Immunoreactive Alpha-inhibin Research Articles

Demonstration of a suppressive effect of inhibin alpha-subunit on the developmental competence of in vitro matured bovine oocytes.

Changes in intrafollicular concentrations of different forms of inhibin (free alpha-subunits and alpha beta dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free alpha-subunit (pro-alpha C) isolated from bovine follicular fluid on maturation of bovine cumulus-oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus-oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml-1) and either inhibin A (0, 0.2 and 1.0 microgram ml-1) or pro-alpha C (0, 2 and 10 micrograms ml-1). Neither inhibin A nor free alpha-subunit affected the cleavage rate of cumulus-oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-alpha C decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = -0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) alpha-inhibin (measured by radioimmunoassay) produced by untreated control cumulus-oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 micrograms ml-1) against two different regions of the inhibin alpha-subunit precursor (pro-region and alpha C fragment) were tested for their ability to neutralize endogenous inhibin alpha-subunit-related molecules produced by cumulus cells; control cumulus-oocyte complexes were treated with normal mouse IgG (20 micrograms ml-1). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-alpha (77% increase; P < 0.05) and alpha C (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus-oocyte complexes. Overall, these results indicate that the inhibin alpha-subunit (pro-alpha C) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.

Immunological localization and ontogenetic development of inhibin alpha subunit in rat brain.

This study examined the immunolocalization and ontogeny of the inhibin-specific alpha subunit in the brain of male rats. Immunohistochemistry using antiserum directed against the mature region of porcine inhibin alpha (1-19, Tyr20) revealed positive reactions in process-bearing cells resembling astroglia in several regions, especially in the dorsal region of the third ventricle, medial and ventral arcuate nucleus, hippocampal dentate gyrus, and layers 1-3 of the cerebral cortex. Generally, inhibin alpha-positive cells in the limbic cortex had larger cell bodies and longer processes than those in the hypothalamus. These inhibin alpha-positive cells were verified to be positive for glial fibrillary acidic protein (GFAP), a differentiated astroglial marker, by double immunolabelling. The expression of inhibin alpha mRNA was higher in the brains of neonatal rats than in those of adult rats, as revealed by reverse transcription-competitive polymerase chain reaction, although the similar changes of immunoreactive inhibin alpha subunit in the brain was not observed. Orchiectomy did not affect expression of inhibin alpha mRNA in the hypothalamic area. This study suggests that inhibin-related peptide is produced by differentiated astrocytes, especially in the hypothalamic arcuate nucleus, the hippocampal dentate gyrus, and the cerebral cortex, and that the expression of inhibin alpha is regulated during brain development.

Endocrinology: Midluteal immunoreactive -inhibin serum concentrations as markers of luteal phase deficiency

The present prospective clinical study was undertaken to determine the usefulness of midluteal phase serum immunoreactive alpha-inhibin concentrations as markers of luteal phase deficiency and whether they are better indicators of biopsy confirmed luteal phase defect than serum progesterone. Consecutive patients (n = 138) with regular menstrual cycles attending our Infertility Clinic (experimental group) and 15 fertile women who were requesting contraception and had regular menstrual patterns (control group) were included. In all women (patients and controls), basal body temperature, midluteal serum concentrations of oestradiol, prolactin, progesterone and immunoreactive alpha-inhibin, and premenstrual endometrial biopsy were used in the same cycle to assess luteal function. Out-of-phase secretory endometria were detected in 15 of the 138 patients. Thus, hormonal concentrations were compared between the following three groups of women: group 1 (n = 15), infertile patients with defective secretory endometria; group 2 (n = 123), infertile patients with normal secretory endometria; and controls (n = 15), fertile women with normal secretory endometria. Midluteal serum concentrations of progesterone, immunoreactive alpha-inhibin, oestradiol, and prolactin of the two groups studied were similar to those of the control group of fertile women. Our results indicate that midluteal serum inhibin determination does not accurately reflect histological maturation of the endometrium and it is not a better indicator of endometrial luteal phase deficiency than midluteal serum progesterone concentration.

Effects of aging on the female reproductive system.

There is an evident decline in human fertility with age. The fundamental physiological question is whether the ovary, the uterus, or both are affected by the changes induced within the body by senescence. Based on clinical studies performed in in-vitro fertilization (IVF) laboratories, there is no doubt that age affects the oocyte. However, the effect of age on the remaining cellular components of the preovulatory follicle have yet not been fully elucidated. In addition, whether the ability of the uterus to have normal implantation of human embryos is age-related is also a matter of controversy. In this review, we analyse the results of our studies that were conducted in order to answer these relevant questions. In the first series of experiments we tested the hypothesis that the function of granulosa cells is affected in older patients (> or = 40 years). In these experiments, we cultured in vitro granulosa-luteal cells from 15 women aged < 40 years and 18 patients aged > or = 40 years undergoing IVF. Immunoreactive alpha-inhibin and progesterone were measured after 48 and 96 h in culture. The measurements showed a significantly higher secretion of inhibin (P < 0.01) after 48 h, and progesterone (P < 0.05) after 48 and 96 h, in the younger as compared to the older group. In addition, we tested the ability of the uterus to sustain normal implantation by an analysis of our oocyte donation programme. In the first approach, we divided all the transfer cycles according to the age of the recipient. We found no differences among groups with regard to pregnancy, implantation or abortion rates. We explored this fundamental question further by performing oocyte donation cycles in which oocytes from the same donor were distributed into a recipient aged < 40 years and another recipient of > or = 40 years. An analysis of 45 cycles in each group of recipients showed a significant (P < 0.05) increase in abortion rates in older as compared to younger patients. When the serum oestradiol concentrations were followed in those women who carried normal single ongoing pregnancies, we found that a significant increase in blood oestradiol concentrations (which probably represents the luteo-placental shift) was detected 2 weeks earlier in younger than in older patients, suggesting a defective vasculature of the uterus that could ultimately be the cause of an increased abortion rate in patients aged > or = 40 years. In conclusion, we firmly believe that senescence affects both the ovary (oocyte and granulosa cells) and the uterus, and these observations should be taken into account in order to advise our patients.

Inhibin functions in male rats.

The role of inhibin in modulating FSH secretion at sexual maturation in male rats was studied using antibodies against inhibin alpha subunit. An immunohistochemical study was performed by immunogold-silver staining; modulation of FSH secretion in sexual maturation was studied by immunoneutralization. The results show that immunoreactive inhibin alpha subunit is localized in the Sertoli cell, especially at young ages. There were no significant differences in testes weights and histology between the antibody-administered group and the control group. Marked FSH increase was observed in the antibody-administered group from 5 to 30 days. No significant difference was detected in the over 50 days group. It would appear that inhibin plays a role in suppressing FSH secretion in infantile male rats, especially before puberty. However, inhibin's role might not be particularly important, as shown by the lack of significant changes in the gonads of antibody-administered group.

Effects of aging on the human ovary: the secretion of immunoreactive α-inhibin and progesterone

To investigate the changes induced by age in the function and secretory pattern of the human ovary. Immunoreactive alpha-inhibin, E2, and P secretion in vivo and in vitro have been compared in two different populations. Prospective study. Women undergoing IVF-ET were divided into two groups according to age: group 1 (32.0 +/- 0.7 years; mean +/- SEM) and group 2 (40.3 +/- 0.3 years). In vitro fertilization program at the Instituto Valenciano de Infertilidad. A total of 33 infertile women with regular menses, undergoing IVF-ET. Follicle aspiration performed by transvaginal ultrasound. Four follicles per patient were aspirated in individual plastic tubes. Granulosa-luteal cells isolated with Percoll columns and cultured in vitro up to 4 days in the presence of hCG. In vitro fertilization parameters, serum levels of E2, immunoreactive alpha-inhibin, and P, as well as the secretion of immunoreactive alpha-inhibin and P by the cultured granulosa-luteal cells. Serum immunoreactive alpha-inhibin levels the day of ovum pick-up were significantly lower in group 2 compared with group 1. Incubation of cells for 96 hours showed a significantly higher ability to accumulate immunoreactive alpha-inhibin in group 1 than 2. Human chorionic gonadotropin stimulated immunoreactive alpha-inhibin production after 96 hours. Cells from younger women displayed a significantly higher ability to secrete P than cells from older women. Human chorionic gonadotropin was able to significantly stimulate P production in group 1. These results confirm previous observations showing a reduced production of immunoreactive alpha-inhibin and steroids of ovaries from older women and suggest that a reduced cellular function, rather than a decrease in the follicular population, is the main mechanism by which these changes are produced.

Human follicular fluid contains pro- and C-terminal immunoreactive alpha-inhibin precursor proteins

Human follicular fluid contains pro- and C-terminal immunoreactive alpha-inhibin precursor proteins

Human follicular fluid contains pro- and C-terminal immunoreactive alpha-inhibin precursor proteins.

The majority of immunoactive inhibin in human follicular fluid (hFF) is devoid of pituitary cell bioactivity and is hypothesized to contain alpha-inhibin monomeric proteins known to cross-react with an inhibin antiserum (Monash 1989). The aim of this study was to define more precisely the nature of these inhibin-immunoreactive proteins using alpha-inhibin sequence-specific antisera. First, a polyclonal antiserum was raised to precursor amino acids 21-35 (PIN-1) and was used in a RIA to measure pro-alpha-inhibin-immunoreactive proteins. Western blotting was used to confirm these findings. Secondly, the binding epitope of the Monash antiserum was defined by peptide analysis to be located within the C-terminus (precursor amino acids 326-341) of alpha-inhibin, and this assay was then used to monitor the presence of C-terminal sequences. Similar levels of pro-alpha-inhibin immunoreactivity (PIN-1 RIA; N-terminus alpha-inhibin precursor) were detected in hFF collected from women with normal menstrual cycles during the follicular phase and from multiple follicles from a woman undergoing ovarian hyperstimulation during an in vitro fertilization (IVF) protocol. Western blotting with the PIN-1 antibody confirmed the presence of immunoreactive proteins of 57,000 and 29,000 mol wt in the follicular fluids of both normal cycle and IVF follicles. However, ovarian hyperstimulation elevated intrafollicular C-terminal immunoreactivity (Monash RIA) compared to that in normal cycle hFF. Furthermore, intrafollicular estradiol and progesterone were significantly correlated to C-terminal activity in follicles from IVF, but not in normal cycles. These data show that 1) both pro- and C-terminal alpha-inhibin proteins are secreted into follicular fluids from normal and IVF cycles, suggesting that alpha-inhibin precursor proteins may be physiologically relevant in the process of folliculogenesis; and 2) IVF and normal cycle follicular fluids differ in their production and processing of inhibin.

Changes in Serum Immunoreactive Inhibin-α during Photoperiod-Induced Testicular Regression and Recrudescence in the Golden Hamster1

Serum immunoreactive inhibin-alpha (irI alpha), FSH, LH, and testosterone (T) were measured in male golden hamsters during short-day-induced testicular regression and during testicular recrudescence following the transfer from short to long days. Serum FSH levels were maximally suppressed within 2 wk of transfer to short days. In contrast to FSH, irI alpha levels were not fully reduced until after 6 wk of exposure to short days, closely paralleling the timing of testicular regression. LH and T levels were also reduced within two 2k, with maximal suppression observed between 6 and 8 wk. Conversely, when males with regressed testes were transferred to long days, serum FSH rose to peak (25 ng/ml) levels by 3 wk and then declined to usual long-day levels. In contrast, serum irI alpha levels rose gradually, reaching adult long-day levels following 10 wk of exposure. Serum LH and T levels rose to peak levels between 5 and 8 wk before declining to adult levels. FSH, LH, and irI alpha levels were also measured after castration in male hamsters maintained on long or short days. Twenty-four hours after castration, levels of irI alpha were reduced in long-day males to levels comparable to those observed in castrated short-day males. Serum irI alpha levels respond slowly to abrupt changes in FSH levels after transfer to either long or short days, suggesting that testicular irI alpha secretion may not be directly and immediately influenced by circulating FSH levels in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

High neonatal triiodothyronine levels reduce the period of Sertoli cell proliferation and accelerate tubular lumen formation in the rat testis, and increase serum inhibin levels.

T3 was injected daily in newborn rats from birth to 16 days of age. Control rats received daily injections of vehicle during the same period. The proliferative activity of the Sertoli cells was studied by means of bromodeoxyuridine incorporation, and tubular lumen formation and nuclear size were taken as markers of Sertoli cell differentiation. T3 treatment strongly reduced the proliferative activity of Sertoli cells from day 7 on, and on day 12, proliferation of Sertoli cells had ceased, while in control rats proliferating Sertoli cells were observed up to day 16. As a result of the reduced Sertoli cell proliferation, the final Sertoli cell number per testis at 23 days of age was reduced by 50% from 38 +/- 1 x 10(6) in control rats to 19 +/- 1 x 10(6) in T3-treated rats. Lumen formation in seminiferous tubules of T3-treated rats began at 12 days of age, while in controls lumen formation was first observed at 16 days. The area of the Sertoli cell nuclei was somewhat larger in T3-treated rats on day 16, but not at any other age examined. Body and testis weights in adult rats at 100 days of age were reduced by 46% and 48% of control values, respectively. The high neonatal T3 levels reduced serum levels of TSH on days 7 and 9, but not at any other age examined. FSH levels were reduced in T3-injected rats on days 5 and 7 and increased on day 23, after cessation of treatment. Immunoreactive inhibin-alpha levels were increased on days 5-9 and reduced on days 16 and 23. These findings indicate that T3 stimulates the production of immunoreactive inhibin by Sertoli cells, but also of bioactive inhibin, as indicated by the reduced FSH levels. It is concluded that the levels of thyroid hormones early in life are important for the terminal differentiation of Sertoli cells and, therefore, for determining adult testis size. The data indicate that this might be a direct effect of T3 on Sertoli cells.

Multiple molecular weight forms of immunoreactive alpha-inhibin in human seminal plasma.

The purpose of these studies was to determine whether the total immunoreactive alpha-inhibin protein concentration in seminal plasma correlated with serum gonadotropin levels or semen characteristics and to identify the forms of alpha-inhibin present in seminal plasma. Thirty-eight serum samples from men being evaluated for infertility were selected for study based on their serum hormone profiles and semen parameters. Serum LH and testosterone levels were normal, but FSH levels ranged from normal to hypergonadotropic (> 20 IU/L). Most semen parameters were within normal ranges, but germ cell numbers ranged from normal to azoospermic. Thus, seminal plasma from these men provided a unique opportunity to examine the antigenic forms of alpha-inhibin in individuals in whom strong correlations between inhibin and FSH levels might be predicted because of the observed ranges of FSH levels and germ cell numbers. Seminal plasma alpha-inhibin was characterized by RIA or Western blotting, using an antiserum directed against the N-terminal of the alpha-subunit of mature [32,000 mol wt (M(r))] inhibin. The antiserum recognized the alpha-subunit of dimeric inhibin as well as free alpha-inhibin and alpha-inhibin precursor proteins. Total immunoreactive alpha-inhibin ranged from 8.21-43.99 nmol/L in seminal plasma. However, alpha-inhibin levels were not statistically correlated with serum FSH levels or any of the measured semen parameters (including germ cell number). In contrast, the immunoreactive alpha-inhibin concentration in seminal plasma was negatively correlated (P < 0.01) with the serum LH level. Western blot analyses revealed that multiple forms of immunoreactive alpha-inhibin are present in seminal plasma. The majority of immunoreactivity was associated with monomeric proteins (ranging from 58,000-95,000 M(r)) that were larger than the alpha-subunit (21,000 M(r)) predicted for mature dimeric human inhibin (32,000 M(r)). The relative amounts of individual forms of immunoreactive alpha-inhibin varied among the patients studied, but could not be correlated with other serum or seminal parameters measured. Our observations demonstrate that various monomeric alpha-inhibin proteins are present in human seminal plasma. It is unlikely that these proteins alone or combined with inhibin beta-subunit proteins have identical biological activities. Thus, until assays specific for each of the various forms of immunoreactive alpha-inhibin are developed, their role as well as that of inhibin in the endocrine or local modulation of testicular function cannot be deduced from RIA data alone.

Multiple molecular weight forms of immunoreactive alpha-inhibin in human seminal plasma

Multiple molecular weight forms of immunoreactive alpha-inhibin in human seminal plasma

Effects of follicle-stimulating hormone on inhibin release by different testicular compartments in the adult ram.

The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Apparent alpha-inhibin subunit immunoactivity in porcine and ovine luteal extracts is due to interference by cytosolic proteases in the assay.

Immunoreactive alpha-inhibin (ir-inhibin) was measured in luteal homogenates and subcellular fractions of ovine and porcine corpora lutea (CL) and in pig granulosa cells (GCs), using a sensitive radioimmunoassay specific for the 1-26 amino acid sequence of the N-terminus of the alpha chain of porcine inhibin (p1-26 alpha-inhibin). Inclusion of N-ethylmaleimide (N-EM) and/or EDTA in the immunoassay had no effect on the measurement of p1-26 alpha-inhibin peptide standards, on ir-inhibin levels in ovine follicular fluid and serum, or on ir-inhibin in subcellular fractions of pig GC. Fractionation of porcine GC homogenates on sucrose gradients demonstrated a major particular peak of ir-inhibin (buoyant density, 1.15-1.21 g/cm3) with variable activity in the cytosol. The particulate ir-inhibin peak was released into the cytosol by pretreatment of GC homogenates with the saponin, digitonin, prior to fractionation. Porcine GC extracts contained a protein (M(r) 45,000) which immunoblotted against p1-26 alpha-inhibin antibody. In the absence of inhibitors of proteolysis, apparent ir-inhibin activity was very high in extracts of sheep and pig CL. However, inclusion of N-EM or EDTA in the radioimmunoassay significantly reduced ir-inhibin levels in porcine and ovine CL extracts in a dose-dependent manner. Measurements of peptide tracer integrity indicated that porcine luteal cytosol degraded 125I-labelled p1-26 alpha-inhibin peptide. Subcellular fractionation studies demonstrated high levels of apparent ir-inhibin in luteal cytosol fractions, with only minor activity peaks associated with particulate fractions; however, this material was not releasable by digitonin. Immunoblotting of detergent extracts of porcine luteal particulate fractions failed to demonstrate alpha-inhibin material, and immunocytochemical localization studies of alpha-inhibin in porcine and ovine luteal sections were negative. Our results are consistent with the intracellular packaging/storage of a form of alpha-inhibin (M(r) similar to that of alpha-inhibin subunit precursor) in the porcine granulosa cell. However, luteinization of the porcine follicle was associated with a dramatic fall in ir-inhibin content, and the loss of immunostaining for alpha-inhibin peptides. We conclude that porcine and ovine CL contain little, if any, authentic inhibin. These studies emphasize the importance of excluding proteolytic artefacts when measuring biological peptides in luteal tissue extracts by radioimmunoassay.

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.

Gonadotropin-releasing hormone, inhibin, and activin in human placenta: evidence for a common cellular localization.

The human placenta produces several hypophysiotropic factors that participate in the control of local hormone secretion. In particular, previous reports showed that inhibin and activin modulate both GnRH release and hormonal effect in cultured human placental cells. The present study evaluated the possible correlations among the synthesis and cellular distribution of inhibin, activin, and GnRH in placental villi at term. mRNA coding for inhibin alpha- and beta A-subunits and GnRH were localized in human placenta at term by in situ hybridization using the corresponding cDNA probes. Autoradiograms revealed that some placental cells express inhibin alpha- and beta A-subunit and GnRH mRNAs. A common localization of the three hormonal mRNAs has been found in the inner part of the villi. Using affinity-purified polyclonal antisera, immunocytochemical studies confirmed that in placental villi at term, immunoreactive inhibin alpha- and beta A-subunits and GnRH had a distribution that was superimposable in several areas. Both the outer layer and the inner trophoblasts contained immunoreactive hormonal products. The present data show that some placental cells at term can produce and release inhibin, activin, and/or GnRH. This complement of hypophysiotropic factors may, thus, represent a local paracrine/autocrine control mechanism within the placenta.

Localization and Secretion of Inhibin/Activin Subunits in the Human and Subhuman Primate Fetal Gonads*

Little is known about the ability of the fetal primate gonads to produce inhibin/activin. We investigated the presence of the alpha-, beta A-, and beta B-subunits of inhibin/activin in fetal human (16-23 weeks gestational age) and rhesus monkey (days 150-157 of gestation; term = 165 days) testes and ovaries by immunocytochemistry. The regulation of alpha-inhibin secretion by gonadotropins was studied in fetal testicular cultures. In the human fetal testis, alpha-subunit immunostaining was found in interstitial and intratubular cells, while beta A- and beta B-subunit immunostaining occurred in clusters of Leydig cells that were clearly demarcated from groups of Leydig cells that were immunonegative. In the late gestational monkey testis, the alpha-subunit was localized in tubular cells, and the beta B-subunit was present in the tubules and interstitium. Testicular cells from midgestation human testes secreted detectable immunoreactive alpha-inhibin in response to FSH and hCG stimulation; alpha-inhibin levels were significantly higher after hCG than FSH. In contrast, levels of alpha-inhibin secreted by rhesus monkey testicular cells were significantly increased by FSH, but not hCG. In the ovary, only weak beta B-subunit immunoreactivity was detected in granulosa cells of a few primary follicles from midgestational human fetal ovaries. In contrast, all three subunits were found in granulosa cells of numerous primary and secondary follicles in the late gestation rhesus monkey ovary. In light of recent evidence that inhibins/activins have actions on gonadal differentiation and growth modulation in vitro, as well as endocrine effects on the fetal pituitary, we propose that these proteins may have intragonadal and endocrine roles in human and subhuman intrauterine gonadal development.

Functional Recovery of the Developing Rat Ovary after Transplantation: Contribution of the Extrinsic Innervation*

These experiments were undertaken to define the neuroendocrine mechanisms underlying the recovery of ovarian function after transplantation to an ectopic site. Both ovaries from 23-day-old rats were transplanted to the region of the neck, next to the jugular vein. Serum gonadotropin and plasma immunoreactive inhibin-alpha levels were determined at several intervals thereafter. Serum estradiol (E2) was measured during the first week posttransplantation. Reinnervation of the ovary by sympathetic and sensory nerves was monitored by immunohistochemistry. Sympathetic nerves were identified as adrenergic by the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, and as peptidergic, by their neuropeptide-Y (NPY) or vasoactive intestinal peptide (VIP) immunoreactivity. Sensory nerves were identified by the presence of substance P (SP) and calcitonin-gene related peptide (CGRP) immunoreactivity. Serum LH and FSH increased, and plasma inhibin levels decreased, within 48 h after transplantation. Serum LH reached maximum levels on day 4, decreasing rapidly thereafter to basal values by day 6. These changes were functionally correlated with the posttransplantation fluctuations in serum E2, which decreased at 48 h, rebounded by day 4, and returned to basal values on day 7. Removal of the transplanted ovaries on day 3 resulted in the disappearance of serum E2 levels on day 4, thus confirming the ovarian graft as the source of E2. In contrast to LH, serum FSH remained significantly elevated for at least 3 weeks after transplantation, then decreased to basal levels after day 21, coinciding with the rise in inhibin secretion. Although a substantial loss of follicles was noted 48 h after transplantation, quantitative examination of the changes on day 4 revealed that approximately 40% of antral follicles were not necrotic. Ovulation and formation of corpora lutea were noted 21 days after transplantation. Reinnervation of the transplanted ovary by TH-, VIP-, NPY-, SP-, and CGRP-containing fibers was first detected 7 days after transplantation. Although VIP reinnervation was sparse and only transiently detected (days 7-21), the density of sympathetic (TH, NPY) and sensory (SP, CGRP) fibers increased 2- to 3-fold between days 7-28, remaining unchanged thereafter. Since apparent completion of this reinnervation coincided with reestablishment of normal levels of both LH and FSH, an additional experiment was performed to determine if the two events were causally related.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in Serum Immunoreactive Inhibin and Follicle-Stimulating Hormone during Gonadal Development in Male and Female Rats1

We have measured serum and ovarian immunoreactive inhibin alpha (irI alpha) and serum FSH in fetal and neonatal rats from 20 days of gestation until 40 days of age. For animals aged 10 days or older, serum measurements were made on intact and gonadectomized animals. Serum irI alpha was detectable in intact male and female rats at all ages studied. In females, irI alpha levels were low until Day 5 and then increased steadily to peak at Day 25. Thereafter they declined until Day 35 to reach levels typical of adult females. There was a significant decrease in irI alpha levels 24 h after ovariectomy at all ages. Serum FSH levels in females were low until Day 7, then increased rapidly to plateau from Days 10-15. The levels then declined until Day 25 and were generally unchanged after that time. There was a significant increase in FSH 24 h after ovariectomy in rats aged 20 days and older, and in younger rats by 48 h after ovariectomy. In male rats, serum irI alpha levels were significantly higher than females until Day 7. The levels increased at Day 7 and then remained relatively constant until Day 20, after which they declined to reach typical adult male levels. Serum irI alpha levels were significantly lower in males than females from Days 25-40. There was a significant decrease in serum irI alpha 24 h after castration at all ages studied. Serum FSH levels in males were low until Day 20, increased at Day 25, and thereafter remained relatively unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of inhibin‐α in the testes of normal men and in men with testicular disorders

Testicular biopsies from normal men and from men with testicular disorders were examined by immunohistochemistry for the presence of the inhibin-alpha subunit using two different antisera. Immunoreactive inhibin-alpha (irI-alpha) was found in Leydig cells in normal, oligospermic, and azoospermic men and in men with Klinefelter's syndrome, and it was also found in a Leydig cell tumour. hCG-treatment apparently increased the amount of immunoreactive inhibin-alpha, particularly in Leydig cells. Sertoli cells also contained irI-alpha but the staining intensity was considerably stronger in testes with impaired spermatogenesis or Sertoli-cell-only syndrome than in normal testes. It is suggested that the serum concentration of irI-alpha and inhibin in humans may, in a complex way, be related to both Leydig and Sertoli cell function, and that the relative contribution from these cells may change in cases of testicular malfunction.