Leydig cell insufficiency in hypospermatogenesis: a paracrine effect of activin-inhibin signaling?

Clinical findings and a variety of experimental models indicate that Leydig cell dysfunction accompanies damage to the seminiferous tubules with increasing severity. Most studies support the idea that intratesticular signaling from the seminiferous tubules to Leydig cells regulates steroidogenesis, which is disrupted when hypospermatogenesis occurs. Sertoli cells seem to play a pivotal role in this process. In this review, we summarize relevant clinical and experimental observations and present evidence to support the hypothesis that testicular activin signaling and its regulation by testicular inhibin may link seminiferous tubular dysfunction to reduced testosterone biosynthesis.

Testosterone and estradiol are co-secreted episodically by the human testis.

In spite of a striking pulsatile pattern of luteinizing hormone (LH) secretion, testosterone (T) fluctuations in peripheral blood in normal adult men are irregular and of low amplitude. To determine whether T secretion by the human testis is episodic, T was measured in blood samples drawn at 15-min intervals for 4 h through a catheter placed in the testicular vein of six men with varicocele-associated infertility. Estradiol (E2) concentrations were also determined in each sample. Each subject released testosterone in well-defined pulses. Gonadal vein T levels ranged from 1 to 1,540 ng/ml. Mean (+/- SE) pulse amplitude was 176 +/- 42 ng/ml, with a frequency of 4.0 +/- 0.3 pulses per 4 h. Testicular vein E2 levels ranged from 0.01 to 6.8 ng/ml. E2 secretory episodes were generally coincident with T pulses, and their amplitudes were highly positively correlated (r = 0.90, P less than 0.01). These results indicate that T secretion by the adult human testis is pulsatile, and suggest a functional relationship between intermittent LH secretion and normal testicular steroidogenesis in men. The failure to appreciate these fluctuations as hormone pulses in peripheral blood may relate to their absolute amplitude and frequency. The concordance between E2 and T pulses suggests that the Leydig cell, under LH control, is the source of most of the E2 secreted by the adult human testis.

Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin.

In primary cultures of rat pituitary cells, inhibin and follistatin reduce steady state levels of FSH beta mRNA to less than 10% of control within 4-6 h, while activin increases this mRNA 2- to 3-fold after 2-4 h of treatment. The effects of these three gonadal polypeptide hormones on the LH beta and common alpha-subunit mRNAs are more gradual and of lesser magnitude. The present study was designed to determine whether inhibin, activin, and/or follistatin act at the posttranscriptional level by altering the stability of the gonadotropin subunit mRNAs. To determine the decay rates of FSH beta, LH beta, and alpha-subunit mRNAs, primary pituitary cell cultures were treated for 1-24 h with either of two transcriptional inhibitors, actinomycin-D or 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB), in the presence or absence of recombinant human inhibin-A, recombinant human activin-A, or purified bovine follistatin. The decay of preexisting gonadotropin subunit mRNAs was followed by Northern blot analysis. Levels of LH beta and alpha-subunit mRNAs remained constant or increased during the 24-h exposure to transcriptional inhibitors; therefore, it was not possible to calculate their half-lives. The stability of these mRNAs was not altered by inhibin, activin, or follistatin. In contrast, FSH beta mRNA turned over rapidly: the estimated half-life was 2.6 +/- 0.19 h (mean +/- SEM of eight determinations) after actinomycin-D treatment and 1.9 +/- 0.14 h (mean +/- SEM of 12 determinations) after DRB treatment. When new RNA synthesis was blocked by either actinomycin-D or DRB, there were no significant effects of inhibin, activin, or follistatin on the stability of FSH beta mRNA (n = 2-4 for each hormone). The decay of FSH beta mRNA in the presence of inhibin or follistatin alone, however, was even more rapid than that determined after the administration of transcriptional inhibitors (P < 0.005). After an initial lag of 1-2 h, the half-life of FSH beta mRNA was 0.88 +/- 0.15 h (n = 4) or 0.62 +/- 0.11 h (n = 3), in the presence of inhibin or follistatin, respectively. The most likely interpretation of these results is that inhibin/follistatin reduces steady state levels of FSH beta mRNA by inducing a labile protein that accelerates the degradation of this mRNA species, and the synthesis of this protein is blocked by actinomycin-D or DRB treatment. It is not clear at present whether inhibin, follistatin, and activin have additional effects on transcription of the gonadotropin subunit genes.

Effect of inhibin from primate Sertoli cells and GnRH on gonadotropin subunit mRNA in rat pituitary cell cultures.

Partially purified inhibin from primate Sertoli cell culture medium (pSCl) suppresses both LH and FSH secretion from cultured rat pituitary cells stimulated with GnRH. To examine the mechanism of action of pSCl, we have measured steady state levels of mRNAs for the gonadotropin subunits in pituitary cell cultures exposed to 10 nM GnRH for 6 h in control or pSCl-containing medium (short term) and after 72-h pretreatment with pSCl or control medium (long term). Messenger RNA levels were determined by Northern analysis using specific cDNA probes for rat FSH beta, LH beta, and the common alpha-subunit. In the long term experiments, pSCl inhibited GnRH-stimulated release of FSH (47.4 +/- 3.3% of control), LH (69.2 +/- 2.3%), and free glycoprotein alpha-subunit (74.2 +/- 4.5%), and intracellular FSH declined to 88.4 +/- 3.5% of control. Concentrations of the subunit mRNAs were all decreased: FSH beta to 54.4 +/- 5.0%, LH beta to 79.6 +/- 9.4%, and alpha to 70.8 +/- 8.7% of control. In the short-term experiments, pSCl also suppressed FSH, LH, and alpha-subunit secretion to 75.9 +/- 3.6%, 79.5 +/- 2.1%, and 90.9 +/- 1.8% of control, respectively. Intracellular LH and alpha-subunit levels were significantly increased in cells treated for 6 h with GnRH and pSCl (155 +/- 18%, 145 +/- 14% of control), while FSH was comparable to control. After 6 h, pSCl selectively reduced the level of mRNA for FSH beta (56.5 +/- 5.8% of control).(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid and profound suppression of messenger ribonucleic acid encoding follicle-stimulating hormone beta by inhibin from primate Sertoli cells.

We showed previously that inhibin, partially purified from cynomolgus monkey Sertoli cell culture medium (primate Sertoli cell inhibin referred to as pSCI), selectively suppressed basal FSH secretion from dispersed rat pituitary cells and decreased total cellular FSH, but not LH content, suggesting a decrease in FSH biosynthesis. In order to investigate the mechanism of action of inhibin at the molecular level, we have now examined the effects of pSCI on steady state levels of the subunit mRNAs encoding LH and FSH and correlated these with release and intracellular content of LH, FSH, and glycoprotein alpha-subunit. Dispersed pituitary cells from 7- to 8-week-old adult male rats were cultured in the presence of pSCI or control medium for 2-72 h. FSH secretion was reduced significantly by 6 h (P less than 0.05) and reached a nadir (38% of control) by 48 h. LH secretion was unchanged, while release of the alpha-subunit was decreased to 89% of control at 72 h (P less than 0.05). Also by 72 h, cell content of both FSH (73% of control) and alpha-subunit (81% of control) were significantly suppressed (P less than 0.001, P less than 0.01), while LH was slightly affected. Total RNA was extracted from the pituitary cell cultures, electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled cDNA probes for the rat alpha-, LH beta-, and FSH beta-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of plasma luteinizing hormone by prolactin in the male rat.

Although a direct effect of PRL on gonadotropins has been previously suggested, it has not been convincingly demonstrated. The secretion of LH and FSH was studied in response to the stimuli of castration and LH releasing hormone administration in adult male rats made hyperprolactinemic with ectopic pituitary glands. Although plasma LH and FSH levels were similar in non-castrate hyperprolactinemic rats vs. controls, LH concentrations 24 h postcastration were less in hyperprolactinemic animals as compared to controls (P less than 0.001). The level of LH achieved was inversely correlated with the PRL concentration generated (r = -0.71; P less than 0.01). LH suppression was evident in hyperprolactinemic rats at 1 and 3 days postcastration but was no longer observable at 7 days postcastration. After LH releasing hormone administration to non-castrate rats the rise in plasma LH was significantly less in the hyperprolactinemic animals (P less than 0.05). These experiments support the hypothesis that PRL directly inhibits LH secretion, presumably at the pituitary level.

Regulation of lutenizing hormone secretion and subunit messenger ribonucleic acid expression by gonadal steroids in perifused pituitary cells from male monkeys and rats.

The mechanisms by which gonadal steroids regulate gonadotropin secretion remain incompletely understood. As previous studies suggest that the pituitary actions of testosterone (T) and estradiol (E) differ in male primates and rodents, we compared the effects of 10 nM T, 0.1 nM E, and 10 nM dihydrotestosterone (DHT) on the LH response to hourly pulses of GnRH as well as the GnRH receptor (GnRH-R) and LH subunit messenger RNA (mRNA) levels in dispersed pituitary cells from intact male monkeys and rats. T suppressed (P < 0.01) and E increased (P < 0.05) GnRH-stimulated LH secretion by rat pituitary cells. With monkey pituitary cells, on the other hand, there was no significant effect of either T or DHT on GnRH-stimulated LH secretion. In E-treated monkey cells, a period of initial enhancement (P < 0.05) was followed by significant suppression (P < 0.05) of LH secretion. GnRH-R mRNA was unchanged by T or E in either rat or monkey cells. T suppressed LHbeta (P < 0.01) and alpha-subunit (P < 0.01) mRNAs, whereas E increased alpha-subunit (P < 0.01), but did not alter LHbeta mRNA levels in rat cells. In monkey cells, however, neither T nor E affected LHbeta or alpha-subunit mRNA levels significantly. Our results identify different regulatory mechanisms by which testicular steroid hormones control LH secretion by the pituitary in male primates and rodents. We propose that the primary site of androgen negative feedback in the male primate is to restrain GnRH pulsatile secretion, whereas in the male rat T also decreases gonadotropin synthesis and secretion by directly affecting the pituitary. E suppresses GnRH-stimulated LH secretion in the primate pituitary, but amplifies the action of GnRH in the rat. Our data also reveal that the action of T to suppress LH secretion and subunit mRNA in male rats is not through decreased GnRH-R gene expression.

Partial characterization of circulating inhibin-B and pro-alphaC during development in the male rhesus monkey.

Gel filtration chromatography and ELISAs for inhibin-B and pro-alphaC were used to examine the circulating forms of inhibin in the neonatal (age 2-6 weeks), juvenile (age 1-2 yr), and adult male rhesus monkey. In all samples, isoforms of inhibin-B of 26-36K and 150K were found. Both forms were significantly greater in the adult. The alpha-subunit assay detected major peaks at 45-60 and 29-31K, and a minor peak of greater than 100K. As for inhibin-B, the major forms of inhibin pro-alphaC were highest in adulthood. Inhibin-B and pro-alphaC were measurable in peripheral plasma at age 1 week, increased with the neonatal rise in plasma FSH, and then decreased but remained detectable through age 1 yr. Values in adult males were higher than at any time during the first year of life. Finally, mean values of plasma inhibin-B and pro-alphaC in five monkeys, based on multiple blood samples drawn between age 1 week and 1 yr, were rank ordered and were found to be highly positively correlated (r = 0.96), suggesting that inhibin levels in the first year of life may be a marker of Sertoli cell number, and may predict the spermatogenic capacity of the testis in adulthood.

Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids.

cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.

Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary cells.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that pituitary adenylate cyclase activating polypeptide suppresses follicle-stimulating hormone-beta messenger ribonucleic acid levels by stimulating follistatin gene transcription.

There is accumulating evidence to suggest that pituitary adenylate cyclase-activating polypeptide (PACAP) may be an important modulator ofgonadotrope function. One of the actions of PACAP identified previously is to decrease FSHbeta messenger RNA (mRNA) levels. In the present series of experiments we demonstrate that PACAP-induced suppression of FSHbeta mRNA correlates with a rise in follistatin mRNA levels in primary pituitary cell cultures. Transient transfection of gonadotrope-derived alphaT3-1 cells with a rat follistatin promoter-luciferase reporter plasmid reveals that PACAP stimulates follistatin gene transcription. PACAP stimulation of LUC activity was maximal at concentrations as low at 1 nM. Furthermore, in alphaT3-1 cells PACAP activation of the follistatin promoter appears to be via the cAMP-dependent protein kinase A pathway. Accordingly, we propose that PACAP stimulates follistatin transcription, which neutralizes activin activity and thereby reduces FSHbeta mRNA. Since PACAP and follistatin are colocalized in multiple tissues including the brain, adrenals, and gonads, our findings may reflect a broadly distributed autocrine/paracrine mechanism for modification of activin effects that is under PACAP control.

Identification of androgen-binding protein from testis cytosol and sertoli cell culture medium of the cynomolgus monkey, Macaca fascicularis.

The biochemical and immunological properties of monkey androgen-binding protein (mABP) from the cynomolgus monkey, Macaca fascicularis, have been examined in testis cytosol and medium of primary Sertoli cell-enriched cultures. mABP in testis tissue was separated from the serum protein testosterone-estradiol-binding globulin (mTeBG) by affinity chromatography on Concanavalin A-Sepharose (Con A). mTeBG from serum extracts was completely retained by the lectin and could be displaced with buffer containing alpha-methyl-D-glucoside. In contrast, mABP from testis extracts either did not interact with the Con A and appeared in the void volume or was partially retained by the column and could be eluted with buffer alone. A third component retained by the Con A may represent mTeBG contamination, a form of mABP which binds to Con A, or both. The specific 5 alpha-dihydrotestosterone (DHT)-binding activity in the void volume of the Con A column was designated mABP and was further studied. [3H]DHT binding to mABP was saturable and of limited capacity (0.163 +/- 19 pmol/mg protein). Scatchard analysis of the data was consistent with a single class of binding sites with an apparent dissociation constant (Kd) at 4 C of 2.6 +/- 0.2 X 10(-9) M. DHT was the most effective competitor of [3H]DHT binding to mABP, followed by 2-methoxyestradiol, testosterone, estradiol, and cyproterone acetate. Concentrated Sertoli cell culture medium subjected to steady state polyacrylamide gel electrophoresis produced a single peak of specifically bound [3H]DHT with a mobility similar to that of other androgen-binding proteins. [35S]Methionine-labeled medium proteins were immunoprecipitated with a rabbit anti-human TeBG antiserum. Two bands, corresponding to mol wt of approximately 46,000 and 48,000, were observed by fluorography, with the lighter component being more intense. After androgen affinity chromatography of radiolabeled medium proteins, these two bands were again observed on sodium dodecyl sulfate-urea-containing polyacrylamide gels. These results demonstrate that 1) mABP may be separated from mTeBG by lectin affinity chromatography, as in humans; 2) hTeBG and mABP are antigenically related; and 3) mABP consists of subunits of different mol wt in unequal ratios.

A comparison of moment to moment and diurnal changes in circulating inhibin and testosterone concentrations in male rhesus monkeys (Macaca mulatta).

The secretion of inhibin by the testis was studied in the rhesus monkey, a species which exhibits marked episodic and diurnal patterns of testosterone (T) secretion. Inhibin and T were measured by RIA in blood samples drawn every 20 min for 24 h from 5 adult male monkeys. The molecular size of circulating inhibin, estimated by gel chromatography, was approximately 31 kDA. Plasma inhibin levels were undetectable in long term castrates. T was secreted episodically at a frequency of 6.0 +/- 0.9 pulses/24 h. The computer algorithm also identified 4.6 +/- 0.8 inhibin pulses/24 h. Of 30 T pulses among the 5 animals, however, only 7 coincided with low amplitude inhibin secretory bursts. Each animal demonstrated a significant diurnal periodicity in T secretion, with mean maximum concentrations at 0108 h (range, 2100-0640 h). By contrast, there was no significant diurnal rhythm for inhibin in any of the animals. The pulsatile administration of GnRH (0.1 micrograms/min, iv, for 3 min every 3 h) was used to activate the pituitary testicular axis in 6 juvenile monkeys. After 5 weeks of GnRH priming, a pulse of GnRH produced an immediate 4-fold rise in serum LH concentrations, followed within 30-50 min by a 5-fold increase in circulating T levels. FSH levels rose 50%. During the 3-h GnRH interpulse interval, however, there was no change in serum inhibin levels. Two GnRH-treated juvenile monkeys underwent bilateral orchidectomy. In each animal, circulating inhibin levels declined rapidly, with estimated first phase half-lives of 23 and 32 min, respectively. In conclusion, circulating inhibin concentrations in male rhesus monkeys exhibit neither the prominent moment to moment changes nor the circadian pattern characteristic of T secretion in this species. The relatively constant inhibin levels cannot be explained by prolonged metabolic clearance. The data are consistent with the proposal that most of the inhibin in the circulation is released across the apical surface of Sertoli cells into the seminiferous tubular fluid with passage into the rete testis from which it is continuously absorbed. The intermittent LH signal, by contrast, appears to make a minor contribution to the release of inhibin from the primate testis into the circulation.

Effects of testosterone on gonadotropin subunit messenger ribonucleic acids in the presence or absence of gonadotropin-releasing hormone.

There is accumulating evidence that the negative feedback actions of testosterone on the pituitary may contribute to the differential regulation of FSH and LH secretion in males. In the present study we measured steady state levels of the mRNAs encoding the gonadotropin subunits in pituitary cell cultures treated with 10 nM testosterone (T) as well as in T-treated pituitary cells perifused with pulses of GnRH to explore further the direct actions of T on the pituitary. T treatment of pituitary cells in monolayer culture for 72 h increased FSH beta mRNA 1.5-fold (P less than 0.05), decreased alpha-subunit mRNA to 45% of the control level (P less than 0.05), and decreased LH beta mRNA to 75% of the control level (P less than 0.05). FSH and uncombined alpha-subunit secretion were increased and decreased by T, respectively, whereas basal LH secretion was unchanged. Treatment with 0.1 nM estradiol, a physiological concentration for males, did not change gonadotropin secretion or subunit mRNA concentrations. Between days 2 and 5 in culture in the absence of steroid treatment, steady state levels of LH beta and alpha-subunit mRNA declined (P less than 0.01) 52% and 61%, respectively, but FSH beta mRNA levels were unchanged. Pulsatile stimulation with 2.5 nM GnRH every 1 h for 10 h increased FSH beta mRNA 2.8-fold (P less than 0.05) and increased (P less than 0.05) alpha-subunit mRNA to 117% of the control level. When cell cultures were pretreated with T for 48 h and then perifused with pulses of GnRH, FSH beta, LH beta, and alpha-subunit mRNA levels were 66%, 74%, and 70% of the value during GnRH alone (P less than 0.05). T treatment also reduced (P less than 0.01) the amplitudes of FSH, LH, and alpha-subunit secretory pulses by 18%, 26%, and 41%, respectively. These data indicate that a portion of the negative feedback action of T is at the pituitary to regulate gonadotropin subunit gene expression. Our data reveal two opposing effects of T on FSH beta mRNA: a stimulatory action, which is GnRH independent, and an inhibitory effect, which is related to the actions of GnRH. These divergent actions of T represent one mechanism through which FSH and LH are differentially regulated.

Developmental changes in testicular inhibin and androgen-binding protein during sexual maturation in the cynomolgus monkey, Macaca fascicularis.

Inhibin was measured by RIA in testicular extracts and plasma of cynomolgus monkeys during four stages of sexual maturation. Immunoactive inhibin levels were compared to those of another Sertoli cell secreted protein, androgen-binding protein (ABP). ABP steroid-binding (bioactive) activity was measured in testes and epididymal segments using the radiolabeled ligand [3H]dihydrotestosterone (DHT). Testicular immunoreactive inhibin concentrations were maximal in late prepubertal monkeys, 2.5-3.5 yr old, while the total testicular content of inhibin progressively increased with age into adulthood. Bioactive testicular ABP concentrations were maximal during the pubertal period of the cynomolgus monkey (3.5-4.0 yr old), while the total ABP content of the testes also increased with sexual maturation. Mean (+/- SE) plasma concentrations of inhibin and testosterone (T) in adults, 6-8 yr old (17.72 +/- 3.5 microliters inhibin equivalents/ml and 7.07 +/- 2.45 ng/ml T, respectively), were significantly higher (P less than 0.05 and P less than 0.001, respectively) than those in early prepubertal, juvenile monkeys, aged 1.5-2.5 yr (5.85 +/- 2.1 microliters inhibin equivalents/ml and 0.27 +/- 0.02 ng/ml T). The increased plasma levels of inhibin and T in adults were associated, respectively, with the increased inhibin and androgen contents of the testes in these same animals. The developmental changes in testicular steady state mRNA concentrations for the inhibin alpha-, beta A-, and beta B-subunits as well as ABP were examined during sexual maturation by Northern blot analysis using heterologous human cDNA probes. Densitometric analysis of the autoradiograms revealed that the inhibin alpha-subunit mRNA concentrations were higher than those of inhibin-beta A and -beta B and ABP mRNA during all stages of pubertal development. Although the relative concentrations of each inhibin subunit mRNA were decreased in the adult animals relative to those in the juvenile monkeys, the total amount of steady state mRNA for the subunits was greater than that in the immature animals. A similar situation existed for the ABP mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of castration on luteinizing hormone and follicle-stimulating hormone secretion by pituitary cells from male rats.

Because the role of the pituitary in the testicular control of gonadotropin secretion remains controversial, we examined the effects of castration on the release of LH and FSH under basal conditions and in response to GnRH stimulation by dispersed pituitary cells in monolayer culture as well as by cells perifused with pulses of GnRH. These effects were compared to changes in LH beta, FHS beta, and alpha-subunit mRNA levels determined by Northern blot analysis. Pituitary cells were prepared from 7-week-old intact rats and rats orchidectomized 2 weeks previously. Castration increased basal FSH secretion from monolayer cultures, interpulse FSH release from perifused pituitary cells, FSH beta mRNA concentrations and serum FSH levels each approximately 2-fold, whereas pituitary FSH contents were similar in cells from intact and castrated rats. Pituitary LH content rose 3-fold, LH beta mRNA rose 5.6-fold, and basal LH secretion increased 6-fold, but serum LH levels increased 22-fold. Thus, the change in FSH synthesis inferred from the increase in FSH beta mRNA was proportional to the increase in FSH secretion both in vitro and in vivo. Whereas the basal release of LH in vitro was also proportional to the change in LH beta mRNA, secretion of LH in vivo exceeded these changes, underscoring the importance of increased GnRH to the serum LH castration response. Castration resulted in an increase in the sum of FSH content and secretion during 10 days in culture in the absence of GnRH, indicating ongoing FSH synthesis. Total LH declined in cells from intact rats, and this decline was prevented by castration; this effect may be due to a castration-related decrease in intracellular LH degradation or increased LH synthesis in the absence of GnRH. Castration also augmented the GnRH-stimulated release of LH and FSH from monolayer cultures 4.5- and 1.8-fold, respectively, and increased the amplitude of GnRH-stimulated LH and FSH pulses 5- and 2-fold in experiments with perifused pituitary cells. The EC50 for GnRH was unaffected by castration.(ABSTRACT TRUNCATED AT 400 WORDS)

A study of the relative roles of follicle-stimulating hormone and luteinizing hormone in the regulation of testicular inhibin secretion in the rhesus monkey (Macaca mulatta).

The purpose of this study was to examine the relative roles of FSH and LH in stimulating testicular inhibin secretion in the male rhesus monkey. Recombinant human (rh) FSH and rhCG were used as the gonadotropic stimuli, and juvenile rhesus monkeys, in which the endocrine activity of the pituitary-testicular axis was being driven in an adult manner with an intermittent i.v. GnRH infusion, were studied. Immunoactive inhibin levels were measured by the Monash RIA. Initiation of an intermittent i.v. infusion of rhFSH (10 IU every 3 h) resulted, after a delay of 5-6 h, in a progressive increase in the concentrations of immunoactive inhibin, which achieved, after 48 h of stimulation, a value twice that observed during vehicle treatment. Gel filtration chromatography revealed that the FSH-induced elevation in immunoactive inhibin was the result of an increase in three distinct mol wt fractions: peak I (100 kDa), peak II (50-60 kDa), and peak III (31 kDa). Although peak III accounted for most of the inhibin immunoactivity in vehicle-treated animals, peaks I and II were most responsive to FSH stimulation. Application of recently developed enzyme-linked immunosorbent assays for inhibin B and pro-alpha-C-related peptides provided additional insights into the nature of the FSH-sensitive forms of circulating immunoactive inhibin. Most notably, the 31-kDa fraction (peak III) was comprised of inhibin B and pro-alpha-C. In contrast to FSH stimulation, an intermittent infusion of rhCG (40 IU every 3 h), which markedly elevated testicular testosterone secretion, failed to increase immunoactive inhibin concentrations. These findings indicate that various forms of immunoactive inhibin are present in the circulation of the rhesus monkey, and that in this species, FSH is the principal stimulus of the secretion of testicular inhibins, including inhibin B. Additionally, they further underline the importance of the FSH-inhibin feedback loop in governing testicular function in primates.

Pituitary follistatin and activin gene expression, and the testicular regulation of FSH in the adult Rhesus monkey (Macaca mulatta).

In rats, FSHbeta gene expression and FSH secretion are increased and decreased, respectively, by pituitary activin and follistatin. Because little information is available on the paracrine control of FSH secretion in the primate, follistatin and activin/inhibin beta(B) messenger RNA (mRNA) levels were measured in pituitaries of adult male rhesus monkeys 6 weeks after castration or sham surgery (n = 5/group). Follistatin mRNA was determined by quantitative RT-PCR assay using oligonucleotide primers designed to span exons 3-5 of the human follistatin gene. Activin/inhibin beta(B) mRNA levels were measured by ribonuclease protection. Orchidectomy resulted in a 100-fold increase in plasma FSH concentrations and a 60-fold rise in those of LH. In castrated monkeys, levels of mRNA encoding FSHbeta, LHbeta, alpha- subunit, and GnRH receptor (GnRH-R) were increased 21-, 2.1-, 1.7-, and 1.7-fold, respectively (P < 0.01). Levels of pituitary follistatin and activin/inhibin beta(B) mRNAs, however, were similar in castrated and intact animals. These data suggest that the paracrine control of FSH secretion in the male differs substantially in primates and rodents. Specifically, the relatively greater postcastration rise in FSHbeta gene expression and FSH secretion in the adult male monkey may result because in this species pituitary follistatin gene expression does not increase after orchidectomy, as it does in the rat.

Interrelationship between the actions of testosterone and primate Sertoli cell inhibin in the control of gonadotropin secretion by cultured pituitary cells.

There is accumulating evidence that the differential regulation of LH and FSH secretion in the male is partly accomplished by the direct actions of testosterone (T) and inhibin on the pituitary. The present study was designed to examine the interaction between T and inhibin, in the presence and absence of GnRH, using dispersed pituitary cells in monolayer culture and cells perifused with pulses of GnRH from intact, 2-week castrated, and castrated T-replaced young adult male rats. The effect of partially purified inhibin from primate Sertoli cell culture medium (pSCI) to suppress basal FSH secretion was similar with pituitary cells from intact and castrated rats. T increased basal FSH secretion in the presence or absence of pSCI but did not alter the dose-dependent suppression of FSH by pSCI with cells from either intact or castrate rats. Castration increased basal FSH and LH secretion, whereas only basal FSH release was increased with cells from T-replaced castrates. T pretreatment increased the action of pSCI to suppress GnRH-stimulated FSH and LH release from perifused pituitary cells. These data indicate that T and inhibin exert opposite but independent effects on basal FSH release. The action of inhibin to suppress basal FSH secretion is not impaired by the absence of T and inhibin subsequent to castration. By contrast, the actions of T and inhibin to suppress GnRH-stimulated gonadotropin secretion are coordinated and interrelated.

Effects of inhibin from primate Sertoli cells on follicle-stimulating hormone and luteinizing hormone release by perifused rat pituitary cells.

We used a pituitary cell perifusion system to investigate the time course and selectivity of the inhibin effect on pulsatile GnRH-stimulated LH and FSH release. Dispersed pituitary cells from 7- to 8-week-old male rats were perifused on a Cytodex bead matrix and stimulated with 10 nM GnRH for 2 min every hour for 8-11 h. The addition of a preparation of inhibin partially purified from primate Sertoli cells reduced pulsatile FSH release within 2 h. After removal of inhibin from the perifusion medium, the effect was reversed within 3 h. GnRH-stimulated LH release was also influenced by inhibin, although the decline in LH was less than that in FSH (80 +/- 3% vs. 68 +/- 4% of control; P less than 0.025). Smaller doses of inhibin suppressed GnRH-induced FSH secretion, but had no effect on LH release. Further, prolonged incubation of pituitary cells with inhibin at the higher dose reduced its FSH inhibitory effect and eliminated the effect on LH. These results indicate that inhibin can reduce both LH and FSH secretion in vitro, although the specificity and magnitude of the effect are a function of both the dose and duration of inhibin treatment. Further, the actions of inhibin and GnRH on the pituitary may be interrelated.