Forms Of Inhibin Research Articles

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle.

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.

Differences in follicular function of 3-month-old calves and mature cows

After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Müllerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Müllerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Müllerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.

Negative feedback regulation of the secretion and actions of gonadotropin-releasing hormone in males.

This minireview considers the state of knowledge regarding the interactions of testicular hormones to regulate the secretion and actions of GnRH in males, with special focus on research conducted in rams and male rhesus monkeys. In these two species, LH secretion is under the negative feedback regulation of testicular steroids that act predominantly within the central nervous system to suppress GnRH secretion. The extent to which these actions of testicular steroids result from the direct actions of testosterone or its primary metabolites, estradiol or dihydrotestosterone, is unclear. Because GnRH neurons do not contain steroid receptors, the testicular steroids must influence GnRH neurons via afferent neurons, which are largely undefined. The feedback regulation of FSH is controlled by inhibin acting directly at the pituitary gland. In male rhesus monkeys, the feedback regulation of FSH secretion is accounted for totally by the physiologically relevant form of inhibin, which appears to be inhibin B. In rams, the feedback regulation of FSH secretion involves the actions of inhibin and testosterone and interactions between these hormones, but the physiologically relevant form of inhibin has not been determined. The mechanisms of action for inhibin are not known.

Serum inhibin B as a marker for spermatogenesis.

Inhibin B generated by Sertoli cells provides negative feedback on FSH secretion. In men, inhibin B seems to be the physiologically important form of inhibin. Serum inhibin B was measured by two-site immunoenzymatic assay in 40 normal men (27 years of age) with sperm concentrations 100 +/- 9.2 x 10(6)/mL, 51 subfertile men (31 years of age) with sperm concentrations 6.8 +/- 0.8 x 10(6)/mL, 16 men with varicocele with sperm concentrations 54.3 +/- 0.8 x 10(6)/mL (31 years of age), men with hypogonadotrophic hypogonadism, men with Klinefelter syndrome, and men with obstructive and non-obstructive azoospermia. In men with normal sperm concentrations (>20 x 10(6) mL) serum inhibin B was 201 +/- 17 pg/mL and FSH 4 +/- 0.5 IU/L. Varicocele patients showed normal sperm concentrations > 20 x 10(6)/mL, normal serum inhibin B (173 +/- 21 pg/mL), and normal FSH levels (4.6 +/- 0.6 IU/L). In patients with sperm concentrations < 20 x 10(6)/mL the inhibin B level was 118 +/- 14 pg/mL and the FSH level was 10 +/- 1.1 IU/L. In all patients, except those with hypogonadotrophic hypogonadism and Klinefelter syndrome. inhibin B and FSH were inversely correlated (r = -.41, p > 0.01). There was a positive correlation between inhibin B and sperm concentrations (r = .34, p < .01). In varicocele men there was a correlation of r = .574, p < .05. Inhibin B may be a marker of exocrine testicular function and may offer an improved diagnosis of testicular dysfunction.

Serum inhibin pro alphaC in infertile men.

Specific assays have been developed for bioactive inhibin dimers, inhibin A and B, and inhibin alpha-subunit precursor pro alphaC. To better understand the role of serum inhibin pro alphaC in infertile men, the authors measured these forms of inhibin in sera from 39 infertile men and analyzed inhibin relationships with serum gonadotropins, testosterone, and estradiol. All subjects had oligozoospermia. Inhibin A levels were undetectable in all subjects. Inhibin B concentrations were 117 +/- 59 pg/mL. Inhibit B concentrations correlated negatively with serum FSH (r = .584, p < .0001) and positively with sperm count (p < .01) and bilateral testicular volume (r = .607, p < .0001). The concentration of pro alphaC was 556 +/- 236 pg/mL (normal range, 446 +/- 28). Inhibin pro alphaC showed no correlation with serum FSH, LH, testosterone, sperm concentration, and bilateral testicular volume. In addition, inhibin pro alphaC was not correlated with inhibin B. Pro alphaC is unlikely to be a useful marker for spermatogenesis in infertile men compared with inhibin B.

Regulation of immunoreactive inhibin A and B secretion in cultured human granulosa-luteal cells by gonadotropins, activin A and insulin-like growth factor type-1 receptor.

Inhibins are gonadal glycoproteins with endocrine effects on pituitary FSH secretion and para/autocrine effects on ovarian and testicular function. The purpose of this study was to investigate the endocrine and para/autocrine regulation of inhibin A and inhibin B secretion in human ovarian granulosa-luteal cells. The cells were obtained from women undergoing in vitro fertilization, and the primary cultures were treated with FSH, LH, human chorionic gonadotropin (hCG), activin A, 8-bromo cyclic AMP (8-BrcAMP), staurosporine (a protein kinase C inhibitor) and an antagonist of IGF action (type-1 IGF receptor antibody alpha IR3). The secretion of inhibins was measured by ELISA assays capable of reliably distinguishing between inhibin A and B. FSH, LH, hCG and 8-BrcAMP increased inhibin A secretion on average up to 180% (P<0.01), 192% (P<0.05), 210% (P<0.01) and 243% (P<0.01) respectively of the control level, while their stimulatory effect on inhibin B secretion was less pronounced (up to 167%, P<0.01; 139%, P<0.05; 127%, P>0.05; 133%, P>0.05 of the controls respectively). alpha IR3 decreased inhibin A and B secretion down to 70% (P<0.01) and 50% (P<0.01) respectively of the control. Staurosporine decreased inhibin B secretion down to 49% (P<0.01) of the control; its effect on inhibin A secretion was not significant. Activin A increased inhibin B secretion up to fourfold of the control (P<0.05) while its effect on inhibin A secretion was insignificant. We conclude that gonadotropins via the protein kinase A signal transduction pathway are the main positive regulators of inhibin A and B secretion in human granulosa-luteal cells. The protein kinase C signal transduction pathway seems to be important especially for inhibin B secretion. Locally produced IGFs are probably important inducers of the production of both forms of inhibin in human ovaries while activins seem to upregulate inhibin B secretion.

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries.

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.

Analysis of follicular fluid hormone concentrations and granulosa cell mRNA levels for the inhibin-activin-follistatin system: relation to oocyte and embryo characteristics

Objective: To explore the potential roles of inhibin, activin, and follistatin in human oocyte development by quantifying their intrafollicular biosynthesis. Design: Prospective, nonrandomized study. Setting: An IVF unit and academic research laboratory. Patient(s): Thirty one patients undergoing IVF. Intervention(s): Human menopausal gonadotropins or human FSH (or both) were administered. Single-follicle aspirates (n = 110) were collected for analysis. Main Outcome Measure(s): Concentrations of dimeric, total and pro-αC inhibin forms; activin A; follistatin; estradiol; and progesterone were measured in follicular fluids. Granulosa-cell mRNA was analyzed for α, βA, and βB inhibin and activin subunits; follistatin; activin receptors; and β-actin. Hormone concentrations and mRNA levels were correlated with oocytes or embryos from the same follicles. Result(s): Levels of progesterone and follistatin were significantly greater in follicles containing MI or MII oocytes than in those containing GV oocytes. Inhibin α-subunit mRNA levels were significantly higher in follicles containing maturing oocytes, the highest-quality oocytes, and oocytes that were subsequently fertilized. In contrast, inhibin α-subunit mRNA levels were significantly lower in follicles from which higher-quality embryos were obtained. Conclusion(s): Inhibin α-subunit biosynthesis is associated with normal oocyte and follicle maturation, but excessive α-inhibin is associated with poor embryo quality. None of the hormones analyzed were associated with oocyte or embryo quality.

Regulation of inhibin production in the human male and its clinical applications.

Investigation of the regulation of testicular function has been enhanced by the ability to measure the dimeric, biologically active form of inhibin in men, inhibin B. This has allowed the demonstration that inhibin B is the afferent arm of the feedback loop from the testis that regulates FSH secretion, and investigation of inhibin B levels during reproductive development and in a variety of physiological and pathological states. Such studies have demonstrated many primary aspects of the relationship between FSH, testicular function (and in particular spermatogenesis) and inhibin B levels in blood, though many more specific questions remain. These include the precise nature of the relationship between inhibin B secretion and Sertoli cell function and how this relationship is influenced by the germ cell types present in the testis and by overall sperm production. When such information becomes available it will allow more accurate interpretation of blood concentrations of inhibin B. Similarly, apical secretion of inhibin B into seminal plasma may have considerable value in aiding assessment of the status of the seminiferous epithelium. Finally, neonatal secretion of inhibin B as a measure of Sertoli cell number and/or as a predictor of adult reproductive function offers novel possibilities for assessment and intervention.

Is inhibin a serum marker for ovarian cancer?

Research endocrinologists are well aware of inhibin and the family of proteins of which it is a part. Inhibin was discovered almost 70 years ago as a product of the gonads that selectively inhibited the secretion of folliclestimulating hormone (FSH) at the level of the pituitary gland. We now know that inhibin is present in at least two biologically active forms (inhibin A and B) as well as in a variety of a subunit precursor forms and has been implicated not only in endocrine functions, but also as a possible local growth regulator in a variety of tissues. In clinical endocrinology, inhibin B appears to be a useful tool in assessing ovarian reserve and the potential for success in assisted reproduction. Research endocrinologists may not be aware, however, of the developing use of circulating inhibin levels as a diagnostic aid in clinical areas outside endocrinology. For example, in the area of prenatal genetic diagnosis, the measurement of inhibin A has been shown to be useful in maternal serum screening for Down’s syndrome pregnancy in the second trimester. In the area of gynecologic oncology, exemplified by the study of Ala-Fossi and colleagues (1) in this issue of the European Journal of Endocrinology, various forms of inhibin are being tested and applied as possible serum markers of ovarian cancer. Inhibin looked promising as a new ovarian cancer marker in the early 1990s when Healy et al. (2) showed that serum levels were elevated in 29% of 143 postmenopausal women with confirmed ovarian cancer. The inhibin antibody (Monash No. 1989) used for the radioimmunoassay in that study was directed exclusively to the a subunit (3) and detected a combination of biochemical forms otherwise known as total inhibin, that included dimeric inhibin A and B as well as a subunits (4). With the advent of more specific inhibin assays, recent studies have been aimed at determining which form (total inhibin, inhibin A, inhibin B, or the a inhibin subunit precursor as measured by the pro-aC assay) might be the best marker of disease. The most coherent and convincing data that serum inhibin levels are useful as a marker of ovarian cancer come from studies of granulosa cell tumors. All forms of inhibin are elevated in the serum of patients with granulosa cell tumors relative to healthy women of the same age (2, 5). This finding has a sound pathophysiological basis since the granulosa cell is the primary origin of inhibin synthesis. There have been subtle differences in the frequency of elevated levels depending on the biochemical form(s) measured: 100% for total inhibin, 89–100% for inhibin B, 90% for pro-aC, and 71–77% for inhibin A (2, 5, 6). Nevertheless it is clear that both a subunit and dimeric forms of inhibin are secreted in abundance in granulosa cell tumors, with total inhibin, a combination of all forms, most reliably increased. There is a consensus that a inhibin is a better serum marker than dimeric inhibin for all cases of epithelial ovarian tumors, and this is further supported by the study of Ala-Fossi et al. (1) in the present issue of this Journal. Inhibin appears to be a suitable serum marker for epithelial tumors of the mucinous type with about 70–80% of patients having elevated total inhibin (2, 5, 7), 55–60% having elevated inhibin B or pro-aC, and 20% having elevated inhibin A levels (5). In contrast, inhibin is not a very good marker in non-mucinous epithelial tumors. While mucinous tumors characteristically include benign ovarian type stroma which is believed to be a source of inhibin (8), most serous, clear cell and endometrioid carcinomas typically lack such ovarian stroma. At best, total inhibin is elevated in 15–35% and pro-aC is elevated in <15%–30% of non-mucinous epithelial ovarian cancer cases (2, 5, 9). The specificity of the inhibin markers for ovarian cancer has yet to be addressed sufficiently in the literature. Few previous studies have examined cases of non-ovarian cancers or benign disease and the published results suggest rather poor specificity of the inhibin markers. The total inhibin elevation has ranged from 7 to 41% in non-ovarian cancers (2, 5) and 28% in benign gynecologic disease (2, 7). In agreement, Ala-fossi et al. (1) found no difference in pro-aC levels in malignant versus benign ovarian disease. Therefore, inhibin’s utility appears to be limited to monitoring of patients with known ovarian cancer. For granulosa cell tumors, inhibin has been shown to decrease after surgery and to increase with disease recurrence (2, 10). In fact, some patients have a documented rise in inhibin 11 months preceding recurrence of granulosa cell ovarian cancer (11). Neither inhibin A nor inhibin B levels were elevated in granulosa cell tumor patients in remission (6). However, it remains to be investigated whether a European Journal of Endocrinology (2000) 142 331–333 ISSN 0804-4643

Production of inhibin forms by the fetal membranes, decidua, placenta and fetus at parturition.

Inhibins are regulators of paracrine and endocrine function during pregnancy, but their intrauterine sites of secretion are not well established. In amniotic fluid, inhibin A-, inhibin B- and inhibin pro-alphaC-containing isoforms were present in high concentrations, whereas in maternal serum, inhibin A and pro-alphaC forms were present in high amounts, with low concentrations of inhibin B. In fetal cord serum, inhibin pro-alphaC was present in all samples, inhibin B was detectable in male but not female fetuses, with no detectable inhibin A in either sex. From cultured explants, both inhibin A and B were secreted by chorion laeve, whereas only inhibin A was secreted by placenta, with both tissues secreting inhibin pro-alphaC. Only low concentrations of both dimeric inhibins and pro-alphaC forms were secreted by decidua parietalis and amnion. The dual perfused placental cotyledon secreted both inhibin A and pro-alphaC into maternal perfusate, but only inhibin pro-alphaC into the fetal circulation and less than to the maternal side. We conclude that trophoblast is the predominant source of dimeric inhibins, but with markedly different secretion depending on its intrauterine location. There was a significant decrease in inhibin A and pro-alphaC in amniotic fluid collected at term active labour compared to elective Caesarean section (P < 0.001). This may reflect a local change in inhibin/activin processing at labour, likely in chorion laeve trophoblast cells, which may be important in the paracrine control of the feto-maternal communication required to maintain pregnancy and initiate labour.

Early follicular phase serum FSH as a function of age: the roles of inhibin B, inhibin A and estradiol

Objective Reproductive aging in regularly cycling normal women is characterized by a gradual decline in ovarian follicle number and a progressive increase in serum follicle stimulating hormone (FSH), particularly over the age of 40 years. The lack of any consistent decrease in circulating estradiol and progesterone has led to the hypothesis that the FSH increase results from decreasing ovarian inhibin production. The aim of this study was to investigate the relationship between serum inhibins A and B, FSH and estradiol in normal women between the ages of 20 and 50 years.Design and patients Serum from 66 regularly cycling subjects, aged 20–50 years, was collected on days 3–5 of the menstrual cycle for this cross-sectional study.Measurements Serum inhibin A and inhibin B levels were measured by specific enzyme-linked immunosorbent assays (ELISAs). Alpha subunit forms were determined by an immunofluorometric assay which detects all known monomeric and dimeric forms of inhibin A and inhibin B and free α subunit. FSH and estradiol levels were measured by immunoassay. Data were log transformed before analysis.Results Serum FSH, inhibin A and estradiol, but not inhibin B, were positively correlated (p < 0.05–p < 0.001) with age between years 20 and 50. Between 40 and 50 years, serum FSH was negatively correlated with inhibin B (r = -0.61, p < 0.001) and α subunit forms (r = -0.47, p < 0.05) and with estradiol (r = -0.39, p < 0.05), but not with inhibin A (r = -0.21, not significant). When log(FSH) was modelled as a function of log(inhibin B) and log(estradiol) with age fitted as a covariate, inhibin B only was a significant independent predictor of FSH (β = -0.30, p < 0.01). Using purified inhibin A and B standards for the three assays, which were calibrated in terms of their α subunit content, serum inhibin A levels were 10–15% of those of inhibin B, with inhibin A + B levels being 22% of total α subunit levels. No significant correlation was observed between total inhibin α subunit and its dimers. The free α subunit, as determined from the difference in levels of total α subunit and inhibin A + B, remained relatively unchanged with age, suggesting that it is not differentially regulated.Conclusions This study shows that, during the early follicular phase, FSH, inhibin A and estradiol but not inhibin B increase with age. Some of the increase in inhibin A and estradiol may be the result of accelerated follicular development with increasing age. Serum inhibin B and estradiol but not inhibin A are inversely correlated with FSH between ages 40 and 50, but only inhibin B is a significant independent predictor of FSH. This supports the postulate that inhibin B is the main form of inhibin regulating FSH at this stage of the menstrual cycle. During the early follicular phase, serum levels of inhibin A are presumably too low to play a significant physiological role or are less active.

Changes in plasma concentrations of inhibin A and inhibin B throughout sexual maturation in the male chimpanzee.

Profiles of circulating plasma inhibin A and inhibin B during sexual maturation in male chimpanzees were investigated by using two-site enzyme-linked immunoassay (ELISA). Plasma concentrations of testosterone and pituitary gonadotropins were also measured. Concentrations of inhibin B, testosterone, luteinizing hormone (LH) and prolactin increased with age throughout prepuberty to adulthood, whereas inhibin A level was low and there were no age-related changes in concentrations of either inhibin A and follicle-stimulating hormone (FSH). Inhibin B showed an inverse correlation with FSH in adult (7 years or order) but not in immature (6 years or younger) male chimpanzees. There was no correlation between plasma levels of FSH and testosterone throughout the period of sexual maturation. However, testosterone levels were positively correlated with inhibin B levels. These results suggest that circulating inhibin B is involved in the regulation of FSH secretion after puberty in adult male chimpanzees, and also that circulating inhibin B is an important form of inhibin as a marker of Sertoli cell function in adult male chimpanzees.

Partial characterization of circulating inhibin-B and pro-alphaC during development in the male rhesus monkey.

Gel filtration chromatography and ELISAs for inhibin-B and pro-alphaC were used to examine the circulating forms of inhibin in the neonatal (age 2-6 weeks), juvenile (age 1-2 yr), and adult male rhesus monkey. In all samples, isoforms of inhibin-B of 26-36K and 150K were found. Both forms were significantly greater in the adult. The alpha-subunit assay detected major peaks at 45-60 and 29-31K, and a minor peak of greater than 100K. As for inhibin-B, the major forms of inhibin pro-alphaC were highest in adulthood. Inhibin-B and pro-alphaC were measurable in peripheral plasma at age 1 week, increased with the neonatal rise in plasma FSH, and then decreased but remained detectable through age 1 yr. Values in adult males were higher than at any time during the first year of life. Finally, mean values of plasma inhibin-B and pro-alphaC in five monkeys, based on multiple blood samples drawn between age 1 week and 1 yr, were rank ordered and were found to be highly positively correlated (r = 0.96), suggesting that inhibin levels in the first year of life may be a marker of Sertoli cell number, and may predict the spermatogenic capacity of the testis in adulthood.

Inhibin Forms in Serum From Postmenopausal Women With Ovarian Cancers

BACKGROUND AND OBJECTIVE Previous studies have shown that serum inhibin as measured by α subunit-directed radioimmunoassay (RIA) and inhibin A ELISA was elevated in postmenopausal women with mucinous and granulosa cell cancers, with the RIA showing a more frequent elevation than the inhibin A ELISA. It was thus hypothesised that these cancers may also produce inhibin B or the free α subunit. The aim of the study was to identify the forms of inhibin found in a range of ovarian cancers using a range of inhibin assays with varying specificities. DESIGN Serum samples obtained from women with ovarian cancer were assayed by inhibin B ELISA and Pro-αC subunit ELISA and compared with inhibin RIA and inhibin A ELISA. PATIENTS Blood samples were obtained from 34 postmenopausal women (>55 years) with no history of endocrine disease and from women with ovarian serous cystadenocarcinomas (n = 66), mucinous cystadenocarcinomas (n = 20), granulosa cell tumours (n = 9 − 11), miscellaneous ovarian cancers (n = 46) and non ovarian cancers (n = 23). MEASUREMENTS Inhibin B and inhibin Pro-αC subunit levels were determined by ELISA and compared to values obtained by RIA and inhibin A ELISA. Cancers were discriminated from controls based on values obtained 2SD above the geometric mean of the control values. RESULTS Granulosa cell tumours were detected by RIA and inhibin B ELISA (100%), Pro-αC ELISA (90%) and inhibin A ELISA (77%). Mucinous tumours were detected by RIA (70%), inhibin B ELISA (60%), Pro-αC ELISA (55%) and inhibin A (20%). Serous tumours were detected by RIA (35%) and the other assays (<15%). Miscellaneous tumours were detected by RIA (41%) and other assays <30%. CONCLUSIONS Ovarian neoplasms may produce a variety of peptides related to the inhibins, including dimeric inhibin A and B. Inhibin B is detected in more ovarian cancers than inhibin A but does not discriminate as well as the α subunit directed assays. The higher discrimination index obtained with the RIA compared to the Pro-αC ELISA suggests that assays detecting all inhibin forms containing the α subunit and not just those detecting the Pro-αC subunit will provide the most useful detection method.

Sexual dimorphism in circulating monomeric and dimeric inhibins in normal boys and girls from birth to puberty.

Inhibins are peptides, mainly of gonadal origin, that suppress FSH production. Dimeric forms of inhibin (A and B) have been proposed as peripheral markers of Sertoli and granulosa cell function. The aim of this study was to establish the relationship between circulating dimeric and monomeric inhibins, and gonadotrophins and sex steroids, in normal boys and girls from birth to puberty. One hundred and forty-six normal children (females: 57; males: 89) were studied. Serum LH and FSH were measured by an immunofluorometric assay. Serum oestradiol and testosterone were measured by radioimmunoassay. Serum inhibin A and B, and Pro-alphaC, were measured by specific two-site enzyme-linked immunosorbent assays. In boys from birth to 6 months of age, the mean serum inhibin B concentration was as high (477 +/- 53.7 ng/l) as that found at puberty (400 +/- 70.2 ng/l). After the first year, inhibin B gradually decreased to reach its lowest concentration (153 +/- 23.6 ng/l) at age 4-6 years. At approximately age 10, it rose progressively to reach pubertal concentrations. Pro-alphaC showed a similar pattern but at lower concentrations. Inhibin A was not detected at any age. In girls from birth to 6 months, inhibin B levels (83.0 +/- 18.3 ng/l) were approximately 50% lower than those found at puberty (181 +/- 25.7 ng/l). After 6 months of age, these levels dropped (17.5 +/- 1.6 ng/l) and remained low until the prepubertal years. Thereafter, they increased to pubertal concentrations. The serum inhibin A concentration after birth (29.9 +/- 8.7 ng/l) was similar to that found at puberty (18.3 +/- 5.7 ng/l); after 6 months, it fell to undetectable levels until the prepubertal years. The sex difference in serum levels of gonadotrophins is associated with sex differences in the levels and proportions of circulating dimeric and monomeric inhibins.

Secretion of inhibin A, inhibin B and inhibin pro-alphaC during the oestrous cycle of the golden hamster (Mesocricetus auratus).

Plasma concentrations of inhibin pro-alphaC, inhibin A and inhibin B were determined by enzyme-linked immunosorbent assay at 6 h intervals throughout the 4-day oestrous cycle of the golden hamster. Plasma concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta were also measured by radioimmunoassay during the oestrous cycle. Plasma concentrations of inhibin A increased from the early morning of day 1 (day 1=day of ovulation) and reached plateau levels at 0500 h on day 2. An abrupt increase in plasma concentrations of inhibin A was found at 1700 h on day 4, when the preovulatory FSH surge was observed. An increase in plasma concentrations of inhibin B occurred on day 1 and reached plateau levels at 1700 h on day 1. The levels remained elevated until 0500 h on day 4 and declined gradually by 2300 h on day 4. Plasma concentrations of inhibin pro-alphaC gradually increased with some fluctuation from day 1 to 1700 h on day 4 and then declined. Significant negative relationships were noted between plasma FSH and both dimeric forms of inhibin from day 1 to day 3. Significant positive relationships were found between plasma oestradiol-17beta and inhibin A or inhibin pro-alphaC throughout the oestrous cycle. In contrast, no significant relationship was found between plasma oestradiol-17beta and inhibin B. These findings suggest that both dimeric forms of inhibin play a role in the regulation of FSH secretion during follicular development. These findings also suggest that inhibin pro-alphaC could be secreted primarily by large follicles, and early atretic follicles could also be responsible for inhibin pro-alphaC secretion. On the other hand, the secretory pattern of dimeric inhibins might shift from inhibin B to inhibin A with follicular development.

Regulation of Biologically Active Dimeric Inhibin A and B From Infancy to Adulthood in the Male

Inhibins are glycoprotein members of the transforming growth factor-β family that have been implicated in the control of spermatogenesis by exerting a negative feedback on FSH secretion. In addition, locally produced inhibins may play a role in paracrine regulation of testicular function. Immunoassays were used to measure the two biologically active dimeric forms of inhibin (inhibin A and B) in serum, seminal plasma, and urine. To better define their actions, inhibins were measured in the male during infancy, sexual maturation, and senescence. Inhibin B but not A was measurable in the serum of male newborns, infants, children, and adults. In adult males, measurable levels of inhibin B were detected in the seminal plasma but not the urine. The circulating levels of inhibin B increased shortly after birth and peaked at 4–12 months of age (210 ± 31 pg/mL). The concentration measured in the serum then decreased to a low of 81 ± 12 pg/mL of inhibin B from 3–9 yr of age followed by a gradual increase beginning ...

Inhibin B levels in azoospermic subjects with cytologically characterized testicular pathology.

Inhibin B, a heterodimeric glycoprotein of gonadal origin, is the most important circulating form of inhibin in human males and an inverse relationship between inhibin B and FSH plasma levels was been recently observed. Azoospermia represents the end-point of different kinds of testicular damage, ranging from a normal spermatogenic pattern (obstructive forms) to the complete absence of germ cells (Sertoli Cell Only Syndrome, SCOS). Furthermore, azoospermia may be related to maturational disturbances at different levels (spermatogonial, spermatocytic, spermatidic). To better define the relationship between testicular damage and inhibin levels and to evaluate the diagnostic value of this hormone in the management of subjects with azoospermia, we performed specific inhibin B assays in a group of azoospermic subjects affected by different kinds of testicular pathology. Eighty-nine azoospermic men were studied by testicular ultrasound examination, fine needle aspiration of the testes and hormonal parameters (FSH, LH and testosterone, inhibin B). Thirty normozoospermic subjects were considered as controls for seminal and hormonal parameters. On the basis of cytological analysis five different testicular appearences were identified in azoospermic patients: (i) Sertoli cell only syndrome (SCOS); (ii) Severe hypospermatogenesis; (iii) Spermatogonial and/or spermatocytic arrest; (iv) Spermatidic arrest; (v) Normal germ line (obstructive forms). No difference in LH and testosterone levels was found among the different groups. A significant negative correlation was present between inhibin B and FSH both in azoospermic men (r = - 0.503, P < 0.0001) and normozoospermic controls (r = -0.361, P < 0.05). Groups characterized by obstructive azoospermia and spermatidic arrest showed inhibin B concentrations similar to normozoospermic subjects (130.7 +/- 73.5, 160.3 +/- 35.1 and 148.5 +/- 46.8 ng/l, respectively), while groups characterized by SCOS, severe hypospermatogenesis and spermatogonial and/or spermatocytic arrest showed mean inhibin B concentrations significantly lower than controls (56.5 +/- 56.0, 57.9 +/- 31.2; 48.9 +/- 16.7 ng/l, respectively). In the group of SCOS, 8 out of 42 subjects (19.0%) showed inhibin B concentrations within the normal range despite high FSH levels. This study demonstrated that, in humans, spermatids play an important role in the mechanism regulating inhibin B secretion by Sertoli cells. The significance of this hormone as a diagnostic parameter in azoospermic patients does not seem to be specific because it does not permit discrimination between obstructive forms or spermatidic arrest. Furthermore, despite an evident clinical, cytological and hormonal pattern for SCOS, inhibin B levels are normal in some of these patients. The significance of this latter result remains to be elucidated.

Combined Inhibin and CA125 Assays in the Detection of Ovarian Cancer

The reproductive hormone inhibin has been used as a diagnostic marker of ovarian mucinous and granulosa cell cancers. The aims of this study were to develop a new inhibin immunofluorometric assay (alphaC IFMA) to replace an inhibin RIA as a diagnostic marker of these ovarian cancers and to assess whether the alphaC IFMA in combination with CA125, which detects serous cancers, leads to an improved biochemical diagnosis of all ovarian cancers. Serum inhibin concentrations were determined in healthy postmenopausal women (n = 165) and women with ovarian cancers (n = 154), using an inhibin RIA and an alphaC IFMA, which detects inhibin forms containing the alphaC subunit as well as the free alphaC subunit. The alphaC IFMA gave a similar or better discrimination of mucinous (90% vs 71%) and granulosa cell (100% vs 100%) cancers compared with the inhibin RIA. Combination of CA125 and alphaC IFMA values by canonical variate analysis or by multiROC analysis showed that the percentage of all ovarian cancers detected was significantly increased compared with either CA125 or alphaC IFMA alone. The alphaC IFMA shows a similar or better specificity compared with the RIA, but with increased sensitivity. In combination with CA125, the alphaC IFMA provides an effective dual test for the detection of the majority (90%) of ovarian cancers.