Abstract
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 α-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 RIA detecting the αN region of the α-subunit, anα -subunit ELISA (Pro-αC) directed to the inhibin forms containing the Pro sequence, and an αC 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 wt 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-αC immunoactivity was identified in serum samples with prominent peaks at 36K and 29K (known Pro-αC 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-αC ELISA may provide an appropriate and specific assay for the measurement of free α-subunit. To compare immunoactivity levels between assays, the inhibins A, B, and Pro-αC standards were calibrated in terms of their αC subunit content, as determined by anα C subunit immunoassay, with the inhibin B standard containing 60% of the αC subunit content compared with either the inhibin A or Pro-αC standard. After adjustments of the various standards for this difference in αC subunit content, a comparison was undertaken of the combined levels of inhibins A, B, and Pro-αC immunoactivity across the hFF and serum chromatograms and compared with levels determined by the α-subunit-directed immunoassays. A high correlation (r = 0.59–0.96) was observed, indicating that the α-subunit immunoactivity in serum consists largely of a composite of presumed known molecular weight forms of inhibins A, B, and Pro-αC. 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 α-subunit immunoactivity in hFF and serum is a composite of presumed known forms of inhibin A, inhibin B, and the α-subunit.
Production and Characterization of Specific Anti-peptide Antiserum Against Free α-subunit of Rat Pituitary Glycoprotein Hormones