Inhibin Activity Research Articles

Inhibin production by sertoli cell cultures

The production of inhibin by cultures of Sertoli cells from 21-day-old rats was assessed by the use of an in vitro bioassay using rat pituitary cells in culture. Sertoli cell culture media (SCCM) caused a dose-dependent suppression of the pituitary cell FSH content which was parallel with that of an ovine testis lymph preparation used as an inhibin standard. SCCM also caused a dose-dependent inhibition of FSH secreted by pituitary cells in response to 10 nM GnRH stimulation. The FSH-inhibitory activity in SCCM was destroyed by heat or trypsin digestion and could not be attributable to the steroid content of the medium, since ether extraction caused no change in the inhibitory activity. The inhibin activity in SCCM was not due to cytotoxicity in the bioassay, since the LH cell content was unchanged and the media produced no change in the release of 51Cr from labelled pituitary cells, a parameter which has been shown to be a useful test of cytotoxicity. Sertoli cell cultures produced inhibin for the 8-day duration of the cultures. The amount of inhibin produced was proportional to the number of Sertoli cells initially plated. If foetal calf serum was included for more than the initial 48 h, the spent medium caused toxic effects in the pituitary cells as evidenced by an increase in 51Cr release from 51Cr-labelled pituitary cells. Similar toxic effects were found if the lyophilized spent media contained cellular debris. A dose-dependent increase in inhibin activity was observed in the presence of graded doses of FSH (0.05–5 μg/ml NIH-FSH-S13).

Antisera to porcine follicular fluid in monkeys: neutralization of human and pig inhibin activity in vivo and in vitro.

Repetitious administration of charcoal-stripped porcine follicular fluid (pFF) to four intact cycling females and six long term castrate rhesus monkeys was associated with a gradual onset of resistence to the FSH-suppressing effects of pFF. After a treatment hiatus of 6 or more months, the pFFinduced selective suppression of FSH in circulation was again demonstrable, but only transiently; that is, within 10–14 weeks after the resumption of therapy, even large doses of pFF were no longer effective in reducing serum FSH in these monkeys. One monkey was studied through seven regimens over 4 yr. Accordingly, our series of investigations was divided into three parts. First, we observed that these changing FSH patterns were apparently due to cyclic fluctuations of pFF antibodies in conjunction with the intermittent schedule of pFF administration. The amnestic response to booster injections occurred within 7–14 days, followed by a gradual decline in the anti-pFF titer during succeeding months. Second, we tested whether these anti-pFF sera would neutralize inhibin activities (selective FSH suppression) in rat pituitary cell cultures. Indeed, the anti-pFF sera were effective in blocking the FSH-suppressing activity of pFF. Again, each successive episode of pFF administration was correlated with an enhancement of the anti-pFF titer, indicated by greater potency of the monkey antisera in the in vitro setting. Thirdly, we assessed whether the monkey's anti-pFF sera would neutralize the inhibin activity in human FF (hFF); thereby establishing cross-reactivity with human inhibin. Clearly, the mixture of anti-pFF serum along with charcoal-stripped hFF prevented the selective suppression of FSH secretion that occurred when control monkey serum was used instead. These findings indicate that porcine inhibin present in charcoalstripped pFF is highly immunogenic in monkeys; further, some of the antibodies generated cross-react with the active inhibin component in hFF. Indeed, the neutralization of inhibin activity in hFF by anti-pFF monkey sera suggests some significant structural similarities between human and pig inhibins; yet, concurrently, the ready immunogenicity of pFF administered to monkeys indicates clear recognition of the antigen as an intrinsically foreign protein. These anti-pFF monkey sera may be useful in the establishment of RIA for porcine and/or human inhibins as well as the preparation of improved reagents for studying the physiological significance of this substance (s) in follicular fluid and circulation.

The specificity of inhibin bioassay using cultured pituitary cells

It has been established that the inhibition of the cellular content of FSH in cultured pituitary cells can be used as a sensitive and precise bioassay for inhibin. During studies on the inhibin content of human seminal plasma, FSH suppression was noted to occur together with LH suppression. Further studies revealed that where combined FSH and LH suppression occurred, cytological changes in the pituitary cells suggestive of toxicity were found, indicating non-specific effects of these substances. Charcoal treatment or gel filtration of seminal plasma removed or separated the toxic substances from the inhibin activity, the latter characterized by FSH suppression parallel to the standard preparation, no LH suppression and no light-microscopic changes in pituitary cells. It is recommended that careful evaluation of all inhibin bioassay systems should be undertaken to detect substances producing non-specific effects and additional guidelines for the assessment of specificity are suggested.

Low‐molecular‐weight Inhibin from Sheep, Human, Rat, and Chicken Testes

Low‐molecular‐weight peptides with inhibin activity have been isolated from sheep, human, rat, and chicken testes by simple gel filtration. These peptides, isolated from avian and mammalian species, suppressed the postcastration rise of serum FSH levels in adult male rats by 34 to 45% and appeared to be biologically and chromatographically similar.

Effect of testosterone, estradiol-17 beta, and 5 alpha-dihydrotestosterone on inhibin production by sertoli cells in culture.

Inhibin activity in charcoal-extracted spent culture medium of Sertoli cells in Primary culture (CSCCM) was monitored by following the temporal inhibition of serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in normal adult male rats. Subcutaneous injection of CSCCM reduced LH and FSH levels within 15 min and maintained the inhibition up to 10 hr. No selective suppression of FSH was observed. The model was used to assess inhibin activity in CSCCM from Sertoli cell cultures incubated with 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol -17 beta (E2) (5 micrograms/ml) for 24 hr. CSCCM from T and E2 treated groups suppressed gonadotropins while the group exposed to DHT failed to elicit any inhibition. DHT seems to inhibit the release of inhibin by Sertoli Cells.

Effect of inhibin like factors on gonadotrophin release by the mouse pituitary in vitro.

The release of gonadotrophins by the mouse whole pituitary in vitro was investigated. Whole tissue from 34 day old male mice was highly responsive to synthetic LRH in short incubations at 37 degrees C. About 15% of FSH and 17% of LH were secreted during a 3 h pulse with 3 ng of LRH. Pre-exposure of the mouse pituitary to fractions containing inhibin activity, inhibited the FSH release induced by LRH. Inhibin from bovine and human seminal plasma and porcine follicular fluid preferentially inhibited FSH secretion, while LH secretion was also reduced by inhibin like factors from bovine follicular fluid and testicular extract. The inhibition of FSH and/or LH release was measured by specific radioreceptor assays and could not be due to destruction of either gonadotrophin or LRH. Purified bovine seminal plasma inhibin was effective at very low concentrations in vitro. The whole incubation system is simple and an estimation of inhibition of bioactive (binding activity) FSH release can be obtained within 24 h.

Regulation of inhibin production by bovine ovarian cells in vitro.

The regulation of ovarian inhibin production was investigated using a rat pituitary cell culture system as a bioassay for inhibin activity. Bovine follicular granulosa cells produced inhibin in vitro provided that the culture medium contained serum. The stimulatory factor(s) present in serum is unlikely to be gonadotrophins, because bovie LH and/or FSH failed to stimulate inhibin production when added to medium devoid of serum. Luteinization of granulosa cells in culture was accompanied by a reduction in their inhibin production and an inverse relationship existed between inhibin and progesterone production by granulosa cells. Bovine corpus luteum cells in culture failed to produce detectable amounts of inhibin. Androgens stimulated granulosa cell inhibin production with testosterone and 5 alpha-dihydrotestosterone being more potent than androstenedione. The androgens did not stimulate inhibin production by luteal cells. Progesterone inhibited granulosa cell inhibin production but oestrogens had no effect. Measurement of steroids and inhibin in fluid from individual follicles indicated that as follicle size increased, concentrations of oestradiol-17 beta increased, testosterone and inhibin decreased and progesterone remained unchanged. The stimulatory effect of testosterone on inhibin production in vitro together with the parallel changes in follicular fluid concentrations of testosterone and inhibin suggest that ovarian inhibin production in vivo may be controlled, at least in part, through androgens modifying granulosa cell inhibin production. The inhibitory effect of progesterone on granulosa cell inhibin production may be more important in regulating ovarian inhibin production at the time of granulosa cell luteinization and CL formation. The stimulatory effect of androgens on granulosa cell inhibin production might also be a means by which androgens promote follicular atresia.

Disappearance of Inhibin‐like Activity in Bull Seminal Plasma Following Castration

Ejaculates from normal and castrated bulls were evaluated for the presence of inhibin‐like activity. Inhibin activity found in the seminal plasma of normal bulls became undetectable after castration. Crude protein concentrates prepared from the ejaculates of castrated bulls failed to produce inhibition of FSH secretion as measured by in vivo and in vitro tests: a) hCG‐induced uterine weight test in mice and b) LH‐RH‐induced FSH secretion in 34‐day‐old male mouse pituitary. The concentrates also lacked the ability to inhibit the binding of iodine‐125 (125I)‐labeled inhibin fraction from normal bull seminal plasma to ovine pituitary membranes. Chromatographic and electrophoretic analyses of the ejaculate from castrated bulls revealed the absence of the band corresponding to inhibin of normal bull seminal plasma.

Inhibin-like activity in extracts of rabbit placentae

Following the demonstration of inhibin activity in the ovary, a search was conducted for such activity in the rabbit placenta. A bioassay was utilized that consisted of 16 hours' incubation of the test substance with cultured rat anterior pituitary cells and determination of gonadotrophins in the recovered media. A crude extract of rabbit placenta had no effect on luteinizing hormone (LH) but a slight and insignificant inhibiting effect on follicle-stimulating hormone (FSH) secretion. Ethanol-extracted low steroid preparation of the rabbit placenta significantly inhibited FSH, but had no effect on LH secretion. On Sephadex G100 chromatography a fraction was identified which had inhibin activity. In a parallel chromatography of human seminal fluid inhibin activity was identified in the same elution volume as that of the rabbit placenta extract. It is suggested that rabbit placenta extract contains a non-steroidal substance that interferes with pituitary secretion of FSH, and that this substance may be inhibin.

Relationship between human follicular fluid inhibin F activity and steroid content.

To examine what relationships exist between human follicular fluid inhibin activity and steroid content, follicular fluid was aspirated from 72 follicles at various stages of follicular development. Twenty samples were collected from cystic ovarian follicles, and 50 samples came from normal ovarian follicles. Estrogen, Δ4-androstenedione, testosterone, and progesterone levels in follicular fluid were measured by RIA. Apparent inhibin activity was estimated in follicular fluid samples after charcoal treatment to remove steroids. Each charcoal-treated sample was assayed in triplicate using rat anterior pituitary cultures. Follicular fluid was added at a dose of 2% in the pituitary culture medium, and the ability of the fluid to inhibit basal FSH secretion in the cultures was estimated after a 24-h incubation. The degree of FSH-suppressing activity in the fluid was estimated and expressed in terms of nanoliters of standard porcine follicular fluid inhibin activity per 10 μ follicular fluid. Charcoal-treated porcine follicular fluid (standard) contained 10,000 U/10 μl and gave a linear log dose-response curve, based on the inhibition of FSH, from 25–350 nl follicular fluid in the pituitary assay. Inhibin activity in human follicular fluid ranged from a lower limit of 18–38 to 1000 U/10 μl. Follicular fluid obtained from noncystic follicles during the follicular phase of the menstrual cycle had significantly more inhibin (84 ± 18 U/10 μl; n = 42) compared to cystic follicles (35 ± 13 U/10 μl P < 0.05; n = 18). Preovulatory follicular fluid contained 87 ± 19 U/10 μl; (n = 8), whereas luteal phase follicular fluid contained 66 ± 29 U inhibin/10 μl (n = 8). If the viability of each follicle was assessed by using the follicular fluid Δ4-androstenedione to estrogen ratio (the more viable follicles had a lower ratio), the following observations could be made. There was a significant negative correlation between the log of the inhibin activity and the log of the androstenedione to estrogen ratio of follicular fluid (r = –0.399; P < 0.05). Follicular phase follicles having an Δ4-androstenedione to estrogen ratio greater than 10 had significantly less inhibin (P < 0.01) compared to follicles having a ratio less than 10. During the luteal phase, follicles contained somewhat lower amounts of inhibin activity, probably because they are atretic, since their follicular fluid showed invariably low levels of estrogen and an Δ4-androstenedione to estrogen ratio greater than 10 (36.2 ± 9.1; n = 11). This Δ4-androstenedione to estrogen ratio was significantly greater (P < 0.001) than the ratio observed in fluid obtained from viable follicular phase follicles (2.08 ± 0.38; n = 28).

Ovarian and peripheral blood inhibin concentrations increase with gonadotropin treatment in immature rats.

The effects of PMSG treatment on ovarian and circulating inhibin concentrations in immature female rats has been examined. Sixty-four hours after injection of 10, 20 or 40 IU of PMSG the animals were anesthetized with ether; ovaries, uteri and blood from the abdominal aorta were collected. Steroid-free extracts of ovary and serum samples were prepared and assayed quantitatively for inhibin activity in an in vitro bioassay system. PMSG treatment elevated (p less than 0.001) both uterine and ovarian wt, and ovarian and peripheral concentrations of inhibin. A dose-related increase occurred ovarian wt, and in peripheral and ovarian content of inhibin. Ovarian inhibin concentration increased with dose of PMSG until the highest dose, where a significant decline and luteinization were seen. Peripheral FSH levels were significantly lowered at all doses of PMSG treatment; in contrasts, LH was significantly elevated, due to cross-reaction of PMSG in the LH assay. These results show that both ovarian and circulating levels of inhibin are related to the degree of gonadotropic stimulation, supporting the view that inhibin is involved in folliculogenesis and in the feedback regulation of FSH.

Binding of an inhibin-like protein from bull seminal plasma to ovine pituitary membranes

A purified basic protein from bull seminal plasma having inhibin activity was labeled with 125I and tested for binding with ovine pituitary membrane fractions. The bound radioactivity could be eluted under acidic conditions and shown to rebind to fresh pituitary membranes. The properties of the eluted labeled inhibin were similar to the unlabeled fraction. The eluted labeled inhibin exhibited specific binding to the membranes, which was displaceable in a dose-dependent manner by an unlabeled active fraction. Only those fractions in the purification scheme which had inhibin activity also competed for the binding. A bovine follicular fluid fraction with molecular weight > 10 000 and inhibin activity also displaced the bound radioactivity from the membranes. Other purified hormones such as ovine FSH, LH or their subunits, prolactin or bovine serum albumin, dialyzed serum or unrelated basic macromolecules such as lysozyme, polylysine, histones, had no influence on the binding of labeled inhibin to ovine pituitary membranes. Synthetic LH-RH also failed to displace the labeled inhibin from the membranes. The binding was sensitive to heat and trypsin treatments. The data are consistent with the direct action of inhibin on the pituitary and demonstrate the existence of binding sites for the active fraction in this target.

Isolation and characterization of a bovine seminal plasma protein inhibiting pituitary FSH secretion

A basic protein with inhibin-like activity was purified from bull seminal plasma by ethanol precipitation, ion-exchange chromatography, gel filtration and preparative disc electrophoresis. The protein migrated rapidly into the acrylamide gel at pH 4.5 but failed to penetrate the gel at pH 8.9. Electrophoresis at pH 4.5 revealed heterogeneity. Its molecular weight by SDS gel electrophoresis was estimated to be approx. 18 000 daltons. It exhibited inhibin activity in both in vivo and in vitro model systems. The partially purified protein fraction was active in suppressing hCG-induced mouse uterine weight in immature mice. It specifically inhibited the castration-induced rise in serum FSH in 34-day-old male rats, blocked the action of synthetic LH-RH in vivo and in vitro in rats and mice respectively.

Morphological studies of outgrowth from glomeruli in tissue culture

The production of inhibin by cultures of Sertoli cells from 21-day-old rats was assessed by the use of an in vitro bioassay using rat pituitary cells in culture. Sertoli cell culture media (SCCM) caused a dose-dependent suppression of the pituitary cell FSH content which was parallel with that of an ovine testis lymph preparation used as an inhibin standard. SCCM also caused a dose-dependent inhibition of FSH secreted by pituitary cells in response to 10 nM GnRH stimulation. The FSH-inhibitory activity in SCCM was destroyed by heat or trypsin digestion and could not be attributable to the steroid content of the medium, since ether extraction caused no change in the inhibitory activity.The inhibin activity in SCCM was not due to cytotoxicity in the bioassay, since the LH cell content was unchanged and the media produced no change in the release of 51Cr from labelled pituitary cells, a parameter which has been shown to be a useful test of cytotoxicity. Sertoli cell cultures produced inhibin for the 8-day duration of the cultures. The amount of inhibin produced was proportional to the number of Sertoli cells initially plated. If foetal calf serum was included for more than the initial 48 h, the spent medium caused toxic effects in the pituitary cells as evidenced by an increase in 51Cr release from 51Cr-labelled pituitary cells. Similar toxic effects were found if the lyophilized spent media contained cellular debris. A dose-dependent increase in inhibin activity was observed in the presence of graded doses of FSH (0.05–5 μg/ml NIH-FSH-S13).

Isolation and characterization of ovine testicular and ovarian inhibin.

Low-molecular-weight (< 1500 daltons) peptides with inhibin activity have been isolated from sheep testes and ovaries by simple gel filtration. These peptides were capable of inhibiting the ovarian weight increase in hCG-primed immature female mice and also of suppressing the post-castration rise of serum FSH levels in adult male rats, suggesting similarities in their biological properties. Both testicular and ovarian inhibin were typsin sensitive and heat stable at 100 degrees C for 30 min and were shown to act by interfering with the production of a hypothetical FSH-RH.

Mechanism of Action of Hypothalamic Hormones in the Adenohypophysis

The observation of a close parallelism between changes of cyclic adenosine 35 monophosphate (cAMP) accumulation and specific hormone release under the influence of 2 stimulatory hypothalamic hormones thyrotropin-releasing hormone (TRH) and luteinizing hormone-releasing hormone (LHRH) and 1 inhibitory peptide (somatostatin) strongly suggest that the adenylate cyclase system is involved in the mechanism of action of these 3 peptides in the anterior pituitary gland. The mechanism (depicted in a figure) is supported by data obtained on the characteristics of the TRH receptor and properties of adenohypophyseal cAMP-dependent protein kinase and its substrates. Estrogen treatment in vivo led to an increase in the number of adenohpophyseal TRH receptors whereas opposite effects were seen after thyroid hormone administration. In vitro pituitary cell primary cultures showed that estrogens stimualted both LH and follicle stimulating (FSH) hormone secretions while androgens had opposite effects on the secretion of the 2 hormones markedly inhibiting LH and stimulating FSH. The effect of progesterone in estradiol-primed cells was biphasic LH secretion whereas the effect on FSH secretion was exclusively stimulatory. Inhibin activity of ovarian and testicular origin was a potent inhibitor of basal FSH release. Recent data indicate that dopamine may be the main or even the only inhibitory substance of hypothalamic origin controlling prolactin secretion. Estrogens were found to stimulate the prolactin secretion directly at the pituitary level and to reverse the inhibitory effect of dopamine agonists on prolactin secretion.

A Rapid Bioassay for Measuring Inhibin Activity

A sensitive and rapid bioassay of inhibin is described. Swiss albino female mice, 27 days old, weighing 25–30 g, were primed with 20 IU hCG, given in 2 equal doses, administered at 0900 h and 1800 h. An increment of 200% in uterine weight could be observed when the animals were autopsied the next day at 1000 h. The validity of the endpoint chosen, as a function dependent on FSH, was shown by the fact that neutralization of endogenous FSH by FSH antiserum led to an inhibition of uterine weight increase. Further injection of the inhibin material caused an actual reduction in the heightened levels of FSH (ng/ml) brought about by hCG: Control, 207 ± 13.6; hCG, 365 ± 28.8 and hCG + inhibin, below the detection level of radioimmunoassay. Inhibin preparation from ovine testicular extract when tested at 3 dose levels showed a dose dependent and significant suppression in uterine weight increase. The assay was statistically validε = 2.5; slope = 23.9; λ = 0.104 and intra- and interassay variation = 8.3% and 11.7%, respectively. Since the mouse uterine weight assay is specific, has greater sensitivity and is uniformly reproducible, it is suggested that this assay procedure is ideally suited to assess the activity of different inhibin test preparations as well as to follow the purification of inhibin activity from crude material.

Studies on Purification of Inhibin from Ovine Testicular Secretions Using an In Vitro Bioassay

An in vitro bioassay based upon inhibition of follicle stimulating hormone (FSH) secretion by rat pituitary cells in culture was used to investigate preliminary steps in the purification of inhibin from ovine rete testis fluid (RTF) and testicular lymph (OTL). Since androgens also suppress gonadotrophin levels in this assay, steroids were removed from test substances. Charcoal adsorption and ethanol precipitation were found to be unsatisfactory because of large losses of inhibin activity. Using RTF protein as the starting material a two‐fold purification was achieved by precipitation with 50% ammonium sulphate. Chromatography of the ammonium sulphate precipitate on DEAE Sephadex resulted in further purification and separation of inhibin from testosterone.

Evidence for inhibin-like activity in bovine follicular fluid.

THE negative feedback action of testicular steroids on circulating levels of luteinising hormone (LH) is well known. It is less clear how the testis influences follicle-stimulating hormone (FSH). The existence of ‘inhibin’, a water-soluble testicular product that inhibits the secretion of FSH, was first postulated in 1932 (ref. 1); recently, inhibin activity has been detected in rete testis fluid2, testicular tissue3, spermatozoa4 and seminal plasma5. Immunisation of rabbits with the inhibin fraction of bull seminal plasma has produced an antiserum which raised plasma FSH levels when injected into male and female rats6. It has been suggested7 that, in the male, inhibin may be produced by the Sertoli cells of the testis. If this is the case, one wonders if inhibin could also be produced by granulosa cells of the ovary. We have, therefore, looked for the presence of a specific FSH-suppressing agent in ovarian follicular fluid by comparing the effects of fluid and peripheral plasma on the levels of FSH and LH in newly castrated male rats.

A convenient and rapid bioassay for inhibin.

A bioassay for inhibin, based on a dose-dependent suppression of HCG-induced ovarian weight increase in intact, immature female rats, is described. The method is specific and has acceptable sensitivity and precision such that it lends itself to statistically valid quantitative assay of inhibin activity. Being a 24-hour assay, it is conveniently and rapid performed as well as being a multiple bioassay for estimating potencies of various preparations.