Forms Of Inhibin Research Articles

Inhibin-A and Inhibin-B in stallions: Seasonal changes and changes after down-regulation of the hypothalamic-pituitary-gonadal axis

The biological function of inhibin is mediated by two heterodimers, inhibin-A and inhibin-B. The relative importance of inhibin-A and –B in male reproductive function varies considerably across species with inhibin-B predominating in many species, whereas inhibin-A appears relatively more important in rams. Research reported to date in stallions has examined total or immunoreactive (ir) inhibin which does not distinguish the two heterodimers. Therefore, the objective of this study was to characterize changes in inhibin-A and inhibin-B concentrations in stallions: 1) across season for a period of one year, and 2) after downregulation of the hypothalamic-pituitary-gonadal (HPG) axis. In Study one, serum samples were obtained monthly from five stallions for a period of one year. Serum concentrations of inhibin-A, inhibin-B, testosterone and estrone sulfate were determined by ELISA. In Study two, stallions were treated with the GnRH antagonist, acyline (n = 4; 330 mg/kg acyline IM) or vehicle control (n = 4; vehicle alone) every five days for 50 days. Plasma concentrations of inhibin-A and –B were determined by ELISA at Days 0, 6, 12, 22, 37, 59, 80, 87 and 104 after initiation of acyline treatment. Testis volume was determined by ultrasonography at weekly intervals. In Study 1, both inhibin-A and inhibin-B showed seasonal changes in concentration with highest concentrations in increasing day length and lowest concentrations in short day lengths. Inhibin-B (overall mean 107.8 ± 4.1 pg/mL) was present at 4.7-fold higher concentrations in serum than inhibin-A (overall mean 23.0 ± 0.7 pg/mL). In Study 2, plasma concentrations of inhibin-B but not inhibin-A were significantly downregulated by administration of the GnRH antagonist, acyline. When the HPG axis was downregulated by acyline, testis volume was strongly correlated with inhibin-B (r = 0.73; P < 0.05) but not inhibin-A (r = 0.22; P = 0.20). In summary, inhibin-B appears to be the predominant form of inhibin in the stallion which undergoes seasonal regulation along with other reproductive parameters and is co-regulated with other endocrine parameters of the HPG axis.

Use of detergent-based buffers allows detection of precursor inhibin forms in an immunoassay format

Inhibin ELISAs are used in monitoring aspects of reproductive function, however these assays are based on the measurements of the mature 30kDa inhibin forms and not precursor forms. In conventional ELISA formats, the 105kDa unprocessed ‘Pro-inhibin’ forms are immunologically inactive, but the immunoactivity can be recovered in the presence of detergents. The immunoactivity of Pro-inhibin forms was assessed in the presence of a range of detergents utilising antibodies to the α-, βA- and βB-subunits of inhibin. In contrast to mature forms, unprocessed inhibin forms showed a 10–40 fold increase in inhibin A and total inhibin immunoactivities under optimised detergent (0.5% SDS/2% Triton X-100) conditions. The suppressed immunoactivity of the Pro-inhibin forms in these immunoassays was attributed to steric hindrance by the respective βA- and α-subunit prodomains. This study details a detergent-based immunoassay that allows detection of previously undetectable precursor inhibin forms.

Changes in circulatory FSH of Barbari goats following treatment with high molecular weight inhibin isolated from buffalo follicular fluid

The objective of the present study was to isolate and purify high MW inhibin (∼129kDa) from buffalo ovarian follicular fluid (buFF) and to investigate its biological activity. Throughout the process of purification, the inhibin fractions were evaluated for bioactivity by a specific, sensitive and uniformly reproducible bioassay in mice. The final biological activity of this preparation was tested in normal cycling adult female Barbari goats. Eight animals, randomly divided into two groups, were synchronized for estrus by administering PGF2α twice at an interval of ten days. Following synchronization, the treatment group (n=4) received (i.m.) 0.4ml (240μg protein total dose) of purified inhibin (MW∼129kDa) of buFF in the morning at 08.00h for the four consecutive days of follicular phase, while the control group (n=4) received only saline (0.4ml). Blood samples were collected from jugular vein immediately before the first injection and subsequent collections were made daily in the afternoon until day 8 of the experiment including four days (0, 1, 2, & 3days) of the next cycle. FSH was assayed in all the samples by ELISA. The peripheral FSH concentration sharply declined from 1.854±0.137 to 0.979±0.02 u/l, 8h after the administration of inhibin on the first day. The value in controls was 2.004±0.132 u/l. For the duration of treatment of four consecutive days, the FSH level in experimental group remained significantly low (p<0.05) compared to control group. After cessation of treatment, the FSH level remained low on day 0 and 1 of the next cycle in the experimental and control animals. However, a significant rebound increase in plasma FSH levels occurred on day 2 & 3 (2.73±0.179 & 1.849±0.128u/l) only in the experimental group compared to control animals (P<0.05). This increment might be caused by the rebound surge of FSH from anterior pituitary which further corroborates the effect of inhibin in treated animals. In conclusion, the present study demonstrates that the high MW form of inhibin (∼129kDa) isolated from buFF has comparable biological activity as revealed by 31–32kDa inhibin from other species.

Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid

Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the anterior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspirated from surface follicle (with diameter> or =5mm). A combination of techniques (viz., gel filtration, SDS-PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS-PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid.

Regulation of inhibin/activin expression in rat early antral follicles

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-αC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione + FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-αC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.

Suppression of Inhibin A Biological Activity by Alterations in the Binding Site for Betaglycan

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.

INHIBINS INHIBINS - QUALITIES, QUALITIES, SIGNIFICANCE SIGNIFICANCE AND HIS ROLE IN PREGNANCY PREGNANCY

&amp;lt;p&amp;gt;Inhibins are glycoprotein hormones of which there are two molecular forms, inhibin A and inhibin B. Classically, inhibin is known to have a negative feedback effect on pituitary follicle-stimulating hormone secretion. The fetoplacental unit produced inhibin throughout pregnancy. Inhibin Ais the predominant molecular form of inhibin in maternal circulation from 4 week of gestation. Although the precise biological function of inhibin Ain pregnancy is unclear, it is evident from recent studies that inhibin A could be a better marker of placental function than human chorionic gonadotropin because of its shorter half-life. The possible clinical application for the measurement of inhibin Ain early pregnancy could be in predicting miscarriage , Down&amp;#039;s syndrome, preeclampsia and fetal growth restriction in the first and/or second trimester before the onset of yhe clinical symptoms. Inaddition, several evidences underline the potential role and the clinical usefulness of their measurements in the diagnosis, prevention,prognosis and follow-up of different gestational pathologies such as:threatened abortion, placental tumors, hypertensive disorders of pregnancy,intrautherine growth restriction,fetal hypoxia.The measurement of inhibin Aand activin Ainto the biological fluids of pregnancy will offer in the future further possibilities in early diagnosis,prediction and monitoring pregnancy diseases.&amp;lt;/p&amp;gt;

Granulosa cell tumors of the ovary: The clinical value of serum inhibin A and B levels in a large single center cohort

In patients with a granulosa cell tumor of the ovary, the value of serum inhibin A and B concentrations for the assessment of disease status was investigated. In 30 consecutive patients with a stage I-III granulosa cell tumor, inhibin A and B concentrations were measured in pre- and post-treatment serum samples. Clinical data concerning diagnosis, treatment and follow-up of these patients were related to serum inhibin A and B concentrations. Serum samples from 41 premenopausal females with cervical dysplasia served as controls. In 30 patients, 13 (43%) recurrences were observed during a median follow-up of 10 years (range 1-31 years). Serum inhibin A and B concentrations were elevated in respectively 67% and 89% of the patients at diagnosis, and in 58% and 85% at recurrence. Inhibin A and B concentrations were normal in all controls. Sensitivity of inhibin A testing for the diagnosis of granulosa cell tumor was 67% with a specificity of 100%, compared to 89% and 100% respectively for inhibin B (ns). Elevations in serum inhibin B concentrations predated recurrences by a median of 11 months. None of the patients in remission showed increased concentrations of inhibin A and B. Inhibin B seems to be the predominant form of inhibin secreted by granulosa cell tumors and appears to reflect disease status more accurately than inhibin A. Measurement of serum inhibin B concentrations may be preferred for the follow-up of granulosa cell tumors.

Production of inhibin A and inhibin B in boars: Changes in testicular and circulating levels of dimeric inhibins and characterization of inhibin forms during testis growth

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin α subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29–31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29–31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa α monomer. Immunohistochemistry indicated that positive immunostaining for the α subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.

Changes in Circulating and Testicular Levels of Inhibin A and B During Postnatal Development in Bulls

We investigated testicular and circulating levels of dimeric inhibins in Holstein bulls from the infantile to postpubertal periods (5 to 50 weeks of age) and examined the relationship between the profiles of circulating dimeric inhibins and FSH. Concentrations of total inhibin and inhibin B in the testis were highest at 4 to 5 weeks of age but decreased gradually as the bulls aged. Testicular inhibin A levels showed a gradual decline to a nadir at 15 to 26 weeks of age, but by 39 weeks, they were high again. The contents of total inhibin, inhibin A, and inhibin B per testis generally increased with age. Fractionation of testicular homogenates obtained from 15-week-old bulls by a combination of immunoaffinity chromatography and SDS-PAGE confirmed the presence of two major molecular weight forms (32 and 45 kDa) of dimeric inhibins in the testes. Circulating levels of total inhibin and inhibin A showed a significant increase in bulls at around 10 to 14 weeks of age compared to the levels between 5 and 7 weeks of age but decreased thereafter. However, immunoreactivity for inhibin B was not detected in the peripheral circulation, probably because of low sensitivity of the inhibin B assays. The concentrations of plasma FSH were high at 5 weeks of age but declined to lower levels between 11 and 40 weeks, and then increased from 41 weeks onward. There was no significant correlation between the plasma levels of FSH and inhibin A or total inhibin. The results clearly indicate that the bull testis produces inhibin A and B and secretes at least inhibin A into the circulation during postnatal development. However, the profile of circulating FSH in bulls shows no reciprocal relationship with the inhibin A or total inhibin profile during the postnatal period.

Gonadotropin Control of Inhibin Secretion and the Relationship to Follicle Type and Number in the hpg Mouse1

Inhibin is secreted in two distinct heterodimeric forms, A and B, but the mechanism for the differential control of these two forms is unclear. To evaluate the relationship between secretion of inhibin forms and folliculogenesis, the effects of gonadotropins on inhibin concentrations were studied in parallel with stereological enumeration of ovarian follicle types in gonadotropin-deficient hypogonadal (hpg) female mice treated with recombinant human FSH (10 IU/day), hCG (1 IU/day), or both for 20 days. Treatment with FSH alone significantly increased blood concentrations of both inhibin A and inhibin B, whereas hCG alone had no effect on either inhibin. The combination of FSH and hCG further increased the concentration of inhibin A but had no effect on the concentration of inhibin B beyond that of FSH. The number of primordial follicles per ovary was significantly reduced in FSH-treated hpg mice, but was not affected by hCG treatment. Antral follicles were absent in the untreated hpg mice, present following treatment with FSH, and were present in only limited numbers following hCG treatment alone. Preovulatory follicles were observed only in the wild-type and combined FSH and hCG treatment groups. These results demonstrate that secretion of both inhibins is associated with the presence of antral follicles. Inhibin A secretion is increased by the presence of preovulatory follicles, whereas the concentration of inhibin B is not affected. The observed effects of gonadotropins on inhibin A and B secretion may be explained by corresponding gonadotropin effects on follicle development.

Role of inhibin in normal and high-risk pregnancy.

Inhibins are glycoprotein hormones of which there are two molecular forms, inhibin A and inhibin B. Classically, inhibin is known to have a negative feedback effect on pituitary follicle-stimulating hormone secretion. The fetoplacental unit produces inhibin throughout pregnancy. Inhibin A is the predominant molecular form of inhibin in maternal circulation from 4 weeks of gestation. Although the precise biological function of inhibin A in pregnancy is unclear, it is evident from recent studies that inhibin A could be a better marker of placental function than human chorionic gonadotropin because of its shorter half-life. The possible clinical applications for the measurement of inhibin A in early pregnancy could be in predicting miscarriage, Down's syndrome, preeclampsia, and fetal growth restriction in the first and/or second trimester before the onset of the clinical symptoms. The source of inhibins, factors controlling inhibin production, the possible functions of inhibin, and the use of inhibin measurement in normal and high-risk pregnancy are reviewed.

Inhibins in normal male physiology.

Inhibin is a dimeric glycoprotein that suppresses follicle-stimulating hormone (FSH) secretion from the pituitary. Two bioactive forms of inhibin exist, inhibin A and B. The availability of specific immunoassays for each of the isoforms has enabled the study of the individual inhibins and their physiological roles. In the male, inhibin B is the circulating form in all species studied to date except the sheep. Inhibin B is produced in the testis, principally by the Sertoli cells. There are temporal changes in inhibin expression and secretion with the changing role of the Sertoli cell in immature and adult testes. Variations in inhibin B production between species reflect the different patterns of maturation. In the adult, inhibin B levels are positively correlated with Sertoli cell function, sperm number, and spermatogenic status and are negatively correlated with FSH. It appears that the regulation of inhibin B production is mediated by a complex interaction between FSH, Sertoli cells, Leydig cells, and germ cells. Inhibin may also play a role at an autocrine or paracrine level in modulating the actions of activin.

Beta A versus beta B: is it merely a matter of expression?

Activins are members of the transforming growth factor (TGF) beta (β) superfamily of proteins that function in a wide array of physiological processes. Like other TGFβ ligands, activins are biologically active as dimers. An activin molecule is comprised of two β-subunits, of which four isoforms have been identified: βA, βB, βC, and βE. The most widely studied activins to date are activin A (βA/βA), activin B (βB/βB), and activin AB (βA/βB). Inhibin is a naturally occurring activin antagonist that consists of an α-subunit disulfide-linked to one of the activin β-subunits, producing inhibin A (α/βA), or inhibin B (α/βB). The development of assays distinguishing between different forms of activins and inhibins, along with knock-in and knock-out models, have provided evidence that the βA- and βB-subunits have independent and separate roles physiologically. Additionally, evaluation of ligand–receptor interactions indicates significant differences in receptor affinity between activin isoforms, as well as between inhibin isoforms. In this review we explore the differences between activin/inhibin βA- and βB-subunits, including expression patterns, binding properties, and the specific structural aspects of each. From the growing pool of knowledge regarding activins and inhibins, the emerging data support the hypothesis that βA- and βB-subunits are functionally differently.

In-vivo concentrations of inhibins, activin A and follistatin in human early pregnancy

The aims of this study were to investigate the relationship between inhibins, activin A and follistatin in first trimester fetal fluids, maternal serum, placenta and decidua, and to investigate if these hormones are present in the circulation of the early second trimester human fetus. Amniotic and coelomic fluid, maternal serum, placental villi and decidual tissue were obtained from normal pregnancies at 8–12 weeks. Fetal blood by cardiocentesis and maternal blood were collected at 14–16 weeks gestation. Placental extracts had higher concentrations of inhibins, activin A and follistatin compared with decidual extracts. In the second trimester, inhibins and follistatin were detectable in fetal blood at 14–16 weeks gestation. Maternal serum concentrations of inhibin A ( P < 0.001) and follistatin ( P < 0.05) were significantly higher than fetal serum whereas inhibin B ( P < 0.01) and pro-alpha C concentrations ( P < 0.001) were higher in fetal serum. Inhibin B concentrations were also higher in male fetal serum samples that had higher concentrations of testosterone. The presence of all molecular forms of inhibins, activin A and follistatin in the first trimester fetal fluids, placental and decidual extracts in the first trimester confirms other reports. In the second trimester, high concentrations of inhibin B with testosterone in the fetal circulation indicate that these hormones may interact in the development of the male fetal gonads.

Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

BackgroundInhibins 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.MethodsThe subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (RACE) RT-PCR from adult rhesus monkey testis RNA.ResultsBoth the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0), which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1). Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained.ConclusionThe data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.

Inhibines, activines et hormone anti-müllerienne : structure, signalisation, rôles et valeur prédictive en médecine de la reproduction

Anti-Müllerian hormone (AMH), inhibins and activins are members of the transforming growth factor (TGFβ) superfamily and are known to have a variety of actions concerning reproduction, hormonogenesis, development processes and differentiation. Inhibins and activins are dimeric glycoproteins that are defined by their actions on the pituitary gonadotroph cells. AMH, inhibins and activins have a vast array of actions usually exerted through paracrine and endocrine mechanisms. The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B seems to be a useful marker of spermatogenesis, but serum and seminal inhibin B levels are not predictive parameters for the selection of azoospermic men as candidates for testicular sperm extraction (TESE). AMH in seminal plasma may be important for sperm production, and is a good marker for sertoli cell development. It might be the only one seminal marker of spermatogenesis in non-obstructive azoospermia. Nevertheless, many of these studies were carried out with small patient numbers, and consequently must be interpreted with caution. In women ongoing assisted reproductive therapy (ART), day 3 inhibin B and AMH levels predict the number of oocytes retrieved, but cannot predict likelihood of pregnancy. Further studies are needed to determine if AMH and inhibin predict ART outcomes better than classical parameters (age, FSH levels and follicular ultrasonography). AMH and inhibin are also specific markers of Sertoli- and granulosa-cell origin in gonadal tumors.

Characterization of plasma inhibin forms in fertile and infertile men.

The reciprocal relationship between plasma FSH and inhibin B generally reflects the state of spermatogenesis but data in some settings indicate further complexity in their relationship. Inhibin circulates as a range of higher molecular weight (mol wt) forms of varying bioactivity such that the serum profile of inhibin forms may differ between normal men and those with varying types of spermatogenic failure. The aim of this study was to establish if the inhibin B mol wt distribution was altered in men with infertility. The mol wt profiles of inhibin B and free alpha-subunit were determined in plasma of fertile (n = 11) and infertile (n = 17) men using a combined immunoaffinity chromatography, preparative SDS-PAGE and electro-elution procedure and fractions assayed using ELISAs for inhibin B, total inhibin (all forms containing the alpha-subunit) and free alpha-subunit. Inhibin B was identified as precursor (60-65 k) and mature (26-30 k) forms in plasma in similar proportions (29%) in fertile men and oligospermic men (25%), but was undetectable in azoospermic men. The free alpha-subunit detected by the pro-alphaC ELISA was identified as both the precursor and processed (pro-alphaC) forms with similar proportions in fertile (8%) and all infertile (4-14%) men. The pro-alphaC ELISA did not detect the precursor forms of inhibin B in plasma while the inhibin B ELISA detected all total inhibin forms following removal of pro-containing forms by immunoabsorption. (i) the proportions of precursor inhibin B and alpha-subunit forms in the circulation are unchanged in men with spermatogenic disorders indicating there is no alteration of the Sertoli cell inhibin secretory pattern; (ii) these fractionation studies indicate that pro-alphaC and inhibin B ELISAs specifically detect the free alpha-subunit and inhibin B forms present in male plasma.

Evidence for a negative intrafollicular role for inhibin in regulation of estradiol production by granulosa cells.

Intrafollicular concentrations of inhibin A and estradiol vary inversely during development of dominant follicles in cattle. Thus, we hypothesized that inhibin has a negative autocrine or paracrine effect on estradiol production by granulosa cells. To examine this hypothesis, a homologous model system was used to test the effects of bovine antibovine inhibin antibodies, bovine inhibin, and a peptide fragment of bovine inhibin (bINH) on capacity of granulosa cells isolated from individual estrogen-active or -inactive dominant or subordinate follicles to produce estradiol during short-term (18 h) serum-free culture. Immunoblot analysis of media demonstrated that granulosa cells basally produce different molecular weight forms of inhibin, similar to those in bovine follicular fluid. Immunoneutralization of endogenous inhibin in culture with different doses (12.5-1000 microg) of highly purified bovine antibovine inhibin antibodies increased estradiol production 2- to 15-fold, compared with controls. Preadsorption of the anti-inhibin antibodies with bINH precursors or bovine pro-alpha(C) suppressed the capacity of anti-inhibin antibodies to enhance estradiol production by granulosa cells, compared with controls. Treatment of granulosa cells with an immunoaffinity-purified preparation of bINH suppressed basal estradiol production 60%, compared with controls. In contrast, treatment of granulosa cells with the bINH peptide increased estradiol production 14-fold, compared with controls. Based on these results, we concluded that both anti-inhibin antibodies and bINH blocked the suppressive local effects of basally produced inhibin on estradiol production during culture of granulosa cells and that inhibin has a negative autocrine or paracrine effect on the in vitro capacity of granulosa cells isolated from dominant or subordinate follicles to produce estradiol.

Inhibin B is the major form of inhibin secreted from testes in male Japanese macaques ( Macaca fuscata).

In order to clarify the cellular source and forms of bioactive inhibin in male Japanese macaques ( Macaca fuscata), circulating concentrations of inhibin A and B, and immunohistochemical localization of inhibin subunits in testis were studied. Plasma concentrations of testosterone were also measured. The present study showed that inhibin B was clearly detected in the plasma of male Japanese macaques. Moreover, concentrations of both inhibin B and testosterone during the breeding (mating) season were significantly higher than those of the non-breeding season. On the other hand, plasma inhibin A was detected neither during the breeding seasons nor during the non-breeding seasons. Positive stainings with alpha and betaB subunit antibodies were observed in the Sertoli cells, however staining with betaA subunit antibody was not observed in the testicular samples. These results indicate that inhibin B is the major circulating inhibin and probably secreting from Sertoli cells in male Japanese macaques.