Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares

Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasound examination at 24-h intervals. The ability of the inhibin antiserum to neutralize the bioactivity of equine inhibin was examined in vitro using a rat pituitary cell culture system. Suppression of secretion of FSH from cultured rat pituitary cells by equine follicular fluid was reversed by the addition of increasing doses of the inhibin antiserum, thereby indicating its bioactivity. Plasma levels of FSH and estradiol-17 beta were higher in mares treated with the inhibin antiserum. The ovulation rate was significantly higher in mares treated with antiserum (100 mL = 3.75 +/- 0.63; 200 mL = 4.50 +/- 0.65) than in control mares (1.25 +/- 0.25). These results demonstrate that inhibin is important in regulating FSH secretion and folliculogenesis in mares. They also show that neutralization of the bioactivity of inhibin may become a new method for the control of folliculogenesis and ovulation rate in mares.     

Variability in the apparent molecular weight of inhibin

An in vitro bioassay based on suppression of GnRH-stimulated FSH secretion by pituitary cells in culture was used to monitor inhibin activity after dialysis, gel filtration or polyacrylamide gel electrophoresis of protein preparations from a variety of gonadal secretions and extracts under native and dissociating conditions. The suggestion that inhibin is a peptide of molecular weight less than 5000 was not confirmed. Although some fractions of low molecular weight suppressed FSH secretion, the amount of activity was low and the dose response curves were not parallel with a standard preparation of inhibin. Under most conditions, inhibin eluted with an apparent molecular weight of about 90 000. However, gel filtration of rete testis fluid protein in 1 M acetic acid resulted in elution of inhibin activity with a lower apparent molecular weight and with polyacrylamide gel electrophoresis in 0.1% (w/v) sodium dodecylsulfate, the apparent molecular weight was 30 000. It is concluded that inhibin is a protein which tends to aggregate and coelute with larger molecules.     

The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin

Three proteins (31, 35 and 39 kDa) with inhibin-like activity have been isolated from bovine follicular fluid with identical NH2-terminal amino acid sequences. These polypeptides are distinct from inhibin, based on their different NH2-amino acid sequence, molecular masses, absence of a subunit structure, absence of inhibin immunoactivity and the failure of inhibin antiserum to neutralize their bioactivity in vitro. Their inhibin-like biological activities based on their ability to suppress FSH cell content by pituitary cells in culture are 5-10% of bovine 31 kDa inhibin.     

Changes in inhibin activity, and progesterone, oestrogen and androstenedione concentrations, in rat follicular fluid throughout the oestrous cycle

Follicular fluid was aspirated from all visible surface follicles of rats at selected times of the oestrous cycle. Fluids from a pair of rat ovaries were pooled and assayed for inhibin activity by the rat anterior pituitary cell culture assay. Serum LH, FSH and progesterone as well as follicular fluid progesterone, total oestrogens and androstenedione were also measured. Follicular fluid inhibin activity was relatively constant throughout the oestrous cycle (30.7 +/- 3.4% inhibition of FSH per 0.1 microliter follicular fluid) except for a well defined surge at pro-oestrus (09:00-16:00 h, peak at 14:00 h = 84.0 +/- 7.2% inhibition of FSH per 0.1 microliter follicular fluid). The follicular fluid was not treated with charcoal before assay because a pilot experiment showed that such treatment did not alter the inhibin activity of follicular fluid. Steroids in follicular fluid were generally lowest on the afternoon of oestrus and the morning of dioestrus I and generally elevated during pro-oestrus.     

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.     

In vivo suppression of pituitary and circulatory follicle stimulating hormone by human seminal plasma inhibin

Human seminal plasma inhibin (HSPI), of prostatic origin, has been shown to bring about a dose dependent suppression in pituitary and circulatory FSH concentrations in intact rats. No significant changes in LH levels either in pituitary or in circulation were observed at the doses used. This has further been substantiated by an immunocytochemical staining. A marked reduction in staining intensity for FSH was observed in the pituitary of inhibin treated rats as compared to the controls. None of the purified inhibin peptides from other sources have so far been reported to act on pituitary FSH in vivo. This study thus, for the first time demonstrates an in vivo effect of inhibin (HSPI) on pituitary FSH concentration and secretion.     

Inhibin production by Sertoli cells in culture.

Studies on the secretion of inhibin and its mode of action were carried out in vitro, utilizing cell cultures. Isolated rat Sertoli cells secreted an inhibin-like heat-labile, non-dialysable substance, Sertoli Cell Factor (SCF), which could selectively suppress FSH secretion by rat anterior pituitary cells. SCF selectively suppressed the basal and GnRH-stimulated FSH release as well as the de-novo synthesis of FSH by acting directly on the pituitary cells. In 1 out of 5 experiments, SCF also suppressed the synthesis of LH, possibly by affecting the overall protein synthesis. Under similar culture conditions, Sertoli cells isolated from animals between 18 and 90 days of age secreted comparable amounts of SCF. In contrast, anterior pituitary cells from adult rats (60-90 days old) were considerably more sensitive to SCF than pituitary cells obtained from younger (18-33 days old) animals, suggesting that decline in circulating FSH level, occurring at approximately 35 days of age, may result from increased pituitary sensitivity to inhibin. Besides identifying the Sertoli cells as the site of inhibin production in the testis, these studies demonstrated direct action of inhibin at the pituitary cell level, resulting in suppression of FSH synthesis and release.     

Molecular weight forms of inhibin a and inhibin B in the bovine testis change with age

To investigate alterations in the molecular weight forms of inhibin in bull testis from the infantile (4-5 wk of age) to postpubertal (49-56 wk of age) periods, testicular homogenates were obtained from animals of various ages and fractionated by a combination of immunoaffinity chromatography and SDS-PAGE. Subsequently, the fractions eluted from the SDS gels were assayed for total inhibin, inhibin A, and inhibin B by fluoroimmunoassay or immunofluorometric assays (IFMAs) and for inhibin bioactivity by an in vitro bioassay. The molecular mass patterns of inhibin A and inhibin B in the testis, as determined by the dimer-specific IFMAs, showed the presence of a peak of approximate 47 kDa until 21-26 wk of age. However, the peak disappeared after 31-32 wk of age. As bulls aged, especially after 31-32 wk of age, inhibin A and inhibin B levels increased in the molecular mass region of 27-34 kDa. Total inhibin showed two peaks, of between 20 and 26 kDa and at approximately 47 kDa, until 21-26 wk of age and a single peak between 20 and 30 kDa after 31-32 wk of age. The eluted fractions corresponding to 29, 31, or 47 kDa gave a dose-response curve that was parallel to the curve generated with 32-kDa inhibin A or 29-kDa inhibin B standard in the IFMA for inhibin A or inhibin B. The fractions corresponding to 29 and 31 kDa suppressed basal release of FSH from rat pituitary cells, but the 47-kDa fraction had a lower FSH-suppressing activity. In the testes of older bulls, immunoblot analysis revealed the presence of a 29-kDa band cross-reacting with inhibin alpha and inhibin betaB antibodies and of a 31-kDa band cross-reacting with inhibin alpha and inhibin betaA antibodies. The 47-kDa band was recognized by the alpha, betaA, and betaB antibodies. Immunohistochemisty of the testis at each age showed that inhibin alpha subunits were found exclusively in Sertoli cells, but the intensity of immunostaining diminished in older bulls, in parallel with the decrease in the testicular concentrations of total inhibin. We conclude that 1) bovine Sertoli cells produce both inhibin A and inhibin B, 2) inhibin production in Sertoli cells during the prepubertal period is characterized by the 47 kDa inhibin-related material that contains precursor forms of inhibin A and inhibin B, and 3) the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, although total inhibin production in Setroli cells decreases.     

An in-vivo method for estimating inhibin production by adult rat testes

The concentrations of inhibin in samples of rat testicular venous and arterial blood and interstitial fluid were measured by an in-vitro bioassay using pituitary cells in culture in which the standard was an ovine testicular lymph preparation (assigned potency 1 unit/mg). Inhibin levels were undetectable (less than 2 U/ml) in both blood samples but reached a mean concentration of 120 +/- 7 U/ml in testicular interstitial fluid. After unilateral efferent duct ligation the rate of inhibin accumulation in seminiferous tubules was determined by the difference in the inhibin content of the ligated and unligated testes. Additionally, the rate of seminiferous tubule fluid production was obtained from the difference in weight between the ligated and non-ligated testes. In the 24 h after efferent duct ligation there were linear increases in inhibin (18.5 +/- 1.0 U/h) and in seminiferous tubule fluid production (26 +/- 1 microliter/h), but there were no changes in serum FSH and LH levels. Experimental induction of bilateral cryptorchidism led to a decrease in the inhibin content of the testis after 10 days. The rate of inhibin accumulation after efferent duct ligation declined more rapidly than the inhibin content, being significantly depressed in cryptorchid testes after 3 days, suggesting that this measurement is a more sensitive index of inhibin production than the determination of testicular inhibin content.     

Development of a radioimmunoassay for human seminal plasma inhibin.

Using a homogeneous inhibin preparation from human seminal plasma with a molecular weight of about 19 000, a sensitive and specific radioimmunoassay (RIA) for inhibin has been developed. None of the purified hormones tested, such as LH, FSH and prolactin from different species, showed any cross-reaction in this RIA. Steroid hormones such as testosterone, dihydrotestosterone, oestradiol-17 beta and progesterone did not interfere with the assay. The antiserum had an affinity constant (Ka) of 2.379 X 10(9). The assay sensitivity was 10-15 ng per tube and the intra- and inter-assay coefficients of variation were 5-7% (n = 6) and 15% (n = 10) respectively. The recovery for inhibin added to the serum of a castrated man was 95-110%. Using this RIA, inhibin levels in various biological fluids and tissues were measured. Normo-spermic semen contained significantly higher levels of inhibin than did oligospermic semen. Human prostate contained a substantial quantity of inhibin. Monkey semen, rat serum, and bovine, ovine and porcine follicular fluids cross-reacted in the RIA, while ram testicular inhibin and bull semen did not do so. In developing (9-28 days of age) male rats, circulating inhibin levels showed an inverse relationship with serum FSH levels. In female rats of this age endogenous inhibin concentrations changed in parallel with those of serum FSH.     

Influence of dose and route of administration of bovine follicular fluid and the suppressive effect of purified bovine inhibin (Mr 31,000) on plasma FSH concentrations in ovariectomized ewes

Ovariectomized Merino ewes were used to develop an in-vivo bioassay for purified bovine inhibin of Mr 31,000. Various doses (0.25, 0.5, 1 or 2 ml) of bovine follicular fluid, given either by the intravenous (i.v.) or intracarotid route (i.c.) resulted in significant linear dose-related suppression of plasma FSH and interval to maximum suppression. Control ewes (1.0 ml steer plasma) showed no significant change in FSH over the same period. Doses of 470 and 2590 U of pure inhibin given i.v. caused a significant suppression of FSH in plasma in all ewes. The in-vivo potency estimate of the high dose (2760 U, 1420-4690 fiducial limits) agreed well with the in-vitro assay of potency. There were no significant changes observed in mean plasma LH after treatment with the higher dose of pure inhibin. There were no rebound effects of treatment with bovine follicular fluid or pure inhibin on FSH concentrations above that of controls. It is concluded that the form of bovine inhibin of Mr 31,000, which is believed to be the predominant circulating form, is biologically active when administered in vivo.     

Species specificity of radioreceptor assay and radioimmunoassay for rat FSH

Species specificity of the radioreceptor assay (RRA) for rat FSH, in which pregnant mare serum gonadotropin (PMSG)-treated immature rat ovary was employed as the receptor, was compared with that of NIAMDD rat FSH radioimmunoassay (RIA). In the RIA system, pituitary preparations from mammals only showed significant crossreaction. Their inhibition curves, however, were not always parallel to the standard curve. On the other hand, in the RRA system, the pituitary preparations from mammals, avians, lizard and amphibians competitively inhibited the binding of radioactive rat FSH to the ovarian receptor. Only the pituitary preparation from dog salmon failed to show any crossreaction in the RRA system. These results indicated that this RRA system would be useful for the measurement of FSH or gonadotropins of the pituitaries from mammals to amphibians.     

Isolation of inhibin from bovine follicular fluid

Bovine follicular fluid was used as a source for the isolation of gonadal inhibin, the activity of which was monitored by the dose dependent suppression of the FSH content of cultured pituitary cells. The procedures presented result in over 3000-fold purification of the starting material and the purified inhibin has an apparent molecular weight of 56000. The purified inhibin can be dissociated under reducing conditions into two subunits with molecular weights of 44000 and 14000 daltons.     

Evidence that testosterone and follicular fluid do not interact in the control of FSH secretion in rams

The hypothesis that testosterone and inhibin interact in the control of FSH secretion in rams was tested. Adult rams were castrated and were simultaneously given testosterone implants and 3-times daily sc injections of 0, 0.4, 0.8 or 1.6 ml charcoal-treated bovine follicular fluid (bFF). After 1 wk, the implants were removed, and the bFF injections continued as before. Blood samples were taken daily for mean LH, FSH and testosterone concentrations, and every 10 min for 12 h in the presence and in the absence of testosterone for assessment of pulsatile LH release. The bFF specifically inhibited FSH secretion from rat pituitary cells in culture. In the presence of testosterone, there were no main effects of bFF on mean plasma FSH or LH concentrations, nor were these values different from their pre-treatment means (P>0.05). Treatment with bFF did not affect LH pulse frequency or amplitude, but the number of rams showing LH pulses was reduced in the 0.8 and 1.6-ml dose groups (P<0.05). Removal of testosterone increased (P<0.05) both gonadotropins. In the absence of testosterone, no main effect of bFF on mean LH or FSH concentrations was observed, although the 1.6-ml dose suppressed the postcastration rise of both LH and FSH. These data suggest that inhibin does not interact with testosterone and that a physiological level of testosterone is sufficient for the regulation of FSH secretion in adult rams.     

The human placenta: a novel source of inhibin

Human placental extracts contain inhibin bioactivity and immunoactivity giving dose response curves parallel to a human follicular fluid inhibin standard. Inhibin bioactivity in vitro was neutralised by preincubation of extracts with antisera raised to pure bovine inhibin. Umbilical cord blood from term infants contained immunoactivity. Human placental inhibin differs from human ovarian inhibin in terms of its biological: immunological ratio.     

Superovulation of immature rats by continuous infusion of follicle-stimulating hormone

Immature female rats were infused s.c. continuously over a 60-h period with partially purified porcine pituitary follicle-stimulating hormone (FSH) preparations differing in degree of purity and having widely divergent luteinizing hormone (LH):FSH potency ratios as defined by radioreceptor assays. Rats infused with the more purified FSH preparation (FSH-A) ovulated a mean of 60-85 oocytes per rat on the morning of the third day (Day 1) after FSH infusion was begun (on Day -2). The same total dose of FSH administered as a single s.c. injection or as twice daily injections over the same 60-h period resulted in ovulation in only a minority of treated rats (3/16), with none achieving ovulation rates approaching those of rats infused continuously. High fertilization rates (80% of ovulated oocytes) were observed in superovulated rats joined with fertile males on the evening of the second day of infusion (Day 0). Of the 67 +/- 7 fertilized ova per rat retrieved from oviducts flushed on Day 1, 52 +/- 8, or 80%, were accounted for as morulae or blastocysts recovered when oviducts and uteri were flushed on the morning of Day 5, demonstrating essentially normal developmental rates and high survival rates in reproductive tracts of superovulated females during the preimplantation period. Infusion of rats with the same dose of a less well-purified FSH preparation (FSH-E) containing 20 times as much LH activity, or injection of rats with a superovulatory dose of pregnant mare's serum gonadotropin (PMSG) (40 IU), were much less effective in causing superovulation, with ovulation rates of 17 +/- 6 and 34 +/- 8 oocytes/rat, respectively, compared to 79 +/- 9 oocytes/rat infused with the FSH preparation (FSH-A) containing lower LH activity.(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)     

Regulation of pulsatile gonadotropin secretion by estrogen, inhibin, and follistatin (activin-binding protein) in ovariectomized rats.

The following study was conducted to examine the effects of estrogen and polypeptides, given either alone or in combination, on pulsatile gonadotropin secretion. One week after ovariectomy, rats received s.c. injections of oil or various doses (0.5, 5, 20 micrograms) of estradiol benzoate (EB) followed 1 day later by i.v. administration of 60 micrograms purified porcine follistatin, 10 micrograms recombinant inhibin, or the appropriate vehicle. Four hours after injection of the nonsteroids, blood was collected at 10-min intervals for 2 h, and the effects on pulsatile hormone release were assessed. Administration of EB alone dose-dependently suppressed mean and trough (lowest point between two pulses) FSH levels and all parameters of pulsatile LH release. Both follistatin and inhibin at the doses employed suppressed mean FSH levels to an equivalent extent (40%). Follistatin, but not inhibin, suppressed FSH pulse amplitude, while neither polypeptide alone influenced FSH pulse frequency or any parameter of pulsatile LH release. The effects of follistatin and EB on mean FSH levels were additive at all EB doses, whereas the effects of inhibin and EB were additive only at the middle EB dose. Follistatin in combination with the lowest EB dose significantly suppressed mean LH levels. These studies are the first to demonstrate that combined treatment with estrogen and the nonsteroids follistatin and inhibin is more efficacious in suppressing FSH release than treatment with either agent alone, thereby indicating that both steroids and nonsteroids are probably important in the physiological regulation of FSH secretion in rats. The additive effects of these compounds on FSH secretion could form the basis for exploring novel contraceptive interventions.     

Effects of flutamide on testicular involution induced by an antagonist of gonadotrophin-releasing hormone and on stimulation of spermatogenesis by follicle-stimulating hormone in rats.

The effects of combined treatment with an antagonist of gonadotrophin-releasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450-500 micrograms/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT+FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.