The biological and immunological characterization of inhibin A and B forms in human follicular fluid and plasma

In a previous study (see Ref. 7), the molecular weight distribution of inhibin activity in fractionated human follicular fluid (hFF) and human male and female plasma/serum was determined by in vitro bioassay using ovine pituitary cells in culture and various specific inhibin A and inhibin alpha-subunit-directed immunoassays. It was shown, however, that the ovine in vitro bioassay detected inhibin B poorly. These findings are extended in the present study by the determination of the molecular weight profile of in vitro bioactivity using rat pituitary cells, which detects both inhibin A and B, a specific inhibin B enzyme-linked immunosorbent assay (ELISA), an RLA detecting the alpha N region of the alpha-subunit, an alpha-subunit ELISA (Pro-alpha C) directed to the inhibin forms containing the Pro sequence, and an alpha C subunit immunofluorometric assay that detects all inhibin forms. The profile in hFF of inhibin in vitro bioactivity, using rat pituitary cells in culture, significantly (P < 0.001) correlated with in vitro bioactivity using ovine pituitary cells (r = 0.85), inhibin A immunoactivity (r = 0.70), inhibin B immunoactivity (r = 0.89), and the combination of inhibin A+B immunoactivities (r = 0.93), with peaks of activity identified at 66K, 55K, 36K and 33K, consistent with presumed known mol mt forms of inhibin. Inhibin B profiles in fractionated serum from women stimulated with gonadotropins and male plasma consisted of two forms (66K and 36K), whereas inhibin A in female serum included, in addition, the 55K form. These findings indicated that higher molecular weight forms of inhibin B are present in biological samples, and their distribution differs from that of inhibin A, suggesting a differential processing of the precursor forms in the circulation. Pro-alpha C immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-alpha C subunit forms) and not with any high mol wt dimeric forms of inhibin. If this observation applies to a wider range of serum samples, the Pro-alpha C ELISA may provide an appropriate and specific assay for the measurement of free alpha-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-alpha C standards were calibrated in terms of their alpha C subunit content, as determined by an alpha C subunit immunoassay, with the inhibin B standard containing 60% of the alpha C subunit content compared with either the inhibin A or Pro-alpha C standard. After adjustments of the various standards for this difference in alpha C subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-alpha C immunoactivity across the hFF and serum chromatograms and compared with levels determined by the alpha-subunit-directed immunoassays. A high correlation (r = 0.59-0.96) was observed, indicating that the alpha-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-alpha C. It is concluded that: 1) inhibin in vitro bioactivity in hFF is largely attributed to the presence of 33-36K and 50-66K forms of inhibins A and B; and 2) inhibin alpha-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the alpha-subunit.