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)     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

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)     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

Stimulatory effect of Sertoli cell culture medium on the FSH release by pituitary halves in vitro

The influence of the medium collected from cultured rat Sertoli cells on the spontaneous and LHRH-stimulated release of gonadotropins by incubated rat pituitary halves was examined. The homogeneity of the cultured population of Sertoli cells taken from 20-day-old rats ranged up to 98%. The cells in culture responded to FSH stimulation with characteristic morphological changes and with increased secretion of estradiol-17 beta. The hemi-pituitaries obtained from sexually mature male rats were incubated for 5 hours in the presence of Sertoli cell culture medium (SCCM) or its fractions obtained by use of ultrafiltration. The SCCM fraction deprived of MW less than 10 kD compounds exhibited a typical inhibin-like activity, whereas crude SCCM as well as its low-molecular-weight fraction stimulated the basal FSH release to about 150% and 175% of the control values, respectively. These fractions exerted an inhibitory effect on the LHRH-stimulated secretion of both LH and FSH. It is concluded that Sertoli cells cultured in chemically defined medium release, apart from inhibin, a non-steroidal, heat-labile substance of MW less than 10 kD which stimulates the basal secretion of FSH and LH and inhibits the LHRH-stimulated secretion of both gonadotropins from incubated rat hemi-pituitaries.     

Expression and regulation of testicular inhibin alpha-subunit gene in vivo and in vitro

Inhibin, a gonadal peptide, selectively suppresses FSH release from the pituitary. The cDNAs coding for ovarian inhibin have been isolated and characterized. However, little is known about testicular inhibin. In this study we have isolated inhibin alpha-subunit cDNA from human testicular cDNA libraries and determined inhibin alpha-subunit mRNA levels in testes. The longest cDNA isolated from human testis was 1380 nucleotides long and contained a nucleotide sequence identical to that of human placental inhibin alpha-subunit and isolated human inhibin alpha-subunit gene, but different from human ovarian inhibin alpha-subunit in two amino acids in the signal peptide. A single 1.5-kilobase species of inhibin alpha-subunit mRNA was identified in the testes of several species. This mRNA was the same size as those in human ovary and placenta. The regulation of inhibin alpha-subunit mRNA in rat testis was next examined. The concentration of testicular inhibin alpha-subunit mRNA peaked between 20-25 days of age and gradually declined thereafter. Hypophysectomy decreased testicular inhibin alpha-subunit mRNA levels. Supplementation of hypophysectomized animals with FSH restored inhibin alpha-subunit mRNA levels to those in intact controls. By contrast, treatment with testosterone had no effect. Similarly, in Sertoli cell-enriched cultures, FSH, but not testosterone, increased inhibin alpha-subunit mRNA levels. We conclude that 1) human testicular inhibin alpha-subunit mRNA is similar to that of human ovary and placenta; and 2) inhibin alpha-subunit mRNA in Sertoli cells is regulated by FSH, but not testosterone, both in vivo and in vitro.     

Adrenomedullin in the rat testis. II: Its production, actions on inhibin secretion, regulation by follicle-stimulating hormone, and its interaction with endothelin 1 in the Sertoli cell

The present study demonstrates the expression of adrenomedullin (ADM) in the rat Sertoli cells and its effect on inhibin production. The regulation of ADM by FSH and its interaction with endothelin 1 (EDN1) in the rat Sertoli cells have also been established. Primary culture of Sertoli cells secreted 414+/-27 pg immunoreactive ADM per 10(6) cells in 24 h and expressed Adm mRNA. In addition, the Sertoli cell was shown to co-express mRNAs encoding for the calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs) 1-3. These may account for the specific binding of ADM to the Sertoli cells. Administration of ADM to Sertoli cells resulted in an enhancement of basal and FSH-stimulated inhibin B production. On the other hand, the production of ADM and the mRNA levels of Calcrl and Ramp2 in the Sertoli cells were suppressed by FSH. The results suggest that ADM, via its control in the secretion of inhibin B, may play a role in regulating spermatogenesis as well as the hypothalamus-pituitary-gonad feedback system. In addition, like in the Leydig cell, ADM and EDN1 were found to regulate the production of each other in opposite directions in the Sertoli cells, suggesting the presence of yet another local regulatory mechanism in the rat testis that may be important in modulating testicular functions regulated by gonadotropins.     

Isolation of porcine follicular fluid inhibin of 32K daltons

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.     

Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background  

Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.     

Kinetics of inhibin secretion in static and superfused Sertoli cell cultures in response to follicle-stimulating hormone

It is generally accepted that inhibin secretion in the testis is regulated by FSH; however, the kinetics of inhibin secretion have not been well defined in vivo and in vitro. We investigated the kinetics of inhibin secretion in response to FSH stimulation in static and superfused Sertoli cell cultures. Sertoli cells from 18-day-old rats were cultured in chemically defined medium for 3 days and were then stimulated for different time periods with FSH (0.1 microgram/ml). In static cultures, media were changed every 2, 4, or 8 h, and the superfusion was carried out at a steady rate of 3 ml/h. Inhibin in the culture media was measured by RIA, using antiserum against synthetic replicate [30Tyr]inhibin alpha-chain-(1-30) and, in some experiments, also by bioassay. The dynamics of inhibin secretion were similar in static and superfused Sertoli cell cultures. A significant increase (p less than 0.01) of inhibin secretion was noted after 5-6 h of FSH exposure. After 8-12 h of continuous FSH presence, the secretion of inhibin reached a maximal level, 5-10-fold higher than basal secretion (no FSH). In the continuous presence of FSH, inhibin secretion remained stable at the high level for up to 54 h. FSH removal caused a delayed (8-h) decrease (p less than 0.01) of inhibin secretion, with return to control basal values after approximately 30 h. When FSH was removed 4 h after its addition, inhibin secretion again increased 5-10-fold between 4 and 12 h, then returned to basal values within 30 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paracrine role of Sertoli cells

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

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Enhancement of in vitro release of FSH from rat pituitaries by a synthetic nonapeptide fragment of human seminal plasma inhibin

A synthetic C-terminal nonapeptide fragment of human seminal plasma inhibin preferentially enhances the basal release of FSH from rat pituitaries incubated in vitro, which indicates a direct action of the peptide on the pituitary. However, in the presence of LHRH, both FSH and LH release was increased particularly at higher doses of the nonapeptide. There was no change in prolactin release at 5 and 50 ng/ml but prolactin release was suppressed significantly at 500 ng/ml.     

Identification in human seminal fluid of an inhibin-like factor which selectively regulates FSH secretion.

Human seminal plasma obtained by centrifugation of human semen contains a factor capable of selectively inhibiting the secretion of FSH both in vivo (reduction of the levels of FSH in rats 24 h after castration) and in vitro (reduction of the FSH released by LH-RH in rat pituitary cell culture). This effect is not due to testosterone, oestradiol-17 beta or progesterone present in the active fractions. The factor has the characteristics of a protein in that its biological activity is destroyed by heat and trypsin digestion. It does not resemble androgen-binding protein. The biological action is not completely specific for FSH as inhibition of LH can be seen with doses usually higher than those which produce inhibition of FSH alone. There is no effect on TSH or prolactin levels in vitro. The factor clearly acts on the release and synthesis of gonadotrophins by gonadotrophs but an effect on the hypothalamus is not excluded. This factor fits the definition of inhibin.     

Type beta transforming growth factor (TGF-beta) is a potent stimulator of the basal secretion of follicle stimulating hormone (FSH) in a pituitary monolayer system

In view of striking similarities between TGF-beta and inhibin, we investigated the possibility that TGF-beta might modulate pituitary hormone release in vitro. Long term incubations of beta transforming growth factor (TGF-beta) with rat anterior pituitary cells for 48 hr stimulates the basal secretion of FSH in a dose-dependent manner. The secretion of LH, TSH, GH, ACTH and PRL is not modified by TGF-beta. The minimal effective concentration of TGF-beta is 10 pg/ml (less than 500 attomolar) and is dose dependent over a range from 1 pg to 10 ng/ml. Treatment of cells with TGF-beta for short incubation times (4 hr) in assays similar to that used for hypophysial releasing factors is not effective, indicating that TGF-beta acts through a cellular mechanism distinct from that of LRF. Inhibin-A, recently characterized on the basis of its capacity to specifically inhibit the secretion of FSH in the 48 hr bioassay system inhibits the stimulatory effect of TGF-beta on FSH-release. Analyses of the dose response curves indicate that the interaction occurs in a typical non-competitive manner. The results suggest that a TGF-beta-like molecule, present in follicular fluid, may be responsible for the FSH-releasing activity ("anti-inhibin" activity) observed by us and others during the process of isolating inhibin from follicular fluids. They also suggest an important role for inhibin and the TGF-beta related molecules in modulating pituitary gonadotropin release.     

Inhibins and activins: chemical properties and biological activity

The long-sought, nonsteroidal, gonadal inhibitor of the secretion of FSH has been isolated, characterized, and the primary structure in several species (human, porcine, bovine, murine) has been deduced. Inhibins are proteins consisting of two subunits (18-kDa alpha- and 14-kDa beta-subunits) linked by disulfide bridges and two forms of inhibins were observed in human, porcine, and murine, but only one in bovine. Each form of inhibin (A and B) has a common alpha-subunit, but a highly homologous, distinct beta-subunit (beta A and beta B). The beta-subunits and the alpha-subunit are linked to form inhibins A and B which exert an inhibitory effect on basal FSH secretion, but the dimer formed by either two beta A-subunits or two distinct beta A- and two beta B-subunits (homoactivin-A and activin, respectively) possess FSH-stimulating activity. Inhibin secreted in response to FSH from the pituitary originates primarily from the granulosa cells of the ovary and the Sertoli cells of the testes, thus demonstrating a reciprocal feedback relationship.