Endocrinology, Vol 129, 1987-1999, Copyright © 1991 by Endocrine Society

ARTICLES

Precursors of alpha-inhibin modulate follicle-stimulating hormone receptor binding and biological activity

AL Schneyer, PM Sluss, RW Whitcomb, KA Martin, R Sprengel and WF Crowley Jr
Department of Medicine, Massachusetts General Hospital, Boston 02114.

Although several forms of monomeric alpha-inhibin have been isolated from follicular fluid, no biological function has yet been ascribed to these posttranslationally processed forms of the alpha-subunit precursor protein. Moreover, previous studies of a FSH receptor binding competitor (FRBC) isolated and characterized from porcine follicular fluid (pFF) suggested certain biochemical similarities between this protein and alpha-inhibin precursors. We, therefore, investigated the hypothesis that alpha-inhibin and/or its precursors might represent autocrine and/or paracrine modulators of FSH action in the ovary, accounting for some of this FRBC activity and thereby exerting some degree of regulation over follicular maturation. Three separate sources of alpha-inhibin proteins were investigated for FRBC activity, including pFF, human FF (hFF), and a 293 cell line into which the full- length human alpha-inhibin cDNA had been stably transfected. Conditioned medium from these transfected cells contained several forms of alpha-inhibin precursors as well as mature alpha-inhibin, but no beta-subunit or intact inhibin. alpha-Inhibin proteins from all three sources, purified by a variety of methods, including immunoaffinity chromatography on an anti-alpha-inhibin column, inhibited FSH binding to both natural tissue FSH receptors as well as recombinant rat FSH receptors expressed in 293 cells. Furthermore, dimeric inhibin and activin, medium from untransfected 293 cells, and non-alpha-inhibin- containing purification fractions were inactive in either assay. In addition, purified recombinant alpha-inhibin proteins were partial in vitro FSH antagonists in a bioassay in which cAMP generation from 293 cells expressing the recombinant FSH receptor is used as an index of FSH biological activity. These same fractions of hFF containing FRBC activity did not bind to LH receptors, thereby demonstrating receptor specificity for this activity. Using sodium dodecyl sulfate- polyacrylamide gel electrophoresis and Western blotting with alpha- inhibin or FRBC antisera, a 57,000 mol wt protein was identified in FRBC-active fractions from all three sources, suggesting that the active moiety was the full-length alpha-inhibin precursor protein or a large mol wt fragment, but not mature alpha-inhibin. Lastly, all FRBC activity from all three sources was extracted by an alpha-inhibin immunoaffinity column and was recoverable upon elution. These results demonstrate that proteins derived from the alpha-inhibin precursor modulate FSH binding to its receptor as well as its biological activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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