Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

On the mechanism of action of lead in the testis: in vitro suppression of FSH receptors, cyclic AMP and steroidogenesis

Previous evidence has shown that prenatal and neonatal exposure to low levels of Pb result in decreased FSH binding and steroidogenesis in the testes at the onset of puberty. The purpose of the present study was to determine by in vitro methods, if Pb acts by interfering directly with hormone binding, cyclic AMP production and steroidogenic enzyme activity. Sertoli cells were isolated from testes of prepubertal rats and cultured in the presence of 2.64 x 10(-4)M of either NaAc (control) or PbAc for 1, 4, 24, 48, 96 or 144 hr. There was no reduction in FSH binding and in FSH-induced cyclic AMP after a 1-4 hr exposure to Pb. After a 24-hr exposure to Pb, the cells exhibited a 10-20% decrease in FSH binding and cyclic AMP production and after 96 hr there was a 75% decrease in these 2 parameters. The inhibition was greater in cells from 16 day old than from 20 day old rats, so that in the former, after a 144 hr exposure the FSH-induced cyclic AMP of the Pb exposed cells was only 3% of the amount produced by the NaAc exposed cells (i.e. a 97% inhibition). After in vitro exposure to Pb for 48 hr, the steroidogenic activity (progesterone conversion to steroid metabolites) of Sertoli cells was significantly reduced and their steroidogenesis was no longer stimulated by FSH. A crude testicular enzyme preparation containing 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) exhibited approximately 25% reduction in activity if the assay buffer contained PbCl2 instead of the equivalent in NaCl. Prolonged in vivo exposure to Pb resulted in approximately 50% reduction in 3 beta-HSD activity. This is the first indication that in the testis Pb may act directly (immediate effect) by suppressing enzyme activities, and indirectly (long term effect) by reducing gonadotropin-receptor binding and the resultant cyclic AMP production.     

Isolation and culture of FSH responsive Sertoli cells.

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.     

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.     

Characterization of rat transferrin receptor cDNA: the regulation of transferrin receptor mRNA in testes and in Sertoli cells in culture

A 3.4 kilobase cDNA complementary to rat transferrin receptor mRNA has been isolated from an adult rat testis cDNA library. The rat transferrin receptor nucleotide sequence was shown to be 82% similar to the human transferrin receptor sequence over the amino acid coding region and over 90% similar in the sequences known to be responsible for iron regulation in the human mRNA. The mRNA was shown by Northern blot analysis to be regulated by iron levels in Sertoli cells in culture. Iron depletion resulted in at least a 5-fold increase in receptor message in Sertoli cells, as well as in an actively growing testicular cell line (S10-7). The level of transferrin receptor mRNA in cultured Sertoli cells was not influenced by hormones; however, chronic administration of testosterone or FSH to hypophysectomized rats resulted in increased transferrin receptor mRNA levels in the testis. Northern blot analysis of mRNAs from testes of rats synchronized at various stages of the cycle of the seminiferous epithelium showed that transferrin receptor mRNA was differentially regulated throughout the cycle. Northern blots of mRNA from germinal cell populations derived from synchronized tests showed that the message was regulated in the nongerminal cell components of the tubule, most likely the Sertoli cell. The comparison of transferrin receptor mRNA levels in normal testes and testes from hypophysectomized rats, as well as in isolated germinal cells and cultured Sertoli cells, suggested that transferrin receptor mRNA levels were considerably higher in Sertoli cells than in other cell types of the seminiferous tubules.     

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro interactions between Sertoli cells and steroidogenic cells

Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells.     

An analysis of the metabolites of progesterone produced by isolated sertoli cells at the onset of gametogenesis

Sertoli cells isolated from 17 day old rats were maintained in culture and incubated with [¹⁴C]-progesterone for 20 h. The cells and media were extracted with ether/chloroform and the extracts chromatographed two-dimensionally on TLC and the radioactive metabolites visualized by autoradiography. Nine of the metabolites (constituting about 88% of total metabolite radioactivity) were identified by relative mobilities of the compounds and their derivatives in TLC and GC systems and by recrystallizations with authentic steroids as the following: 20α-hydroxypregn-4-en-3-one, 3α-hydroxy-5α-pregnan-20-one, 5α-pregnane3α,20α-diol, 17β-hydroxy-5α-androstan-3-one, 5α-pregnane-3,20-dione, 17-hydroxypregn-4-ene-3,20-dione, testosterone, 5α-androstane-3α,17β-diol and androst-4-ene-3,17-dione. Over 71% of the metabolite radioactivity was due to 20α-hydroxypregn-4-en-3-one, the major metabolite. 5α-reduced pregnanes constituted about 12% and C₁₉ steroids comprised about 2.9% of the radioactivity of the metabolites. Calculation of relative steroidogenic enzyme activities from initial reaction rates suggested the following activities in μunits/mg Sertoli cell protein: 20α-hydroxysteroid oxidoreductase (20α-HS0; 7.71), 5α-reductase (4.77), 3α-HS0 (3.57), 17α-hydroxylase (0.93), 17β-HS0 (0.34) and C₁₇-C₂₀ lyase (0.34). The relatively high rate of steroidogenic enzyme activities in the Sertoli cells of young rats may indicate that Sertoli cells are less dependent on Leydig cell steroidogenesis than has been assumed. Since nearly all the metabolites of progesterone and testosterone are now identified, it is possible to construct a picture of Sertoli cell steroidogenic activity.     

Regulation of testicular and Sertoli cell gamma-glutamyl transpeptidase by follicle-stimulating hormone

Hormonal deprivation achieved by hypophysectomy or gonadotropin-releasing hormone (GnRH)-antagonist treatment of immature rats resulted in markedly lower testicular gamma-glutamyl transpeptidase (GGT) activity than in the testes of age-matched controls. When begun 15 days after hypophysectomy, follicle-stimulating hormone (FSH) treatment significantly increased testicular GGT above that in testes from hypophysectomized controls in a time- and dose-dependent manner. In contrast, testosterone propionate had only a small effect. Testicular GGT was higher in adult hypophysectomized rats treated with FSH from the time of surgery than in untreated hypophysectomized rats; testosterone propionate treatment had no effect. GGT activity in Sertoli cells isolated from GnRH antagonist-treated or hypophysectomized immature rats was also lower than in cells from control rats. FSH treatment from the day of hypophysectomy resulted in Sertoli cell GGT values equivalent to those from intact controls. These data indicate that FSH regulates GGT activity in rat testis and Sertoli cells.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Immunocytochemical staining of isolated rat Sertoli cells for anti-FSH beta

Sertoli cells are a primary target for the action of follicle-stimulating hormone (FSH) in the testis. The purpose of this investigation was to verify ultrastructurally that FSH binds to receptors on the plasma membrane of isolated rat Sertoli cells. A relatively pure aliquot of Sertoli cells was obtained by first dissociating testicular tissue from immature rats with collagenase and then centrifuging the suspension in Percoll density gradients. Pre-embedding staining with the peroxidase anti-peroxidase (PAP) complex technique using anti-FSH beta as the primary antiserum localized endogenous receptor-bound FSH on the plasma membrane of isolated Sertoli cells. Staining was considered to be specific since membranes of Sertoli cells derived from hypophysectomized rats were not stained when subjected to the same procedure. Cytoplasmic vesicles in Sertoli cells from experimental, control, and hypophysectomized groups also stained with PAP. Staining of these structures appeared to be specific since it was obliterated by preabsorption of anti-FSH beta with FSH. Preabsorption with luteinizing hormone (LH) did not affect the staining of cytoplasmic vesicles. The results of this investigation provide the first evidence for ultrastructural localization of specific binding sites for anti-FSH beta on the cell membrane of isolated Sertoli cells using an unlabeled antibody technique, and they further support the contention that Sertoli cells are a primary target for the action of FSH.     

LH contamination may explain FSH effects on rat Leydig cells

Treatment of immature, hypophysectomized male rats with 50 micrograms ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17 beta-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose-response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10(6) Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.     

Paracrine role of Sertoli cells

Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH.     

Rat testis 17 beta-estradiol: identification by gas chromatography-mass spectrometry and age related cellular distribution

The aromatization of testosterone into 17 beta-estradiol (E2) was assessed in purified Leydig and Sertoli cells from rats aged 10-80 days. E2 was identified by gas chromatography-mass spectrometry (GC-MS) and measured both by radioimmunoassay (RIA) and GC-MS associated with stable isotope dilution. A potent competitive inhibitor of the aromatase activity, 4-hydroxyandrostenedione (4-OH-A) was used to test the enzymatic specificity. The basal aromatase activity was present in both cell types whatever the age of the animals. The basal E2 levels did not vary in Sertoli cells while a gradual increase was noted in Leydig cells until day 40, followed by a slight decrease in mature rats. In 10-day old animals, the aromatase activity was localized in Sertoli cells and highly stimulated by FSH; on day 20, both Sertoli and Leydig cells synthesized E2 although E2 from Sertoli cell origin was still predominant. Starting on day 20 until adulthood, the aromatase activity was under LH control in Leydig cells with a maximum around 40 days. The FSH and LH effects were mediated by cyclic AMP.     

Effect of uncoupling treatments on FSH-induced hyperpolarization of immature rat Sertoli cells from Sertoli cell-enriched cultures

Sertoli cell-enriched cultures isolated from immature rat testes by enzymic treatments were investigated by intracellular microelectrode recordings. The hyperpolarization of cells induced by FSH was independent of the age of the rats (7-37 days) and was unchanged by exposure to a hormone-free medium or to a glycine buffer of pH 3. It was reduced by treatments which decreased the electrical coupling between cells either by an increase of intracellular calcium [i.e. calcium ionophore (A 23187, 5 x 10(-6) M), general anaesthetic (heptanol, 3.5 mM) and uncoupler of oxidative phosphorylations (carbonylcyanide m-chlorophenylhydrazone-CCmP, 10(-6) M)] or by a decrease of extracellular calcium [i.e. 0Ca + EGTA (1 mM) medium]. These effects were partly or totally reversed by a recovery period in a drug-free medium. Similar results were obtained by an exposure to trypsin (0.05%) followed by a second mechanical dispersion, but new cell hyperpolarization was induced by a new exposure to FSH. This electrophysiological study suggests an initial effect of FSH on the junctional complex between Sertoli cells, then the control by calcium of this complex.     

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.