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Follistatin is an activin-binding glycoprotein that decreases FSH secretion in vitro and in vivo in rats. The mechanism by which follistatin acts is unclear, but it has been suggested that it may bind endogenous activin and neutralize its effects. In this study, we wished to test the ability of follistatin to suppress FSH secretion in vivo in primates whose FSH secretion has been stimulated by activin or by GnRH. Six prepubertal male monkeys were injected intravenously with human recombinant follistatin at the dose of 90 micrograms/kg or 180 micrograms/kg plus activin (90 micrograms/kg) or GnRH (10 micrograms/kg). Frequent blood samples were drawn for 12 hours following each injection. Bio FSH and LH levels were measured in those samples. GnRH and activin each stimulated FSH bioactivity. Both doses of follistatin significantly inhibited the activin-induced increase in FSH (p0.05). The GnRH-induced increase in FSH was not affected by follistatin. LH levels were not affected by follistatin in any of the studies. These data suggest that follistatin can suppress the activin-induced increase in FSH in primates and is consistent with the hypothesis that follistatin can block the physiological effects of endogenous activin in primates. This effect is likely to be due to the binding of follistatin to activin either in the peripheral circulation or at the pituitary level.

Inhibin and activin were first described as feedback inhibitors of pituitary function. However, a range of data now supports the hypothesis that these factors play a major role in the paracrine regulation of gonadal function (1, 2). Activin seems to act as a mitogen and morphogen during development. Both inhibin and activin, and their receptors and binding proteins, are also expressed during the normal cycling of the adult ovary (3) and testis. The exact response to these hormones, however, may vary with the developmental stage of the testis or ovary and the stage of the seminiferous or follicular cycle.

This report describes the development of a specific and sensitive assay for inhibin B and its application to the measurement of inhibin B concentrations in plasma during the human menstrual cycle. A monoclonal antibody raised against a synthetic peptide from the betaB-subunit was combined with an antibody to an inhibin alpha-subunit sequence in a double antibody enzyme-linked immunosorbent assay format. The validated assay had a limit of detection of 10 pg/mL and 0.5% cross-reactivity with inhibin A. Using this immunoassay, we found that the plasma concentration of inhibin B rose rapidly in the early follicular phase to a peak of 85.2 +/- 9.6 pg/mL on the day after the intercycle FSH rise, then fell progressively during the remainder of the follicular phase. Two days after the midcycle LH peak, there was a short lived peak in the inhibin B concentration (133.6 +/- 31.2 pg/mL), which then fell to a low concentration (20 pg/mL) for the remainder of the luteal phase. In contrast, the inhibin A concentration was low in the early follicular phase, rose at ovulation, and was maximal during the midluteal phase. The concentration of inhibin B in individual follicular fluid samples was 20- to 200-fold higher than the concentration of inhibin A and was highest in follicular fluid samples from the early follicular phase. Inhibin B appears to be the predominant form of inhibin in the preovulatory follicle. The different patterns of circulating inhibin B and inhibin A concentrations observed during the human menstrual cycle suggest that these forms may have different physiological roles.

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.

Twenty day-old rats were used to prepare Sertoli cell-enriched cultures which were grown for 4 days in serum-free medium supplemented with human transferrin, epidermal growth factor, and insulin. Proteins in the medium were first fractionated by anion-exchange HPLC. Forty-five to 50 fractions were collected, and each was examined by electrophoresis in sodium dodecyl sulfate-containing polyacrylamide gels. Using silver nitrate as a stain, more than 30 proteins were routinely identified. Rat androgen binding protein and rat transferrin were measured by immunoassays. When the cultures were supplemented with either testosterone, FSH, or both hormones and the media fractionated by HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these proteins could be tentatively categorized as follows: unresponsive to added hormones; responsive primarily to testosterone; responsive primarily to FSH; or responsive to testosterone or FSH but maximally responsive to both hormones. This report provides a method for identifying proteins in Sertoli cell-enriched cultures and has allowed us to show for the first time that many are responsive to hormones. The procedure takes advantage of the large loading capacity of the HPLC column to allow fractionation of liter quantities of medium. The high resolving power of the column permits preparation of fractions which are highly enriched with one or a limited number of proteins. The purification of many of these proteins is now feasible. The approach described is generally applicable for isolation of proteins from media from a variety of cells.

We have investigated the effects of insulin and somatomedin-C/insulinlike growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 micrograms/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM-C and insulin, respectively. Insulin and Sm-C had no additive effect on these parameters. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 micrograms/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [3H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.

Specific RIAs for rat transferrin (rTF) and androgen-binding protein (rABP) were used to determine whether the secretion of these proteins was coordinately regulated in the Sertoli cell under a variety of conditions. Sertoli cell-enriched primary cultures were prepared from the testes of 20-day-old rats, and rTF and rABP were assayed in medium from the same culture. There was a strong effect of cell density on both rABP and rTF secretion per cell, with increased secretion per cell at high densities. Human TF (hTF), FeSO4, and desferrioxamine had little or no effect on rTF secretion. The age of the animal at the time of preparation of cells for culture had a strong effect on the pattern of rTF and rABP secretion in vitro; however, the effects of animal age, time in culture, and medium supplementation differed for the two proteins. In cultures prepared from 20-day-old animals, insulin, epidermal growth factor, and testosterone stimulated both rTF and rABP secretion, although to different extents. Retinoic acid was required for the stimulation and maintenance of rTF secretion, but had no effect on rABP secretion in the presence of insulin, hTF, and epidermal growth factor. Conversely, FSH and isoproterenol stimulated rABP, but not rTF, secretion. These data suggest that the secretion of rABP and rTF by Sertoli cells is under differential control.

The pattern and hormonal control of rat androgen-binding protein (rABP) secretion in vitro was investigated using animals of different ages to initiate primary cultures of Sertoli cells. Sertoli cells were isolated from testes of 7- to 31-day-old rats and cultured for periods of up to 30 days in serum-free medium, medium supplemented with insulin, transferrin, and epidermal growth factor (3F), or 3F plus FSH, testosterone, progesterone, hydrocortisone, and vitamin E (8F). The amount of rABP secreted by Sertoli cells during the first 24 h in culture (initial rate) exhibited an age-dependent pattern which reflected the apparent in vivo activity of these cells. Between 7 and 25 days of age, the initial rate of rABP secretion per Sertoli cell increased 20-fold; a further 2-fold increase occurred between 25 and 35 days of age. The pattern of rABP secretion exhibited by Sertoli cells cultured for several weeks was dependent not only on added factors (3F or 8F), but also on Sertoli cell age, expressed as the total of animal age plus time in culture (total age). In cultures of Sertoli cells isolated from very young animals (7-10 days old), the rate of rABP secretion increased until 20 days (total age), but declined thereafter. This early increase in rABP secretion was augmented by, but not dependent on, hormone additions. In contrast, Sertoli cells isolated from older animals always showed decreasing rates of secretion with time in culture. Furthermore, Sertoli cells from very young animals retained the capacity to respond to hormones in vitro with increased secretion of rABP and maintenance of cell viability. This responsiveness decreased with age, similar to the loss of hormone response seen in vivo after puberty. In conclusion, culture conditions were established which permitted the study of FSH-dependent and independent regulation of rABP secretion and of the acquisition of hormone resistance at the time of puberty. The initial rate of rABP secretion in culture (first 24 h) correlates with the age of the animal from which the cultures are obtained. The pattern of rABP secretion during subsequent long term culture is determined by the total age (animal and culture age), with increasing rates of secretion up to 20 days and decreasing rates thereafter. This inherent pattern of rABP secretion as well as loss of responsiveness of the Sertoli cell to hormonal stimulation appear to be programmed early in development.

Summary Sertoli cells synthesize and secrete a number of cell-specific products including androgen binding protein (ABP), as well as “serum proteins” such as transferrin. The secretion of these proteins is regulated by extra-testicular hormones such as FSH and insulin; Leydig cell-produced steroids and proopiomelanocortin-derived peptides; and the presence of peritubular myoid cells and/or factors secreted by these cells. Many of the Sertoli cell proteins are secreted in a cyclic fashion during the different stages of the spermatogenic cycle suggesting communication between Sertoli cells and developing germ cells. The availability of quantitative measurements for Sertoli cell-specific proteins such as ABP make it feasible to follow Sertoli cell function in vivo by measuring these products in serum. A bidirectional secretion of proteins by Sertoli cells is proposed to explain the presence of specific peptides in the male reproductive tract and blood.

Studies demonstrating calcitonin receptors on Leydig cells have suggested that these cells may be one of the many sites affected by this peptide. To investigate this possibility, the effect of synthetic salmon calcitonin on the TM3 Leydig cell line (derived from immature mouse Leydig cells) and on primary Leydig cell-enriched preparations was examined. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in TM3 cells. In addition, the hormone stimulated the basal secretion of testosterone in both TM3 cell- and Leydig cell-enriched cultures and potentiated the action of hCG on Leydig cell-enriched cultures. Synthetic salmon calcitonin also increased the concentration of androgen and estrogen receptors in cultured TM3 Leydig cells by 2- and 4-fold, respectively, when added to the culture medium (1 micrograms/ml). The fact that 8-bromo-cyclic AMP decreased both androgen and estrogen receptor concentrations suggested that the effect of calcitonin on sex steroid receptors is not mediated by its effect on cyclic AMP in these cells. The possibility that the action of calcitonin on steroid receptors might be mediated by another messenger such as calcium (Ca2+) was therefore considered. Progressively lowering the concentration of Ca2+ in the culture medium of the cells from 1.5 mM to less than 0.01 mM decreased the concentration of both androgen and estrogen receptors. Returning the Ca2+ concentration to normal levels (1.5 mM) restored steroid receptor levels. Receptor levels were also decreased when the extracellular Ca2+ concentration was lowered to 0.5 mM, and treatment with the Ca2+ ionophore, A23187 (1 microM), restored receptor levels to normal. The calcium channel blocker, verapamil, decreased the androgen receptor concentration but unexpectedly increased the concentration of estrogen receptors. It was concluded that calcitonin stimulates cAMP formation and testosterone secretion, and increases the concentration of sex steroid receptors. These observations provide evidence that the previously demonstrated calcitonin receptors on Leydig cells may be coupled to several biologic responses in this cell type.

Four forms of acid phosphatase have been found in the testicular tissue of many mammalian species, but their exact cellular site has remained obscure. In this work, acid phosphatases have been studied in different reproductive organs of the male rat, in somatic cell lines derived by cloning from both rat and mouse testes, in primary cultures of rat Sertoli cells, and in isolated spermatogenic cells of the mouse. Among the reproductive organs, preputial glands show the highest specific activities with p-nitrophenyl phosphate as substrate, followed by the testicular tissue and the different regions of the epididymis. By contrast to that in other tissues, testicular activity with p-nitrophenyl phosphate is not influenced by tartrate and is activated markedly by cobalt (Co2�). Among the somatic cell lines, the highest hydrolysis rates are obtained with napbtbyl substrates in the epithelial (TR-1) and myoid (TR-M) cell lines and marginally lower rates in the Leydig (TM3) and Sertoli (TM4) cell lines. With thymolphthalein phosphate, the latter two cell lines show very low activity. These activities are not influenced by different hormones and growth factors in the culture medium. The most marked Co2+�activated reaction with p-nitrophenyl phosphate is found in advanced stages of germinal cells and residual bodies. Primary cultures of Sertoli cells, prepared from rats 10 to 30 days of age, show a slight decrease in acid phosphatase levels; however, the activities are not influenced markedly by addition of follicle-stimulating hormone (FSH) and/or testosterone to the culture medium. Chromatofocusing of somatic and germinal cell homogenates resulted in two tartrate-sensitive activity peaks (El, ElI), which probably correspond to lysosomal enzymes, and a double-peak of tartrate-resistant activity (Elli). The epithelial and myoid cell lines also have a minor tartrate-resistant activity (EV) with a low isoelectric point (p1). The germinal cells and residual bodies as well as the Sertoli cell line each have a separate tartrate-resistant enz,�yme (EIV) that is activated markedly by Co2+ and several other divalent metal ions (Mg2+, Mn2+, Ni2+, Zn2 ). It is concluded that the latter enzyme may have a special function in processing the structures of germinal cells before and after spermiation, while others, (El-ElIl, EV) are obviously more widely distri buted forms of acid phosphatase.

Angiotensin-converting enzyme (ACE) has been studied in different reproductive organs of the male rat, in somatic cell lines clonally derived from both rat and mouse testes, and in isolated spermatogenic cells of the mouse. Among the various reproductive organs only testis and epididymis show high levels of enzyme activity. The testicular activity is found mainly in the isolated germinal cells and residual bodies, whereas somatic cell lines contain negligible levels of activity even after addition of hormones and growth factors. Testicular homogenates, spermatogenic cells, epididymal spermatozoa, and spermatozoan cytoplasmic droplets, when fractionated by anion exchange chromatography, contain one major and one minor activity peak, whereas epididymal homogenates and epididymal secretions reveal an additional major activity peak together with the minor peak. All forms of ACE have a similar response to different modifiers, and are equally sensitive to the specific inhibitor N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl-L-proline (Enalapril). The testicular enzyme could provide a useful marker for spermatogenic maturation and/or cytoplasmic processes both in testis and epididymis. The separate epididymal peak is a secretory enzyme that may be responsible for the processing of spermatozoan plasma membrane constituents during epididymal transit, or may have a role in attacking some biologically active compounds.

The indazole carboxylic acid derivative, tolnidamine, has marked antispermatogenic activity in several animal species. In this study, we assessed the effect of tolnidamine on rat Sertoli cell function both in vivo and in vitro, using androgen binding protein (r ABP) as a marker. Groups of six male rats were killed 2, 4, 8, 16, 32, 64 hours and 5, 8, and 12 days following tolnidamine administration (250 mg/kg by oral gavage). There was a progressive reduction in both testicular and epididymal weights. Serum FSH levels did not change and LH showed a transient increase between 64 hours and 8 days. Except for an initial increase at 2 hours, there were no changes in serum testosterone. Epididymal rABP concentration and content declined as early as 8 hours, with the lowest values occurring at 5 and 12 days. By 16 hours, there was an increase in testicular rABP, which was also evident at 8 days and 12 days. Within 16 hours after tolnidamine, there was a rise in serum rABP, which persisted until the end of the experiment. When another indazole carboxylic acid derivative, lonidamine, was administered (250 mg/kg), similar changes were evident in epididymal and serum rABP at 32 hours, but the rapid decrease in testicular rABP suggested a different mechanism of action. In another experiment, single oral doses of tolnidamine (50, 100, 250, and 500 mg/kg) were administered to other groups of rats and the animals were killed after 24 hours and 5 days. With increasing doses of tolnidamine, there was a reduction in epididymal rABP concomitant with an increase in testis and serum rABP levels. Since rABP levels in the testis and epididymis change following treatment with tolnidamine, we wished to determine whether this agent had a direct effect on Sertoli cells; this possibility was investigated in vitro. Sertoli cells were prepared from 20-day-old animals and cultured in serum-free medium containing insulin, transferrin, epidermal growth factor, and various concentrations of lonidamine or tolnidamine. By 7 days, there were no significant effects on rABP secretion into the medium with either agent. It can be concluded that a single dose of tolnidamine has a minimal effect on the pituitary Leydig cell axis since there was only a transient increase in serum LH with no alterations in testosterone. In contrast, this drug had a dramatic and rapid effect on seminiferous tubule function as manifested by disruption of the dynamics of rABP secretion. The reduction in epididymal rABP may be best explained by inhibition of the passage of tubular fluid to the epididymis. Obstruction of efferent ductules was unlikely because there was no increase in testicular weight. The increase in testicular rABP was presumably related to accumulation secondary to reduced secretion into, or transport from, the seminiferous tubular lumen. The rise in serum rABP is best explained by increased testicular rABP concentration. The failure to observe a change in rABP concentration during incubation of cultured Sertoli cells with increasing doses of tolnidamine implies a lack of a direct effect of tolnidamine on rABP synthesis, and suggests that this drug primarily alters the kinetics of secretion from the intact tubule.

In vitro formation of haploid spermatids has not been convincingly demonstrated in mammals. To investigate this problem we selected defined segments of rat seminiferous tubules containing late pachytene and diakinetic primary spermatocytes (Stages XII and XIII of the cycle) for culture in a chemically defined medium. After 2 days, most spermatocytes completed both meiotic divisions, and by 6 days the tubular epithelium developed morphologic characteristics of Stage V in which the newly formed spermatids had acrosomic systems characteristic of step 5 spermiogenesis. The seminiferous tubules also differentiated biochemically as evidenced by increased production of proteins characteristically secreted by Stage V. Since this in vitro differentiation of the germinal epithelium occurred in the absence of testosterone and FSH, we conclude that late pachytene spermatocytes and their associated Sertoli cells have all the information required for both meiotic divisions and early spermiogenesis.

The present paper examines the steroidogenic responsiveness of immature porcine Leydig cells in primary culture. Both testosterone (T) and dehydroepiandrosterone sulfate (DHAS) secretion were measured under basal conditions and after stimulation with human chorionic gonadotropin (hCG) (25 ng/ml). In medium supplemented with insulin, transferrin, epidermal growth factor (3H) and 0.1% calf serum, cells survived 3–5 days in culture. The production of steroids (under hCG stimulation) is poor on day 0–1 of the culture. On day 2–4 basal T and DHAS levels are 1.9 and 17.0 ng/10 6 cells/24 h. The addition of hCG stimulated T and DHAS production 19- and 6-fold respectively and the average productions were 37 and 109 ng/10 6 cells/24 h. Increasing the serum to 0.5% did not change the viability of the cultures, but increased hCG stimulated T and DHAS production (183 and 188 ng/10 6 cells/24 h). The addition of α-tocopherol (vitamin E) to 0.1% calf serum led to a 4-fold increase in stimulated T production (142 ng/10 6 cells/24 h) and maintained full cell viability for more than 5 days. Measurement of 3β-ol steroid dehydrogenase activity indicates that the amount of enzyme is 4 times higher at day 2 than at day 0 and 1 (with or without hCG), suggesting a spontaneous maturation of the cells in culture. This might explain the increased T production with time in culture. In cumulative experiments (24 h) the cells do not seem to be desensitized to hCG stimulation following prolonged exposure to 25 ng hCG since the daily steroid production is increasing with time in culture. However, kinetic studies show that steroidogenesis is not linear over a 24 h period. In cumulative experiments the steroid production stops between 12 and 16 h following hCG exposure (5 and 100 ng/ml) and resumes following a medium change. These results suggest that some inhibitory compounds are accumulated in the medium and are controlling the Leydig cell function. Moreover high doses of hCG (100 ng/ml) result in a lower production of steroids and an earlier plateau in the case of DHAS. These results demonstrate that porcine Leydig cells can live and differentiate in hormone- and vitamin-supplemented medium and that auto-feedback mechanisms inhibiting steroid accumulation take place under in vitro conditions.

Androgen binding protein (ABP) was isolated from rat testes and epididymides by affinity chromatography. Rat ABP is a glycoprotein with a native molecular weight of 85,000 daltons. This protein is comprised of 45,000 and 41.000 dalton components (rABP h and rABP l in a ratio of 3:1. rABP h and rABP l have similar polypcptide chains with identical binding sites. Part of the difference in these protomers is due to their carbohydrate compositions. A major portion of rABP is secreted by Sertoli cells into the seminiferous tubular lumen and from there, it is transported to the epididymis. The remainder is secreted into the blood. Evidence is reviewed supporting the postulate that most of rABP that enters the blood is released from the base of the Sertoli cells. A kinetic analysis of the disappearance of rABP from blood following removal of the testes plus the epididymides versus removal of only the testes suggests that the epididymides can release ABP into the blood during androgen deprivation. A study of hypophysectomized rats supports this postulate. Results are also presented which suggest that progestins, in addition to testosterone and FSH. stimulate ABP release into the blood.

In order to evaluate the direct effect of gossypol on testicular cells, we used primary cultures of rat Leydig and Sertoli cells. No alteration in Leydig cell survival, morphology, or testosterone production was seen during three days of culture with up to 3 microgram/ml gossypol. With higher concentrations (3 to 7 microgram/ml) of gossypol, there was a reduction in cell survival but no change in androgen secretion. In contrast, there was a marked change in Sertoli cell morphology after five days of gossypol treatment. Large vacuoles and electron dense granules appeared in the cytoplasm, but these effects were reversed within six days of removing gossypol from the medium. There was a significant decrease in androgen binding protein (ABP) secretion by Sertoli cells in the presence of gossypol. We also tested the effect of gossypol on the growth of three established cell lines. Two Sertoli-derived cell lines, TM4 and TR-ST, were more sensitive than a Leydig-derived cell line (TM3). These results suggest that, of the somatic cell types in the testis, the Sertoli cells are most sensitive to gossypol.

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.

Vitamin E (α-tocopherol) is known to be required for testicular function but its action on specific testicular cells has not yet been studied. The present study used procine Leydig cell cultures, in a hormone-supplemented medium, to study the effect of vitamin E (vit E) on Leydig cells. It was seen that the addition of vit E to the medium led to an increase in cell survival, lengthening the life span of the cultures from 3–4 days to more than a week. The Leydig cells maintained their LH/hCG receptors and responsiveness throughout this period as evidenced by an increase in testosterone (T) and prostaglandin secretion. The hCG stimulated T levels were synergistically increased in the presence of vitamin E, while basal levels of T secretion were not changed. Other secretory products of Leydig cells are prostaglandins E2 and F2α. It was found that the addition of vit E inhibited both the basal prostaglandin levels and the stimulated levels by 90%. Maximal effects on all of these parameters were seen at 10 ng/ml vit E. It is obvious that vit E plays a critical role in maintaining porcine Leydig cells in primary cultures beyond the first 3 days. This vitamin seems to be involved both in steroidogenesis and in prostaglandin production in the Leydig cells. The exact mechanism of the action of vit E these two biosynthetic pathways remains to be determined.

Adrenocorticotropin (ACTH), beta-endorphin, and the melanocyte-stimulating hormones (MSHs), which are products of a common precursor, pro-opiomelanocortin (POMC), are present in a variety of tissues other than pituitary. The recent detection of immunoreactive POMC-derived peptides in the male reproductive tract raised the possibility that these hormones might regulate reproductive function. To determine whether the low concentrations of POMC-derived peptides in the male reproductive tract are synthesized locally and are not contaminants from blood, we have demonstrated POMC-like gene expression in both testis and epididymis. The identification of cells in testis capable of synthesizing POMC mRNA was established by showing the presence of this mRNA in mouse Leydig cell lines (TM3 and I10A). The hybridizing species of POMC-like mRNA in the testis, epididymis, and Leydig cell lines (TM3 and I10A) were approximately 150 bases shorter than those in the pituitary or hypothalamus but were similar in size to that in the amygdaloid nucleus of rat brain. The concentration of POMC-like mRNA in the testis is almost as high as that in the hypothalamus. This finding is quite unexpected because the concentrations of POMC-derived peptides in the testis were 2-3 orders of magnitude lower than those in the hypothalamus. The demonstration of a POMC-like gene expression in male reproductive tissues suggests that POMC-derived peptides are synthesized in Leydig cells and epididymis. These observations are consistent with the postulate that POMC-derived peptides may exert paracrine and/or autocrine effects in these organs.

The effects of synthetic salmon calcitonin on primary Sertoli cell-enriched cultures and on an established cell line (TM4 cells, derived from immature mouse Sertoli cells) were studied. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in both cell systems. In addition, this peptide stimulated the secretion of rABP in primary Sertoli cell-enriched cultures prepared from rat testis. Calcitonin also increased the total concentration of both androgen and estrogen receptors in TM4 cells. Because cAMP analogs decreased androgen and estrogen receptor concentrations, the effect of calcitonin on sex steroid receptors may not be mediated by its effect on cyclic AMP in these cells. The possibility that the action of synthetic salmon calcitonin on the receptors might be mediated by a change in cellular Ca2+ was investigated. Lowering extracellular Ca2+ concentrations from 1.5 mM to less than 0.01 mM markedly reduced the concentration of androgen and estrogen receptors; restoration of Ca2+ to 1.5 mM returned receptor levels to normal. When the receptor concentrations were decreased by lowering extracellular Ca2+ concentrations to 0.5 mM, treatment with the calcium ionophore, A23187, restored receptor levels to normal. Although the calcium channel blocker, verapamil, decreased receptor levels, calcitonin partially counteracted its effect. Trifluoperazine, an inhibitor of calmodulin, also diminished androgen and estrogen receptor, levels in the cytosol of TM4 cells. It was concluded that calcitonin stimulates the formation of cyclic AMP and the secretion of rABP by Sertoli cells. This peptide also increases the concentration of androgen and estrogen receptors, possibly by a mechanism that is, in part, Ca2+ -mediated. These results, along with those on Leydig cells, suggest that calcitonin could be a regulator of testicular function.

Estrogen and androgen receptors were measured in the cytosol prepared from established cell lines of Sertoli, Leydig, myoid, and endothelial cells as well as in primary Sertoli cell-enriched cultures. Estradiol-binding sites with characteristics of estrogen receptors were identified in established lines of Sertoli cells from rat [43 +/- 7 (mean +/- SEM) fmol/mg protein; Kd = 1.1 nM] and mouse (34 +/- 4 fmol/mg protein; Kd = 0.9 nM). The binding sites were estrogen specific, since only estradiol and diethylstilbestrol, but not testosterone, progesterone, or dexamethasone, competed with [3H]estradiol for binding sites in the cytosol prepared from the cell lines. Exposure of the cells to estradiol (10 nM) resulted in accumulation of estrogen receptors in nuclei, with maximal uptake by 30 min. The estrogen receptor concentration was very low or undetectable (less than 10 fmol/mg protein) in primary cultures of rat Sertoli cells that were cultured for 3 days. However, after 15 days in culture, the estrogen receptor concentration increased and reached levels similar to those in the established Sertoli cell lines. No estrogen receptors were measurable in myoid or endothelial cells. By contrast, androgen receptors were identified in all five cell lines and in primary Sertoli cells cultured for 3 and 15 days. The content of both estrogen and androgen receptors in the mouse Sertoli cell line increased as a function of cell density. We conclude from these studies that androgen receptors are present in all of the testicular somatic cell lines examined and in primary Sertoli cells; estrogen receptors are present in Sertoli cell and Leydig cell lines, but not in myoid and endothelial cell lines; the low estrogen receptor concentration in Sertoli cells cultured for 3 days increases 4-fold after 15 days in culture; and cell density is a major regulator of the concentrations of estrogen and androgen receptors in the Sertoli cell line.

Cultures of porcine Leydig cells (>80% pure) were used to study the regulation of LH/hCG receptors and Leydig cell responsiveness over several days. These cells contained approximately 60,000 receptors/cell, with an apparent Kd of 1.5 × 10-9 M. Binding of hCG to receptor was time and dose dependent (saturation at 4–6 h), and maximal binding was maintained for 24 h. After removal of hCG, radioactivity was released into the medium. This spontaneous release (50% released by 8 h) was unaffected by the presence of cold hormone in the medium. About 80% of the released material was inactive in a binding assay and had the properties of monoiodotyrosine. Throughout the period of culture, hCG receptor was maintained at or above the original level. The addition of hCG during the culture caused a dose-and time-dependent down-regulation of receptor. After removal of the hormone, hCG receptors increased, reaching 50% of control within 24 h. Most of the bound radioactive hormone could be released by a brief acid treatme...

Proopiomelanocortin and its derivative peptides alpha MSH and beta-endorphin are produced by Leydig cells. beta-Endorphin or another testicular opiate is believed to suppress Sertoli cell hypertrophy. The goal of this study was to determine the effects of another proopiomelanocortin-derived peptide on Sertoli cells. The activities of both alpha MSH and des-acetyl alpha MSH have been compared, since this latter peptide has been identified in testicular extracts. Both alpha MSH and des-acetyl alpha MSH stimulated cAMP accumulation in the media of primary Sertoli cell cultures when incubated in the presence of a phosphodiesterase inhibitor, FSH or forskolin. Both peptides shifted the FSH dose-response curve to the left, making the cells more sensitive to this gonadotropin. The apparent potencies of alpha MSH and its des-acetyl derivative, as measured in Sertoli cells, were similar. We conclude that the MSHs are one of a group of modulators regulating Sertoli cells via the cAMP system, and Sertoli cells are equally responsive to alpha MSH and des-acetyl alpha MSH, unlike central nervous system and melanocytes which show differential responses to these peptides.