Purine modulation of the hormonal response of the rat Sertoli cell in culture

To investigate the possibility that purines modulate the response of testicular cells to gonadotropin, binding of adenosine analogs and biological responses to adenosine were evaluated in Sertoli cell-enriched cultures. The adenosine analog cyclohexyladenosine bound specifically to a crude particulate fraction prepared from such cultures. Binding was saturable, and steady state studies showed the presence of a high affinity binding site (Kd = 2.1 +/- 0.3 nM; n = 4) and a receptor density of 200-300 fmol/mg protein. The bound radioactive ligand was displaced by N6-phenylisopropyladenosine (PIA), adenosine, and methylisobutylxanthine. In addition to the presence of a specific binding site, purines modulated the biological function of the Sertoli cell. The adenosine analog PIA inhibited both the FSH-dependent cAMP response and the FSH-stimulated androgen aromatization. Under all experimental conditions, the IC50 of PIA was 1-3 nM, and maximal effects were observed at 10-100 nM PIA. Adenosine itself inhibited the FSH-dependent response of the Sertoli cell, but was less potent than PIA. In addition, purine inhibition of the FSH response was antagonized by methylisobutylxanthine, while the nonxanthine phosphodiesterase inhibitor Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)2-imidazolidinone] was without effect. Purine modulation was evident not only when cells were stimulated with FSH, but also when the androgen aromatization was augmented by the beta-adrenergic agonist isoproterenol, cholera toxin, and forskolin. On the contrary, the purines had no effect when cells were stimulated with (Bu)2cAMP. The data reported are consistent with the presence of purine inhibitory receptors in Sertoli cell-enriched cultures and show that purines can regulate the response of the immature Sertoli cell in vitro.     

Long term treatment with adenosine analogs modifies the responsiveness of immature rat Sertoli cell in culture

A1 inhibitory adenosine receptors are present in cultured Sertoli cells. Activation of these receptors by short term exposure to adenosine agonists attenuates the adenylate cyclase activity and reduces FSH stimulation of androgen aromatization to estrogen. In the present study it was investigated how long term activation of the adenosine inhibitory system affects the responsiveness of the Sertoli cell. Sertoli cells from 15- to 17-day-old Sprague-Dawley rats were incubated with medium containing adenosine deaminase (1 IU/ml) in the presence or absence of 100 nM N6-2-phenyl-isopropyl-adenosine (PIA) for 24-48 h. At the end of this pretreatment medium was changed, and cell responsiveness was measured in terms of cAMP and estrogen production. In control cells, FSH-stimulated cAMP and estradiol production were inhibited by PIA, with an EC50 of 0.70 +/- 0.13 nM. This inhibitory effect was reduced in cells that had been pretreated for 24-48 h with 100 nM PIA. The PIA concentration-response curve of pretreated cells was shifted to the right, with a 4-fold increase in the EC50. Similar effects were also evident when adenosine itself or nonmetabolizable adenosine analogs other than PIA were used in the pretreatment. In addition to these changes in the inhibitory responses, PIA pretreatment increased the response of the Sertoli cell to FSH and forskolin in terms of both cAMP accumulation and estradiol production. Potentiation of the hormonal response was due to an increase in basal and maximal stimulation without significant changes in the total stimulation. This effect was dependent on the concentration of PIA used during the pretreatment. The increase in estradiol production was also evident when cells were stimulated with (Bu)2cAMP, suggesting that adenosine analog pretreatment affects steps distal to cAMP accumulation. Moreover, the responses to both the PIA inhibitory signal and FSH stimulation were restored to control levels when pretreated cells were incubated in fresh medium in the absence of PIA for 24 h. The long term PIA effects were also blocked by pretreatment in the presence of the A1 receptor antagonist 8-[4-([([ (2-amino-ethyl)amino]carbonyl)methyl]oxy)phenyl]1,3- dipropylxanthine. These results indicate that the A1 adenosine system present in the Sertoli cell becomes refractory after prolonged exposure to adenosine analogs. Furthermore, PIA pretreatment produced a potentiation of the Sertoli cell response to stimulatory signals by affecting several steps of the cAMP-dependent pathway.     

Involvement of phosphodiesterase in the refractoriness of the Sertoli cell

Gonadotropin treatment of the Sertoli cell produces a marked refractory state of the cell to subsequent hormonal stimulation. Because FSH also stimulates the phosphodiesterase activity of these cells, the possible involvement of an altered cAMP catabolism during refractoriness was investigated in an in vitro model. Sertoli cells, after 3 days of culture in a defined medium, were exposed to FSH or isoproterenol for 1-24 h. After this pretreatment, cells were stimulated for 1 h with a maximal FSH dose, and the responsiveness was measured in terms of cAMP accumulation. Sertoli cells previously treated with hormone entered a refractory state, a second exposure being ineffective in elevating intracellular or extracellular cAMP. Addition of the phosphodiesterase inhibitor 3-isobutyl-methylxanthine in the second incubation partially restored the ability of the cell to accumulate cAMP in the presence of hormone. This phosphodiesterase inhibitor also caused an apparent decrease in the potency of FSH to induce the refractory state. Such an impairment of response developed in the intact cell in 4 h, and was accompanied by a partial desensitization of the adenylate cyclase and an increase in phosphodiesterase activity. The stimulation of phosphodiesterase activity, but not the desensitization of adenylate cyclase, was inhibited by cycloheximide. The inhibition of protein synthesis also prevented the onset of the refractory state of the intact Sertoli cell. Pretreatment of the Sertoli cells with either FSH or isoproterenol rendered the cell refractory to a second stimulation with either agonist; in contrast, the adenylate cyclase desensitization in the homogenate was apparent only for the agonist employed in the preincubation. These results indicate that phosphodiesterase regulation is involved in the control of Sertoli cell responsiveness to hormone. Thus, the net decrease in cAMP production of the FSH-treated cells is the result of a decreased adenylate cyclase stimulation and an increased cAMP catabolism mediated by phosphodiesterase. The latter phenomenon appears to be the predominant cause of the partial refractoriness induced by low doses of gonadotropin.     

Glucagon-stimulated cyclic AMP production and formation of estradiol in Sertoli cell cultures from immature rats

Addition of glucagon to the incubation medium of cultured Sertoli cells isolated from immature (19-day-old) rats resulted in a time- and concentration-dependent stimulation of cAMP accumulation measured both in the cells and in the medium. Maximal intracellular levels of cAMP were reached after 30 min, after which the levels decreased. In the medium cAMP levels reached a plateau after 6 h. The magnitude and kinetics of the responses were comparable to those observed with FSH in the same culture preparations. 1-Methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, greatly potentiated the magnitude of the effects of glucagon and FSH. Glucagon stimulated adenylate cyclase activity in isolated membrane preparations from similar cultures, and the concentration causing half-maximal stimulation (EC50) was approximately 300 ng/ml. Glucagon also stimulated aromatization in cultured Sertoli cells to the same extent as FSH. It is concluded that cultured Sertoli cells isolated from immature rats contain receptors for glucagon, coupled to adenylate cyclase, and that glucagon also stimulates aromatization of testosterone to estradiol.     

Forskolin and phosphodiesterase inhibitors stimulate rat granulosa cell differentiation

The effect of forskolin (an adenyl cyclase activator) and 1-methyl-3-isobutylxanthine (MIX, a phosphodiesterase inhibitor) on granulosa cell steroidogenesis and LH receptor formation was studied in vitro. Granulosa cells from immature hypophysectomized, estrogen-treated rats were cultured for 2-3 days in androstenedione-supplemented media in the absence or presence of FSH or forskolin (10(-7)-10(-4) M). Some cultures were also treated with forskolin with or without MIX (0.125-1.0 mM) or theophylline (1.25-10 mM). Forskolin (3 X 10(-6)-10(-4) M) stimulated the production of estrogen, progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) and cAMP in a dose-related manner to levels similar to or higher than that elicited by FSH alone. Similarly, forskolin and FSH both increased LH/hCG receptor content in cultured granulosa cells, although forskolin was only 50% as effective as FSH. Treatment with MIX alone increased basal levels of cAMP, accompanied by elevations of estrogen and progestin biosynthesis without affecting LH/hCG receptor content. In contrast, theophylline treatment only increased cAMP and progestin accumulation. Furthermore, MIX potentiated the stimulatory effects of forskolin and FSH on cAMP and progestin production. In contrast, MIX inhibited FSH- and forskolin-stimulated estrogen production. Thus, activation of adenyl cyclase and inhibition of cAMP breakdown in the cultured rat granulosa cells enhance steroidogenesis and LH receptor formation, reinforcing the concept that cAMP is a (but may not be the only) second messenger in the hormonal regulation of granulosa cell differentiation.     

Effects of adenosine analogs on glucagon-stimulated adenosine 3',5'-monophosphate formation in Sertoli cell cultures from immature rats

In the present study, we have examined the effects of two adenosine analogs, (-)N6-(R)phenyl-isopropyl-adenosine (PIA) and 2-chloro-adenosine, on glucagon- and FSH-stimulated cAMP production in Sertoli cell cultures isolated from immature (19-day-old) rats. Both FSH and glucagon caused a 5- to 10-fold stimulation of cAMP levels in the spent media from Sertoli cell cultures during an 18-h incubation. Addition of 1 microM PIA significantly inhibited both FSH- and glucagon-stimulated cAMP levels. In the presence of a maximal concentration of glucagon (2.5 micrograms/ml), PIA caused a concentration-dependent inhibition of cAMP formation, and the concentration of PIA causing half-maximal inhibition of cAMP formation (IC50) ranged from 0.5-1 nM. When Sertoli cells were incubated with increasing concentrations of glucagon (1.28 ng/ml to 4.00 micrograms/ml) in the absence and presence of either PIA (1.0 microM) or 2-chloro-adenosine (10.0 microM), the responses to glucagon, measured as cAMP formation, were almost completely abolished. 1-Methyl-3-isobutylxanthine (MIX), a well known inhibitor of cAMP phosphodiesterase activity, is also an inhibitor of adenosine binding to receptors on the cell membrane. When Sertoli cells stimulated with glucagon (2.5 micrograms/ml) were incubated in the absence and presence of MIX (0.1 mM) and increasing concentrations of PIA (0.025-10,000 nM), the presence of MIX reduced the inhibitory activity of PIA by almost 2 orders of magnitude (IC50 without MIX, 0.5 nM; IC50 with MIX, 20 nM). Thus, the present study shows that adenosine analogs inhibit agonist-stimulated cAMP formation in cultured Sertoli cells, and that MIX reduces this effect. This indicates that cultured Sertoli cells from immature rats contain A1-receptors for adenosine mediating inhibitory effects on adenylate cyclase.     

Cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, induces cytodifferentiation of follicular granulosa cells cultured in serum-free medium

Cultured granulosa cells from intact immature rats produced large amounts of progestins in response to phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX). MIX alone stimulated up to 9 times higher amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) when compared with FSH (100 ng/ml)-stimulated steroidogenesis. Combined action of MIX and FSH did not yield more activity than MIX action alone. MIX activity was demonstrable exclusively in serum-free culture medium (Dulbecco modified Eagle's medium/F-12) supplemented with insulin, transferrin, hydrocortisone, and fibronectin. Serum severely inhibited MIX induction of 20 alpha-OH-P production. MIX also caused dramatic cell shape changes which occurred within 14 h of exposure to the drug. The cells assumed a spherical shape and remained so as long as MIX was maintained in the culture medium. Upon removal of the drug, the cells respread and acquired the morphology of luteinized cells. In contrast to FSH action, MIX failed to induce the appearance of functional LH receptors. However, similar to FSH effect on immature granulosa cells, MIX successfully induced aromatase enzyme throughout 12 days in culture. MIX alone caused only a minute increase in the accumulation of cAMP. cAMP content in cells induced with FSH (100 ng/ml) was 46 times higher than the cyclic nucleotide levels measured under maximal effective doses of MIX. The discrepancy between the intensive steroidogenic activity of MIX and its inability to substantially raise cAMP content suggests a possible cAMP-independent mechanism for steroid production in the ovarian cells.     

Regulation of follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate of a phosphodiesterase isoenzyme of the Sertoli cell

The regulatory role of FSH on phosphodiesterase was studied in immature Sertoli cells in culture. Cells were prepared from 15-day-old immature rats, cultured for 3 days in defined medium, and then stimulated for 24 h with gonadotropin or with other factors known to regulate Sertoli cell cAMP. All agents that increased cAMP intracellular levels also had an effect on the total phosphodiesterase activity of cell homogenates, FSH and dibutyryl cAMP being the most potent stimulators. Further, stimulation was more evident when phosphodiesterase was measured at cAMP concentrations below micromolar. With 1 microM cGMP as substrate, no modification of the activity could be detected. Reversal of phosphodiesterase activation was observed at 24 or 40 h when dibutyryl cAMP was removed from the incubation medium. Separation of the isoenzymes present in the soluble fraction of Sertoli cell made it possible to demonstrate a selective stimulation of one isoenzyme. FSH and dibutyryl cAMP increased 10- and 50-fold, respectively, the activity of a high affinity cAMP phosphodiesterase, while the Ca2+-dependent cGMP hydrolyzing enzymes were not apparently affected. The enzyme regulated by FSH and dibutyryl cAMP had an apparent Km for cAMP of 2 microM, was Ca2+ and calmodulin insensitive, and migrated on sucrose density gradients with a sedimentation coefficient of 5.5S. These results indicate that FSH, after stimulation of intracellular cAMP, induces an increase in a high affinity phosphodiesterase and, therefore, an increased cAMP turnover in the Sertoli cell. This, in turn, might be a relevant phenomenon in the control of the responsiveness of this cell to gonadotropin.     

Effects of follicle-stimulating hormone on cultures of Sertoli cell preparations.

Sertoli cell-enriched preparations were obtained by sequential enzyme treatment of testes of 18-20 day old rats, and were maintained in culture in a chemically defined medium. The addition of highly purified follicle-stimulating hormone (FSH) to cells immediately after preparation, or after 48 h in culture, elicited an increase in the level of cyclic adenosine 3',5'-monophosphate (cAMP) when incubated in the presence of 3-isobutyl-1-methylxanthine (MIX). Luteinizing hormone (LH) had no effect on the cAMP levels. The cells cultured in the presence of FSH or dibutyryl cAMP for 48 h incorporated more [3H]leucine into trichloroacetic acid (TCA)-insoluble material than did cells cultured in the basal medium. Cycloheximide abolished the amino acid incorporation into protein precipitated by TCA. The data demonstrate that the Sertoli cell is a target cell for FSH action, and indicate that added dibutyryl cAMP can duplicate the enhancement of amino acid incorporation into protein elicited by FSH.     

Androgen/antiandrogen modulation of cyclic AMP-induced steroidogenesis during granulosa cell differentiation in tissue culture

FSH stimulation of granulosa cell differentiation is believed to be mediated by the intracellular cyclic AMP (cAMP) level. However, steroidogenic enzyme induction in the differentiating granulosa cell is subject to direct modulation by androgenic steroid: in granulosa cell cultures established from ovaries of oestrogen-pretreated, prepubertal rats, potentiating effects of testosterone (T) on FSH induction of oestrogen synthetase (aromatase) and progesterone (P) biosynthesis can be blocked by including a stoichiometric excess of antiandrogen (hydroxyflutamide, SCH 16423) in the culture medium. In this study we used the same experimental model to determine effects of T and SCH 16423 on the induction of steroidogenesis by endogenous cAMP and an exogenous cAMP analogue, 8-bromo-cAMP (8brcAMP). Granulosa cells were cultured in medium containing variable FSH concentration (3–300 ng/ml) with a fixed (100 μm) dose of 3-isobutylmethylxanthine (MIX), or containing a fixed (minimally effective: 10–15 ng/ml) dose of FSH with MIX concentration variable (50–800 μM). By relating steroidogenic endpoints at 48 h to the acute cAMP response (accumulations in the medium) at 1 h, it was deduced that aromatase induction was saturable under conditions where FSH-sensitive cAMP production and the induction of P biosynthesis showed further, proportionate increases.Although T (0.1μM) did not alter acute FSH-responsive cAMP production, its presence throughout the 48 h culture was required for full expression of FSH-induced steroidogenesis in the cell monolayers. When the aromatase response (but not the P response) was ‘supersaturated’ by endogenous cAMP (i.e. culture with FSH plus MIX), SCH16423 was unable to antagonize the potentiating effect of T on aromatase induction while it continued to block T-potentiated P biosynthesis. Steroidogenic induction by cholera toxin (100 ng/ml) was also subject to similar modulation by T and SCH16423. However, the phosphodiesterase-resistant cAMP analogue 8brcAMP (3 mM) not only induced each response (albeit submaximally in the case of aromatase) in the absence of T, but its effects tended to be refractory to androgen/antiandrogen modulation. Accumulations of cAMP in the medium from 48 h cultures which had been incubated with FSH (100 ng/ml) were increased 2–3-fold by the additional presence of T (0.1 μM). This long-term stimulatory effect of T on FSH-dependent cAMP accumulation was blocked by culture in the presence of SCH16423 (10 μM). Thus, androgen potentiation of steroidogenic enzyme induction during FSH-stimulated granulosa cell differentiation may involve a suppression of cAMP catabolism exerted by way of the androgen-receptor system.     

Granulosa cell aromatase bioassay for follicle-stimulating hormone: validation and application of the method

Stimulation of rat granulosa cell aromatase activity by FSH has recently been used as a sensitive biological end point to develop an in vitro FSH bioassay. The present report provides a detailed validation and application of this assay. In the presence of androstenedione and diethylstilbestrol, FSH stimulated estrogen production in a dose-dependent manner. Although addition of high doses of a phosphodiesterase inhibitor [1-methyl-3-isobutyl xanthine (MIX)] decreased maximal estrogen production, treatment with 0.125 mM MIX increased the sensitivity of granulosa cells to FSH, presumably by minimizing endogenous cAMP breakdown. Addition of insulin and human CG (hCG) further synergistically enhanced granulosa cell sensitivity to FSH. Although inclusion of gonadotropin-free serum obtained from hypophysectomized male rats decreased the assay sensitivity, pretreatment of serum with polyethylene glycol [(PEG) 10-14%] resulted in a dose-dependent decrease in the serum-interfering effect. Studies using exogenous [125I]iodo-rat FSH or RIA measurement indicated recovery of 94-98% FSH after pretreatment of serum with 12% PEG. In the presence of the PEG-pretreated gonadotropin-free serum (4%), ovine, rat, and human FSH preparations induced parallel dose-response curves for estrogen production with minimal detectable doses of 0.12 ng, 0.12 ng, and 0.12 mIU/culture, respectively. In contrast, treatment with GH, PRL, TSH, and ACTH did not affect estrogen production. The apparent stimulatory effect of high doses (greater than 60 ng/culture) of LH and hCG could be attributed to FSH contamination or intrinsic FSH activity in these preparations. Changes in serum bioactive FSH levels were studied in adult male rats after GnRH administration. GnRH (5 micrograms/rat) treatment significantly elevated FSH levels within 30 min after injection. Maximal increases (approximately 2.8-fold) in serum bioactive FSH were observed between 60-120 min. At 8 h after treatment, FSH levels decreased to control levels. Comparison between granulosa cell aromatase bioassay and RIA results indicated no apparent changes in the bio- to immuno- ratio of FSH after GnRH treatment. In conclusion, extreme sensitivity of the bioassay allows the measurement of circulating levels of bioactive FSH. Since rat granulosa cells respond to FSH preparations from different species, the in vitro assay should also provide valuable information on FSH levels in many animal species including those lacking a specific RIA. Measurement of serum levels of bioactive FSH should provide insight regarding the role of FSH in various physiological and pathological conditions.     

Age-related and testicular regression-induced changes in adenosine 3',5'-monophosphate responses in cultured hamster Sertoli cells

Sertoli cells prepared from adult hamsters with maximally regressed testes responded to FSH with an increased accumulation of intracellular cAMP similar to that of Sertoli cells from an immature animal. That is, the age-dependent decline in responsiveness of Sertoli cells to FSH was reversed during testicular regression. To determine whether this testicular regression-induced restoration of FSH responsiveness was mechanistically a reversal of the age-dependent decline in response, the ability of cholera toxin, forskolin, catecholamines, and pertussis toxin to stimulate cAMP accumulation in Sertoli cells cultured from hamsters undergoing testicular regression was assessed and compared to the responses of Sertoli cells from 18- and 36-day-old hamsters. The age-related decline in responsiveness was evident not only with FSH but also when cells were stimulated with isoproterenol, cholera toxin, forskolin, or pertussis toxin singly or in combination. While the FSH response of Sertoli cells from regressed testes was restored to values indicative of an immature Sertoli cell, the response of the cultured cells to cholera toxin, forskolin, catecholamines, or pertussis toxin either singly or in combination suggested that the adenylate cyclase system of Sertoli cells from regressed testes was unchanged from its "adult" activity. Thus, our original hypothesis that testicular regression/recrudescence was a mirror image of sexual maturation was an oversimplification. However, since FSH is the physiological regulator of Sertoli cell function, and its response is restored to levels found in the immature animal during testicular regression and declines to adult levels during testicular recrudescence, our original hypothesis that testicular recrudescence mimics sexual maturation (i.e. the animal goes through "puberty" each time it goes through a regression/recrudescence cycle) is still tenable. However, at the molecular level, differences in mechanism exist.     

Two mechanisms of inhibition by prostaglandin E2 of hormone-dependent cell cAMP in the rat collecting tubule.

The effect of exogenous prostaglandin E2 (PGE2) on hormone-dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated by microradioimmunoassay in collecting tubules microdissected from the cortex (CCT) or outer medulla (MCT) of the rat kidney. Two phosphodiesterase inhibitors were used: either a xanthine derivative (isobutyl-methylxanthine (IBMX, 1 mM] active on all forms of phosphodiesterase or Ro 20-1724 (50 microM) active on the phosphodiesterase type III. A prostaglandin synthesis inhibitor was added to all media. In the presence of IBMX, 0.3 microM PGE2 inhibited by 39.1% the response induced in the CCT by the beta-adrenergic agonist isoproterenol (1 microM). Under the same experimental conditions, arginine vasopressin (AVP)-stimulated cAMP accumulation in CCT or MCT was not affected by PGE2. In the presence of Ro 20-1724, 0.3 microM PGE2 did not modify the response to 1 nM AVP in CCT but inhibited this response in MCT samples (mean inhibition: 52.7%). The inhibition by PGE2 was dose dependent with a maximum at 0.3 microM, observed for all concentrations of AVP tested (from 50 pM to 1 nM) and did not affect the concentration of AVP inducing half-maximal cAMP accumulation. In a second experimental series performed in the presence of adenosine deaminase, an A1-adenosine agonist [theta)-N6-(R-phenylisopropyl)adenosine (PIA, 0.1 microM] also decreased the response to 1 nM AVP in the MCT. The addition of an A1-adenosine antagonist relieved the effect of PIA but did not modify the inhibition observed with PGE2. Thus PGE2 decreased the synthesis of cAMP in beta-adrenergic sensitive cells in rat CCT and might affect the catabolism of AVP-dependent cAMP level rather than its synthesis in rat MCT.     

Cellular localization of rat testicular aromatase activity during development

The aromatase activity from purified testicular sources (Leydig and Sertoli cells) of immature (5- and 15-day-old) and adult rats (60-day-old) was investigated by the tritiated water release method in isolated Leydig and Sertoli cells that were morphologically and functionally characterized. Electron micrographs of Sertoli cell preparations from different ages showed no marked changes, except that tight junctions between Sertoli cells normally present in 60-day-old rats were not observed in 5-day-old and rarely found in 15-day-old animals. Leydig cells underwent ultrastructural changes along with development, such as the appearance of thicker nuclear heterochromatin and laminar-like mitochondria. The 15-day-old rat interstitial tissue possessed less than 10% of Leydig cells morphologically similar to those present in the adult, whereas the rest were probably transition cells, since they did not show typical Leydig cell structure but were able to bind [125I]iodo-hCG, as evaluated by autoradiography. The number of LH/hCG-binding sites increased with age in Leydig cells, but was not detectable in Sertoli cells. The highest number of FSH-binding sites in Sertoli cells was observed in the 15-day-old animals. Minor FSH binding was found in Leydig cell preparations, which was consistent with the known LH contamination of the human FSH tracer preparation. cAMP production increased significantly in Leydig cells only after hCG treatment and in Sertoli cells after FSH stimulation. Both types of cells were shown to have the capacity for aromatization. The aromatase activity increased in the Leydig cell but decreased in the Sertoli cell during testicular development. The highest aromatase activity was found in adult rat Leydig cells, and the enzyme activity was significantly higher (2-fold) in purified Leydig cells than in crude interstitial cell preparations. Estradiol production in response to hCG stimulation in vitro was not different from the basal value in 5-day-old rat Leydig cells, but increased significantly in 60-day-old rat Leydig cells. In conclusion, 1) Leydig cells are the major site of estrogen synthesis in adult rat testis; and 2) the low aromatase activity observed in immature rat Leydig cells could partially explain the differential response of the mature and immature rat testis to hCG-induced desensitization.     

Stimulation of adenosine 3',5'-monophosphate production in rat Sertoli cells by alpha-melanotropin-stimulating hormone (alpha MSH) and des-acetyl alpha MSH

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.     

Biochemical actions of follice-stimulating hormone in the sertoli cell of the rat testis.

The sequenc of biochemical events associated with the action of follicle-stimulating hormone (FSH) in the testis has been investigated using a Sertoli cell-enriched testis model system. The Sertoli cell-encriched testis, created by irradiation of male rats in utero, is devoid of germinal elements but contains a normal complement of supportive Sertoli cells. Comparison of the Sertoli cell-enriched testis with normal testis, demonstrates that the two types of testes contain equal numbers of FSH specific receptors, judged by the binding of labeled hormone. In addition, FSH over a concentration range from 6 X 10(-11) to 6 X 10(-9)M will stimulate the production of adenosine 3',5' monophosphate (cAMP) in the Sertoli cell-enriched testis in a manner indistinguishable from that of the normal testis. Incubation of Sertoli cell enriched testis also results in the activation of soluble cAMP-dependent protein kinase. This response to FSH is dependent upon the age of the animal and disappears at about 32 days of age. While sensitivity to the hormone can still be detected in mature Sertoli cell-enriched animals by the addition of the phosphodiesterase inhibitor 1-methyl-3-isobutyl-xanthine, no detectable increase in phosphodiesterase activity is apparent after 30 days of age. Injection of FSH into Sertoli cell-enriched animals results in an increase in total testicular protein synthesis as well as in the production of the Sertoli cell-specific protein, androgen-binding protein within 30 minutes. Furthermore, while hypophysectomy of Sertoli cell-enriched animals result in a decline of the testicular concentration of androgen-binding protein, the injection of FSH will stimulate and maintain the levels of androgen-binding protein in such animals. These results demonstrate that the Sertoli cell-enriched testis is capable of carrying out the sequence of biochemical events previously described for FSH in the normal testis and therefore, suggest that the Sertoli cell is the primary target cell for FSH action.     

Cholinergic control of cyclic nucleotide metabolism in human thyroid cells

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages

Indirect (hypotonically treated culture) and direct (coculture) approaches were used to study the influence of germ cells on androgen-binding protein (ABP) and 17 beta-estradiol secretion by Sertoli cells prepared from 10-, 15-, 20-, and 45-day-old rats. Using these approaches, the mechanisms that govern Sertoli cell-germ cell membrane recognition were shown to be unaltered by the hypotonic treatment, to vary with the age of the Sertoli cell donors, and to be FSH/(Bu)2cAMP dependent in the younger animals (10-20 days old). Hypotonic treatment had no effect on estradiol levels at all ages studied, but resulted in a marked fall of ABP production by Sertoli cells from 20 days onward. However, the relative stimulation of ABP induced by FSH/(Bu)2cAMP (ABP levels in FSH/(Bu)2cAMP/hypotonically treated Sertoli cell cultures vs. ABP in hypotonically treated cultures) was markedly increased at 20 and 45 days, indicating that germ cells influence Sertoli cell responsiveness to FSH/(Bu)2cAMP. The addition of crude germ cell preparation to the Sertoli cell cultures stimulated ABP and inhibited estradiol levels at all ages studied. Moreover, the addition of germ cells to hypotonically treated cultures induced a decrease in the relative ABP response to FSH and (Bu)2cAMP, thus confirming (see above) that germ cells may be involved in the mechanism of Sertoli cell refractoriness to FSH that occurs with advancing age in the rat. Germ cell-stimulated ABP production was higher in adult Sertoli cells than in immature cells. That Sertoli cell responsiveness to germ cells varies with age is further supported by the data showing that whereas spermatocytes stimulated ABP and inhibited estradiol productions at all ages studied, early spermatids only influenced these parameters from 20 days onward. It is concluded that germ cell control over Sertoli cell function in vitro may be of important physiological significance during Sertoli cell maturation in mammals.     

Follicle-stimulating hormone and androgens increase the concentration of the androgen receptor in Sertoli cells

The influence of FSH and androgens on androgen receptor levels in primary Sertoli cell cultures from immature rats is studied in a monolayer binding assay and by sucrose density gradient centrifugation using the synthetic radiolabeled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) as a ligand. Preincubation of Sertoli cells for 4 days with FSH, testosterone, or 5 alpha-dihydrotestosterone results into a 2- to 3-fold increase in mibolerone binding, as measured 18 h after removal of the agonists. The combination of androgens and FSH has additive effects. The action of FSH can be mimicked by (Bu)2cAMP, and the activity of the androgens can be blocked by the antiandrogen cyproterone acetate. The mibolerone-binding protein has the ligand specificity, affinity, and sedimentation behavior characteristic for an androgen receptor. Using a DEAE-cellulose filter disc assay and 5 alpha-dihydrotestosterone as a ligand, androgen-binding protein (ABP) was measured in the media of the studied Sertoli cell cultures. Despite some similarity in the hormonal control of ABP and the androgen receptor, there are distinct differences in the ligand specificity of the two androgen binding proteins, which exclude that ABP might interfere with the receptor measurements. The effects of androgens and FSH on the androgen receptor are evident at concentrations equal to or lower than those required to provoke a measurable increase in ABP secretion. It is concluded that FSH and androgens control androgen receptor levels in Sertoli cells.