Abstract

In order to identify the molecular weight forms of bioactive and immunoactive inhibin in human plasma, plasma/serum was sequentially fractionated by immunoaffinity chromatography (using immobilised inhibin α subunit antiserum), reversed phase HPLC and preparative SDS-PAGE. The electroeluted gel fractions were assayed for inhibin in vitro bioactivity and immunoactivity, the latter by RIA. Initial experiments examined human follicular fluid as an inhibin-rich source. Bioactive and immunoactive fractions of 30, 35, 53, 65 and ∼120 kDa were identified in addition to bio-inactive, immunoactive fractions of 26 kDa and 32 kDa. These molecular weights correspond to those of known inhibin forms and are attributed to differing degrees of glycosylation of the inhibin α subunit and variable processing of the α and β inhibin subunits.

Fractionation of male plasma pools revealed the presence of higher molecular weight immunoactive forms (55–120 kDa) as well as 28–31 kDa forms although the molecular weight distribution of activity between pools varied. To assess if the molecular weight pattern was modified by storage and/or subsequent fractionation, protease inhibitors were added initially to plasma and fractionated as above. The molecular weight distribution of immunoactivity was largely unaffected by the treatment, indicating that minimal processing had occurred. Postmenopausal serum itself showed low to undetectable activity. The addition of recombinant human 31 kDa inhibin to postmenopausal serum resulted in a molecular weight profile of inhibin immunoactivity consistent with the presence of 31 kDa inhibin. Fractionation of a serum pool from women undergoing gonadotrophin stimulation, in which inhibin levels were elevated, showed a range of bioactive and immunoactive inhibin forms over the 30–120 kDa range. A good correspondence between activities was observed.

It is concluded that: 1. inhibin exists in plasma/serum as a range (28–120 kDa) of molecular weight forms. 2. In female serum, the majority of inhibin isoforms appear to be bioactive. 3. This fractionation procedure provides a basis for investigating the forms of inhibin in plasma and provides a means of assessing the specificity of new assay methods.

Journal of Endocrinology (1995) 144, 261–269

ABSTRACT

The mode of action of a recently isolated gonadal protein, termed FSH-suppressing protein (FSP) or follistatin, on basal and gonadotrophin-releasing hormone (GnRH)-stimulated release of FSH and LH and on pituitary cell content of FSH and LH was examined in rat pituitary cell cultures and compared with the previously reported effects of inhibin. Pituitary cells were cultured for 3–9 days in the presence of graded doses of FSP and the basal release rates and changes in cell contents of FSH and LH determined during this period. FSP suppressed both the basal release rate and the cell content of FSH with median inhibitory concentrations (IC₅₀) of 135 and 161 pmol/l respectively. The corresponding effects of FSP on LH basal release rate and LH cell content (IC₅₀ = 200 pmol/l) were limited compared with the effects on FSH. The effect of FSP on GnRH-stimulated release of FSH and LH during 4 h was determined in cells which had been preincubated with FSP for 3 days, and the GnRH-stimulated release of FSH and LH analysed as a percentage of the respective gonadotrophin available for release. FSP antagonized GnRH action with dose-related increases in the GnRH median effective (stimulatory) concentrations for FSH and LH release (EC₅₀ values = 56 and 400 pmol/l respectively) and a suppression in the maximum release of FSH and LH by excess GnRH (IC₅₀ values =142 and 150 pmol/l respectively). The effect of FSP on FSH cell content after 3 days in culture was insensitive to the neutralizing effects of an inhibin antiserum. The pattern of FSP-induced suppression of FSH basal release and cell content and its inhibition of GnRH-stimulated release of FSH and LH was similar to that observed with inhibin, although FSP is 10–20% as potent as inhibin. When inhibin and FSP were assayed either individually or as a 1:1 (v/v) mixture, the experimentally determined biological activity of the mixture did not differ significantly from that predicted from the individual activities.

It was concluded from the similarity in pituitary responses to FSH and inhibin that FSP is a weak inhibin agonist. Its action can be discriminated from that of inhibin by an inhibin antiserum; however, FSP and inhibin may share a common underlying mechanism of action since their effects are additive.

Journal of Endocrinology (1990) 124, 417–423