, a histochemical test for delta 5,3 beta-HSD activity was performed, and cells which showed a positive reaction were defined as Leydig cells, in contrast to Sertoli cells which did not manifest enzyme activity. Testosterone and oestradiol levels in culture media were determined by radioimmunological assays. Sertoli cells in co-culture showed a tendency to organize themselves as in vivo, forming a kind of pseudo-wall of the tubule. This process becomes more evident with the time of culture. Co-cultures secreted more androgens than Leydig cells alone and more oestradiol than Sertoli cells alone. This influence was strengthened by the presence of follicle stimulating hormone (FSH) in the culture medium, which was not the case in cultures of Leydig and Sertoli cells cultured separately.     

Expression of thyrotropin-releasing hormone receptors in rat testis and their role in isolated Leydig cells

Thyrotropin-releasing hormone (TRH) was initially discovered as a neuropeptide synthesized in the hypothalamus. Receptors for this hormone include TRH-receptor-1 (TRH-R1) and -2 (TRH-R2). Previous studies have shown that TRH-R1 and TRH-R2 are localized exclusively in adult Leydig cells (ALCs). We have investigated TRH-R1 and TRH-R2 expression in the testes of postnatal 8-, 14-, 21- 35-, 60-, and 90-day-old rats and in ethane dimethane sulfonate (EDS)-treated adult rats by using Western blotting, immunohistochemistry, and immunofluorescence. The effects of TRH on testosterone secretion of primary cultured ALCs from 90-day-old rats and DNA synthesis in Leydig cells from 21-day-old rats have also been examined. Western blotting and immunohistochemistry demonstrated that TRH-R1 and TRH-R2 were expressed in fetal Leydig cells (in 8-day-old rats) and in all stages of adult-type Leydig cells during development. Immunofluorescence double-staining revealed that newly regenerated Leydig cells in post-EDS 21-day rats expressed TRH-R1 and TRH-R2 on their first reappearance. Incubation with various doses of TRH affected testosterone secretion of primary cultured ALCs. Low concentrations of TRH (0.001, 0.01, and 0.1 ng/ml) inhibited basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion of isolated ALCs, whereas relatively high doses of TRH (1 and 10 ng/ml) increased hCG-stimulated testosterone secretion. As detected by a 5-bromo-2′-deoxyuridine incorporation test, the DNA synthesis of Leydig cells from 21-day-old rats was promoted by low TRH concentrations. Thus, we have clarified the effect of TRH on testicular function: TRH might regulate the development of Leydig cells before maturation and the secretion of testosterone after maturation. This research was supported by grants from the National Natural Science Foundation of China (nos. 39870109 and 30370750).     

Photoperiodic variations of somatic and germ cell populations in the Soay ram testis

Seasonal changes in the Leydig, Sertoli and stem cells (undifferentiated A0 and cyclic A1) spermatogonia and the daily spermatid production were analysed in the testes of adult Soay rams exposed to short days (8L:16D) or long days (16L:8D) for 12 weeks. The total numbers of Leydig, Sertoli and stem cells (A0 + A1) were not affected by the treatments, but the size of the Leydig and the Sertoli cells, the efficiency of spermatogenesis (i.e. the number of male gametes produced by an A1 spermatogonium) and the daily sperm production were all significantly reduced in the rams exposed to long days. There was a positive correlation between the concentration of FSH and testosterone and many of the cytological changes consistent with a causal role for these hormones in mediating the effects of photoperiod on the testicular function in the ram.