Forms Of Inhibin Research Articles

Placental, Maternal Serum and Amniotic Fluid Molecular Weight Forms of Inhibin A and Pro-αC

Previous studies have identified the presence of unidentified small molecular weight (mol wt) forms of inhibin and the pro-αC region of the inhibin α subunit in serum from women during late pregnancy. The aim of this study was to investigate if these gestational-related changes in mol wt forms arose from changing placental production. Pooled placental extracts, derived from normal healthy singleton pregnancies in the 1st, 2nd and 3rd trimesters of pregnancy, were fractionated by a combined immunoaffinity chromatography, preparative PAGE and electroelution procedure. Inhibin A, inhibin B and the pro-αC region of the inhibin α subunit were determined in the eluted fractions by specific ELISAs, with the profiles of immunoactivity characterized in terms of molecular size and percentage recovery. Inhibin B was undetectable in all samples. Mol wt peaks of 36k, 75K and 97K for inhibin A and 29k, 55K and 97K for pro-αC were detected in placental extracts across all three trimesters. The relative abundancy of small mol wt inhibin A forms (<30K) present in the placenta increased significantly in the third trimester placenta, increasing from 0.3 per cent in the first trimesters to 6 per cent in the third trimester (P=0.01, chi-squared test). The relative abundances of various mol wt forms of pro-αC was similar at all three gestations (P=0.67). In serum, small mol wt inhibin A and pro-αC forms accounted for 23.4 per cent and 37.4 per cent of inhibins, respectively, in the third trimester.These data suggest that the presence of small mol wt forms of both inhibin A and pro-αC in maternal serum is only partially attributed to placental production and/or secretion. We conclude that inhibin A and pro-αC inhibins in maternal serum are processed in late pregnancy by more than one mechanism to form low mol wt circulating forms of, as yet, undetermined structure.

Localization and secretion of inhibins in the equine fetal ovaries.

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

Circulating Inhibin Forms in Patients with Hydatidiform Mole

Circulating inhibin forms have not been evaluated in patients with hydatidiform mole (HM) before evacuation of the mole. Serum inhibin A, inhibin B, pro-αC and hCG levels were determined by enzyme-linked immunosorbent assay in HM before and after evacuation of the mole (inhibin B only before evacuation). Before evacuation, inhibin B levels in HM were undetectable, and inhibin A levels were similar to those in normal women at the corresponding weeks of pregnancy. Pro-αC and hCG levels were higher in molar pregnancy than in normal pregnancy. After evacuation, pro-αC levels significantly fell in the patients with spontaneous resolution, but not in those who developed persistent trophoblastic disease (PTD). Both pro-αC and hCG levels were higher in the patients with PTD than in those with spontaneous resolution after evacuation, reflecting continued stimulation of the corpus luteum by lower, but still effective hCG levels, persistent trophoblastic tissues secreting pro-αC or both.

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

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

Molecular weight forms of inhibin A, inhibin B and pro-alphaC in maternal serum, amniotic fluid and placental extracts of normal and Down syndrome pregnancies.

Inhibin A, an established prenatal marker of Down syndrome (DS), exists in the maternal circulation in a number of isoforms. The present study explored whether specific inhibin A isoforms may be selectively increased in DS, offering the prospect of improved marker performance. Second trimester maternal serum, placental extracts and amniotic fluid (AF) pools from both normal and DS pregnancies were fractionated by a combined immunoaffinity (IA) chromatography, preparative polyacrylamide gel electrophoresis (Prep-PAGE) and electroelution procedure. Inhibins A, B and pro-alphaC were determined in the eluted fractions by specific enzyme-linked immunosorbent assays (ELISAs) and the profiles of immunoactivity (IA) characterized in terms of molecular weight (MW) and percentage recovery. The MW patterns of inhibin A and pro-alphaC in maternal serum and AF were similar between DS and control pregnancies, both showing peaks between 25-40 k and approximately 65 k. AF contained, in addition, a higher proportion of <30 k inhibins A and B, and <25 k pro-alphaC forms. There were large differences in the inhibin forms present in DS placentae, with more 70 k and less 30-40 k inhibin A than in controls. The present data suggest that the processing, cleavage or secretion of inhibin MW forms by the DS placenta differs from normal. However, these differences are not reflected in maternal serum and so improvements in serum screening will not be afforded by measuring specific inhibin A isoforms.

Effects of oestradiol and progesterone on secretion of gonadotrophins and health of first wave follicles during the oestrous cycle of beef heifers

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.

Effects of oestradiol and progesterone on secretion of gonadotrophins and health of first wave follicles during the oestrous cycle of beef heifers

Antral follicle development in cattle is initially FSH dependent and then LH dependent. The aim of the present study was to determine the effects of oestradiol- and progesterone-induced suppression of FSH and LH on growth and differentiation of first wave follicles. Cyclic heifers (n = 45, n = 6-10 per group) received the following i.m. injections or treatments beginning 30 h after oestrus: (i) saline (controls); (ii) 0.75 mg oestradiol benzoate (ODB); (iii) insertion of a progesterone-releasing intravaginal device (PRID) for 42 h (progesterone); (iv) 0.75 mg oestradiol benzoate plus PRID (ODB plus progesterone); (v) 0.75 mg ODB plus injection of 1 mg Ovagen(TM) at 33, 39 and 45 h after onset of oestrus (ODB plus FSH). In Expt 1, follicle development was monitored by ovarian ultrasonography once a day. In Expt 2, heifers were ovariectomized. Emergence of the first follicle wave and dominant follicle selection were delayed in ODB plus progesterone-treated heifers compared with controls. Interval to nadir FSH concentration was shorter in ODB-, progesterone- and ODB plus progesterone-treated heifers compared with controls. Frequency of LH pulses was unaffected in ODB- or ODB plus FSH-treated heifers, decreased in progesterone-treated heifers and further decreased in ODB plus progesterone-treated heifers. Intrafollicular oestradiol concentrations were lower in the largest follicle from ODB plus progesterone-treated heifers compared with control (66 h) heifers, but follicle diameter and concentrations of insulin-like growth factor binding proteins (IGFBPs) and inhibin forms were unaffected. Treatment with ODB decreased follicular oestradiol concentration in smaller follicles in the cohort. It is concluded that growing cohort follicles are uniformly responsive to increased FSH concentration but differentially responsive to suppressed FSH and LH release, which is consistent with an LH-mediated survival advantage of the largest follicle in the cohort before cessation of the growth of remaining follicles in the cohort occurs.

Properties of inhibin binding to betaglycan, InhBP/p120 and the activin type II receptors

Activin-stimulated FSH synthesis and release by the pituitary gonadotrope is antagonized by gonadally derived inhibins. The two isoforms of inhibin, inhibin A and B, bind to the activin type II receptors, though at a lower affinity than the activins, but do not stimulate intracellular signaling. Theoretically, therefore, inhibins can prevent activin signaling through competitive binding if present at higher concentrations than the activins. In reality, the inhibins have been shown to antagonize activin signaling when the two ligand types are present at equimolar concentrations. These observations led to the hypothesis that inhibin binding proteins or co-receptors exist that either increase the affinity of the inhibins for the activin receptors or propagate inhibin-specific intracellular signals. Two candidate inhibin co-receptors, betaglycan and InhBP/p120, interact with activin receptors and augment inhibin antagonism of activin action. Here, we report the effect of betaglycan and InhBP/p120 on both inhibin A and inhibin B binding to the activin receptors ActRIIA and ActRIIB2. InhBP/p120 did not bind inhibin A or B when expressed alone or in combination with activin receptors, requiring a re-examination of the role of this protein in inhibin biology. Both inhibins bound the activin type II receptor, ActRIIB2. Inhibin B had a higher affinity for this receptor than inhibin A but an approximately 10-fold lower affinity than that of activin A. Inhibin A and B bound betaglycan with high affinity; however, only inhibin A binding to ActRIIB2 was significantly enhanced in the presence of betaglycan. Both inhibin isoforms showed slight but significant binding to ActRIIA, yet this binding was potentiated in the presence of betaglycan. Additionally, the complex formed between the inhibins, ActRIIA, and betaglycan was resistant to disruption by activin A, whereas activin A potently competed for inhibin binding to ActRIIB2 and betaglycan. Collectively, these data show that the inhibin isoforms have different affinities for the activin type II receptors but bind betaglycan with high affinity. A recently developed model of inhibin action proposes that inhibins form a high affinity, activin-resistant ternary complex with activin type II receptors and betaglycan, thereby providing a mechanism for inhibin antagonism of activin signaling. Importantly, the results presented here clearly show that this model does not apply equally to both forms of inhibin nor to the different activin type II receptor isoforms. Thus, it appears that the mechanisms of inhibin action may vary depending on the ligand and receptor types involved.

Short term fluctuation in peripheral concentrations of inhibin B in fertile men

Objective: Inhibin is a glycoprotein hormone of gonadal origin which was first identified by its ability to negatively regulate FSH. The first specific assay for the measurement of serum inhibin B was available in 1996. After the first demonstration that inhibin B is the physiologically relevant inhibin form in man, this assay was used to study the changes in serum inhibin B levels in a number of pathophysiological conditions. In childhood, Sertoli cell proliferation and Follicle stimulating hormone (FSH) control inhibin activity, germ cells are a major determinant of inhibin B production in adulthood. After puberty, in the presence of FSH, the prime regulator of inhibin B levels is the spermatogenic status and inhibin B production is directly proportional to the ‘amount’ of spermatogenesis, as shown by the direct correlation between serum inhibin B and sperm count. Once full spermatogenesis is ongoing, changes in inhibin B levels reflect mainly the status of germ cell proliferation and development and depend only secondarily on FSH. Spermatogenic activity requires sufficient testosterone (T) concentration. Testosterone is produced by the Leydig cells under the influence of luteinizing hormone (LH). In the adult, serum inhibin B shows a clear diurnal variation closely related to that of testosterone.

The final stages of dominant follicle selection in cattle

The final stages of ovarian follicle growth in cattle are typically characterized by the ultrasound-detectable emergence of a cohort of small (3–5 mm in diameter) antral follicles, followed by a selection process during which the number of follicles continuing to grow decreases. Finally, only one follicle (the dominant follicle; DF) shows an enhanced growth rate and estradiol synthesis when it attains 8.5 mm compared to its closest competitor (the largest subordinate follicle; SF). Cohort emergence is caused by a transient FSH rise, while DF selection occurs during declining FSH indicating differential FSH dependence of DF and SF. In order to elucidate the mechanisms underlying DF survival or SF atresia, this review aims to (i) describe follicular changes in the local production and regulation of members of the inhibin family of proteins and the insulin-like growth factor (IGF) system in relation to FSH deprivation leading to DF selection, and (ii) develop a model for DF selection outlining the putative involvement of inhibins, activin and follistatin on the one hand, and bioavailable IGFs regulated by IGF binding proteins (IGFBPs) and IGFBP proteases on the other hand. It is concluded, that the first indications of differential FSH dependence are seen within 33 h of the FSH peak, and high amounts of precursor forms of inhibin and free activin, and low amounts of the lower molecular weight (MW) IGFBPs are related to follicle survival in terms of enhanced growth and estradiol synthesis, and suppression of granulosa cell apoptosis. In addition, maintenance of low amounts of intrafollicular IGFBP4 may constitute an important mechanism in the future DF to attain FSH independence.

Inhibins/activins as diagnostic markers for ovarian cancer

It is widely recognised that the early detection and subsequent assessment of recurrence of ovarian cancers are key steps for successful treatment. Available serum markers (e.g. CA125) are sensitive for some epithelial carcinomas (e.g. serous, endometrioid, clear cell), however, these markers are less sensitive for granulosa cell tumours and mucinous carcinomas. Serum inhibin is an ovarian product which decreases to non detectable levels after menopause, however, certain ovarian cancers (mucinous carcinomas and sex cord stromal tumours such as granulosa cell tumours) continue to produce inhibin which provides a basis for a serum diagnostic test. Studies from this and other laboratories have investigated the suitability of inhibin as a diagnostic marker by identifying which inhibin (inhibin A (αβA), inhibin B (αβB), free α subunit) or activin (βAβA) form is associated with these cancers. Available data show that inhibin assays which detect all inhibin forms, i.e. assays which detect the α subunit both as the free form and as an αβ subunit dimer provide the highest sensitivity/specificity characteristics as an ovarian cancer diagnostic test. This review will discuss the data supporting these observations and show recent studies in which a new α subunit monoclonal antibody-based ELISA is used as a potential diagnostic test. Furthermore, based on the high sensitivity/specificity characteristics of the respective assays for the various types of ovarian cancer, the combination of the inhibin assay with CA125 detects the majority of all ovarian cancers.

Characterization of Inhibin Forms and Their Measurement by an Inhibin α-Subunit ELISA in Serum from Postmenopausal Women with Ovarian Cancer

The aim of this study was to characterize the molecular wt forms of inhibins A and B and its free alpha-subunit present in serum from women with ovarian cancer as a basis for developing improved monoclonal antibody-based inhibin assays for monitoring ovarian cancer. Three new inhibin alpha-subunit (alphaC) ELISAs were developed using monoclonal antibodies directed to three nonoverlapping peptide regions of the alphaC region of the inhibin alpha-subunit. To characterize serum inhibin molecular wt forms present in women with ovarian cancer, existing inhibin immunoassays (inhibin A, inhibin B, and pro-alphaC) and the new alphaC ELISAs were applied to sera from women with granulosa cell tumors and mucinous carcinomas previously fractionated using a combined immunoaffinity chromatography, preparative SDS-PAGE, and electroelution procedure. The distribution and molecular size of dimeric inhibins and alpha-subunit detected were consistent with known mol wt forms of inhibins A and B and inhibin alpha-subunit and their precursor forms present in serum and follicular fluid from healthy women. The alphaC ELISAs recognized all known forms of inhibin and the free inhibin alpha-subunit, although differences between alphaC ELISAs were observed in their ability to detect high mol wt forms. To assess which of the alphaC ELISAs was preferred in application to ovarian cancer, the alphaC ELISAs were applied to serum from a range of normal postmenopausal women (n = 61) and postmenopausal women (n = 152) with ovarian (serous, mucinous, endometrioid, clear cell carcinomas, and granulosa cell tumors) and nonovarian (breast and colon) cancers. Despite differences in their ability to detect high mol wt forms of inhibin, the alphaC ELISAs showed similar sensitivity (i.e. proportion of cancer patients correctly detected) and specificity (proportion of controls correctly detected) indexes in the detection of mucinous carcinomas (84% and 95%) and granulosa cell tumors (100% and 95%) compared with earlier inhibin RIA or polyclonal antibody-based immunofluorometric assays. A combination of the alphaC ELISAs with the CA125 assay, an ovarian tumor marker that has a high sensitivity and specificity for other ovarian cancers (serous, clear cell, and endometrioid), resulted in an increase in sensitivity/specificity indexes (95% and 95%) for the all ovarian cancer group. These new monoclonal antibody-based inhibin alphaC ELISAs now provide practical and sensitive assays suitable for evaluation as diagnostic tests for monitoring ovarian cancers.

Changes in molecular weight forms of inhibin A and pro-alpha C in maternal serum during human pregnancy.

Maternal serum pools obtained from healthy women throughout normal pregnancy were fractionated by a combined immunoaffinity chromatography, preparative PAGE, and electroelution procedure. Inhibin A and the pro-alpha C region of the inhibin alpha-subunit were determined in the eluted fractions by specific ELISAs, and the profiles of immunoactivity characterized in terms of molecular weight and percent recovery. The molecular weight patterns of inhibin A and pro-alpha C in serum during early pregnancy (<19 wk gestation) showed peaks between 25-40K and approximately 60K, consistent with the presence of known mature and larger precursor inhibin forms. However, during late pregnancy (>19 wk gestation), an increase in the proportion of smaller molecular weight forms (from 2% to approximately 25%) of inhibin A and pro-alpha C of unknown structure were observed in the less than 30K and less than 25K regions, respectively. To assess whether this change in molecular weight distribution in late pregnancy was related to the method of serum collection, serum and plasma from women during early and late pregnancy were collected and snap-frozen. Three pools [one from early pregnancy (12-15 wk), two from late pregnancy (28-39 wk)] of serum and plasma were then fractionated as described above. No differences in molecular weight patterns of inhibin A and pro-alpha C were observed between serum and plasma pools obtained in early pregnancy. However, in late pregnancy there was a reduction in the proportion of low molecular weight forms between serum (25% inhibin A, 35% pro-alpha C) and plasma (12% and 17%, respectively), but not to the low levels seen in early pregnancy. Incubation of iodinated 30K human inhibin A with serum or plasma obtained from early or late pregnancy showed no evidence of cleavage, suggesting that 30K inhibin A is not the cleavage precursor. It is speculated that the formation of small molecular weight forms of both inhibin A and pro-alpha C is attributed to proteolytic changes, in part induced in the circulation during late gestation and in part by the placenta before secretion. It is concluded that inhibin A and pro-alpha C are processed in late pregnancy by more than one mechanism to form low molecular weight circulating forms of unknown structure.

Regulation of follicle-stimulating hormone secretion by estradiol and dimeric inhibins in the infantile female rat.

Plasma and ovarian levels of the dimeric forms of inhibin and plasma estradiol-17beta were investigated and compared with changes in plasma gonadotropins from Postnatal Day (PND) 5 to PND 30 in the female rat. The inhibin subunit proteins were localized in follicular granulosa cells of the ovary. Plasma immunoreactive inhibin levels were low until PND 15 and increased thereafter. Plasma levels of inhibin B (alpha and beta(B) subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A (alpha and beta(A) subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = -0.67, P < 0.0002), ovarian inhibin A (r = -0.48, P < 0.01), plasma inhibin B (r = -0.48, P < 0.05), and ovarian inhibin B (r = -0.54, P < 0.01). Plasma estradiol-17beta levels were elevated from PND 5 through PND 15, then fell sharply through PND 30. Plasma estradiol-17beta was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin alpha, beta(A), and beta(B) subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17beta likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.

Are serum inhibin concentrations new markers of placental tumours in the course of chemotherapy?

The study was conducted to evaluate whether the detection of serum molecular forms of inhibin (A and B) could be useful for the diagnosis, prognosis and follow-up of placental tumours. A total of 17 patients with hydatidiform mole (n = 13), invasive mole (n = 1) or choriocarcinoma (n = 3) were studied; serum concentrations of inhibins A and B, human chorionic gonadotrophin (HCG) and its free beta subunit (HCGbeta) were measured before chemotherapy (after mole evacuation for eight patients) and also during the course of chemotherapy (for 10 patients). After evacuation or before chemotherapy for refractory disease, serum inhibin A and B concentrations were found to be increased in 10/17 and 4/17 patients, when HCG and HCGbeta were high in all patients. In 10 patients with a follow-up during treatment, nine had a high concentration of inhibin A which correlated with those of HCG and HCGbeta. Normalization of inhibin A was faster than that of HCG and HCGbeta for three and six patients respectively. There was no correlation between changes of inhibin B and HCGbeta concentrations. Our results suggest that inhibins A and B are not useful markers and that HCG determination still remains the most useful marker for diagnosis and follow-up of placental tumours.

Inhibin B in male reproduction: pathophysiology and clinical relevance.

The recent availability of specific inhibin assays has demonstrated that inhibin B is the relevant circulating inhibin form in the human male. Inhibin B is a dimer of an alpha and a betaB subunit. It is produced exclusively by the testis, predominantly by the Sertoli cells in the prepubertal testis, while the site of production in the adult is still controversial. Inhibin B controls FSH secretion via a negative feedback mechanism. In the adult, inhibin B production depends both on FSH and on spermatogenic status, but it is not known in which way germ cells contribute to inhibin B production. The regulation of inhibin B production changes during life. There is an inhibin B peak in serum shortly after birth only partly correlated with an increase in serum FSH, probably reflecting the proliferating activity of the Sertoli cells during this phase of life. Afterwards, inhibin B levels decrease and remain low until puberty, when they rise again, first as a consequence of FSH stimulation and then as a result of the combined regulation by FSH and the ongoing spermatogenesis. In the adult, serum inhibin B shows a clear diurnal variation closely related to that of testosterone. The administration of FSH increases the secretion of inhibin B in normal men, but is much more pronounced in males with secondary hypogonadism. The treatment of infertile men with FSH, however, does not result in an unequivocal inhibin B increase. There is a clear inverse relationship between serum inhibin B and FSH in the adult. Serum inhibin B levels are strongly positively correlated with testicular volume and sperm counts. In infertile patients, inhibin B decreases and FSH increases. In general, there is very good correlation with the degree of spermatogenetic damage, with the arrest at the earlier stages having the lowest inhibin B levels. However, for unknown reasons, there are cases of Sertoli-cell-only syndrome with normal inhibin B levels. Inhibin B and FSH together are a more sensitive and specific marker for spermatogenesis than either one alone. However, the inhibin B concentrations are not a reliable predictor of the presence of sperm in biopsy samples for testicular sperm extraction. Suppression of spermatogenesis with testosterone and gestagens leads to a partial reduction of inhibin B in serum but it is never completely suppressed. In contrast, testicular irradiation in monkeys or humans leads to a rapid and dramatic decrease of inhibin B, which becomes undetectable when germ cells are completely absent. In summary, although inhibin B is a valuable index of spermatogenesis, the measurement of serum inhibin B levels is still of limited clinical relevance for individual patients.

Serum Inhibin B als ein Marker der Spermatogenese

Inhibin B wird von Sertolizellen produziert und reguliert die FSH-Sekretion über einen negativen Feedback. Beim Mann scheint Inhibin B die physiologisch bedeutsame Form zu sein. Wir bestimmten Serum-Inhibin B unter Verwendung eines zwei-Seiten-immunoenzymatischen Assays bei 40 Normalpersonen (Alter: 27,37 ± 0,83 Jahre, Mittelwert ± Standardfehler) mit einer Spermienkonzentration von 100,0 ± 9,2 × 106/ml, bei 51 subfertilen Patienten (30,86 ± 0,94 Jahre) mit einer Spermienkonzentration von 6,8 ± 0,8 × 106/ml, bei Männern mit Varikozele (n = 16) mit einer Spermienkonzentration von 54,3 ± 17,1 × 106/ml und einem Alter von 30,69 ± 1,52 Jahren, sowie von Männern mit Kallmann-Syndrom, Klinefelter-Syndrom, obstruktiver und nicht obstruktiver Azoospermie. Wir verglichen Serum-Inhibin-B-Konzentrationen mit Serum FSH- und Spermienkonzentration. Bei Männern mit normaler Spermienkonzentration (> 20 × 106/ml) betrugen die Konzentrationen von Inhibin B im Serum 201,5 ± 17,1pg/ml und von FSH 4,1±0,5 IU/l. Die Patienten mit Varikozele zeigen ebenfalls eine Spermienkonzentration > 20 × 106/ml und liegen mit ihren Serum-Inhibin-B-Werten im Bereich von 173,7 ± 21,3pg/ml; die FSH-Werte liegen bei 4,6 ± 0,6 IU/l. Bei Patienten mit Spermienkonzentrationen < 20 × 106/ml betrug die Inhibin-B-Konzentration 118,2 ± 14,8 pg/ml und die FSH-Konzentration lag bei 10,1 ± 1,1 IU/l. Bei allen Patienten, außer jenen mit hypogonadotropem Hypogonadismus und Klinefelter-Syndrom, war die Korrelation zwischen Inhibin B und FSH invers (r = - 0,41, P > 0,01). Die Spermienzahl korrelierte positiv mit den Serum-Inhibin-B-Konzentrationen (r = 0,340, P < 0,01). Eine Korrelation von r = 0,574, P < 0,05 ergab sich bei der Gruppe mit Varikozele. Aus den Untersuchungen lässt sich schlussfolgern, dass Inhibin B ein Marker der exokrinen testikulären Funktion ist und eine sehr wesentliche Hilfe zur Diagnosestellung testikulärer Funktionsstörungen darstellt.

Development of an inhibin α subunit ELISA with broad specificity

Inhibin immunoassays with a sufficiently broad specificity to detect all α subunit-containing forms are of value in detecting and monitoring various ovarian cancers. Assays to date with this specificity are not readily amenable to wide diagnostic application. The objective of this study was to develop a sensitive two-site ELISA using α subunit-directed monoclonal antibodies (Mabs) able to detect all forms of inhibin to replace a previously described α subunit-directed immunofluorometric assay (IFMA). In this study, the major inhibin epitopes in the two polyclonal antisera used in the αC IFMA were initially identified and Mabs were raised to these regions. These Mabs in conjunction with the inhibin α subunit R1 Mab (Groome) were used to develop α subunit ELISAs with high sensitivity. Application of these assays to human serum and human follicular fluid following fractionation by an immunoaffinity/preparative PAGE/electroelution procedure which separated inhibins according to their molecular weights, indicated that the specificity of the various ELISAs differed between Mab combinations with preferences noted for either the α subunit or dimeric forms. A combination of Mabs in an ELISA was identified which provided data which matched that obtained with the αC IFMA and which may be useful as a replacement inhibin assay in clinical studies.

Serum activin A and follistatin in disorders of spermatogenesis in men.

Inhibin, activin and follistatin are glycoprotein hormones produced by the gonads. Recent studies have shown that inhibin B is the predominant form of inhibin in the circulation in men. The objective of this study was to investigate circulating levels of activin A and follistatin in disorders of spermatogenesis in men and their relationship with FSH and inhibin B. Serum from five different groups of men was prospectively collected and stored at -20 degrees C. The groups were men with: (i) proven fertility (controls) (n=20), (ii) primary testicular failure (n=15), (iii) obstructive azoospermia (n=10), (iv) oligospermia (n=10) and (v) miscellaneous sperm dysfunction (n=40). WHO criteria (1992) were used for semen characterisation. Serum concentrations of 'total' activin A, follistatin, FSH and inhibin B were measured using specific two-site enzyme immunoassays. Activin A levels were significantly lower than in the controls in the obstructive azoospermia group and higher in the miscellaneous sperm dysfunction group. Serum follistatin levels did not significantly vary in any group compared with the controls. Circulating levels of FSH were higher than in the controls in the primary testicular failure and obstructive azoospermic group. Levels of inhibin B were lower than in the controls in all disorders of spermatogenesis studied. This study demonstrates that activin A and follistatin are in the circulation in males and activin A levels are significantly lower in obstructive azoospermia and higher in miscellaneous sperm dysfunction than in controls. The mechanism involved in altering the levels of activin A in these conditions is not clear. However, high follistatin:activin A molar ratios (>2.5) in all groups suggests that all activin A in the circulation is bound to follistatin in males.

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, Mullerian 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, Mullerian 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 (Mullerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Mullerian 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.