A recently described two-site enzyme immunoassay incorporating a pre-assay oxidation step was validated and used to measure serum concentrations of dimeric inhibin in five normally cycling women and in 13 women undergoing gonadotrophin therapy. Recombinant human inhibin A (standard) gave an assay response curve which was parallel to those for human serum samples and recovery of exogenous inhibin added to serum samples before assay was quantitative (109 +/- 8%, n = 11). During the normal menstrual cycle dimeric inhibin concentration increased from 9.0 +/- 2.0 pg/ml during the early follicular phase to reach a mid-cycle peak of 55.3 +/- 11.1 pg/ml coincident with the pre-ovulatory gonadotrophin surge. After falling to 27.9 +/- 5.7 pg/ml 1 day after the luteinizing hormone surge, inhibin then rose in parallel with serum progesterone to reach a peak value of 115.6 +/- 19.3 pg/ml during the mid-luteal phase, before falling to 14.1 +/- 4.9 pg/ml by the onset of next menses. During the follicular phase, dimeric inhibin concentrations were closely correlated with those of serum oestradiol (r = 0.69; P < 0.001), whereas during the luteal phase they were most closely correlated with serum progesterone concentrations (r = 0.73; P < 0.001). Daily treatment with human menopausal gonadotrophin promoted a progressive increase in serum dimeric inhibin concentration which increased approximately 20-fold in 6 days. In the same period total alpha-inhibin (measured by radioimmunoassay) increased approximately 5-fold, while serum oestradiol increased approximately 30-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

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Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified Mr 32,000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated. As in rats, both crude and highly purified inhibin preparations promoted a dose (P less than 0.001)- and time (P less than 0.001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P less than 0.001) increased by the presence of either bFF (+75%) or highly-purified inhibin (+64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 microliters specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17 beta (1 pmol/1-10 nmol/l) nor monomeric alpha-subunit of bovine inhibin (2.5-40 ng/ml) significantly affected basal or GnRH-induced release of LH. These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat.

The main objective of this study was to determine whether activin A concentrations in peripheral blood fluctuate during the normal human menstrual cycle and pregnancy. Blood samples were collected longitudinally from five regularly cycling volunteers (22-30 yr) throughout a spontaneous menstrual cycle and cross-sectionally from normal pregnant women attending the antenatal clinic (8-38 weeks gestation: 3-20 subjects/time point). Total (i.e. bound plus free) activin A concentrations were measured using a recently developed two-site enzyme immunoassay that employs an analyte denaturation/oxidation step to eliminate interference due to endogenous activin-binding proteins. During the menstrual cycle, mean serum activin A levels varied in a biphasic manner (by ANOVA, P = 0.02), with highest levels around midcycle (approximately 220 pg/mL) and the late luteal/early follicular phase (approximately 310 pg/mL) and nadirs in both midfollicular (approximately 125 pg/mL) and midluteal (approximately 120 pg/mL) phases. Between the mid- to late luteal phase, the activin A level increased progressively (approximately 2.5-fold; P < 0.05), whereas inhibin A, estradiol, and progesterone all decreased progressively (approximately 10-fold; P < 0.001). During pregnancy, serum activin A levels were much higher than those in nonpregnant subjects, with a value of 2.12 +/- 0.31 ng/mL recorded in week 8. Levels remained at approximately 2 ng/mL between weeks 8-24, but increased thereafter to reach 25.5 +/- 6 ng/mL by week 38, a value approximately 100 times greater than that during the normal menstrual cycle. Serum activin A levels during pregnancy were significantly correlated with inhibin A (r = 0.69; P < 0.001), estradiol (r = 0.55; P < 0.001), and progesterone (r = 0.74; P < 0.001) values. Gel permeation chromatography indicated that all of the detectable activin A in human follicular fluid, pregnancy serum, and term placental extract eluted with an apparent molecular mass between 70-200 kDa, indicating that little, if any, free activin (molecular mass, 25 kDa) is present in these samples. Although these results support a possible endocrine role for circulating activin A during the human menstrual cycle and pregnancy, the observation that all detectable activin A is associated with binding protein(s) raises questions about its relative bioavailability for action on peripheral target cells.

The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte-cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity. Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF). Oocyte-cumulus culture media were collected after in-vitro insemination. The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium. Hormone concentrations were compared with oocyte quality and fertilizing capacity. The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size. Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality. Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes. There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity. This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation. Higher concentrations of activin A produced by good quality oocyte-cumulus complexes suggest that activin A may play a role in oocyte maturation.