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Biology of Reproduction, Vol 46, 155-161, Copyright © 1992 by Society for the Study of Reproduction


ARTICLES

Inhibin and inhibin alpha-chain precursors are produced by immature rat Sertoli cells in culture

AD Hancock, DM Robertson and DM de Kretser
Department of Anatomy, Monash University, Clayton, Victoria, Australia.

Stimulation of Sertoli cells from immature rats with dibutyryl cyclic (dbc) AMP resulted in a decrease in the ratio of inhibin biological (B):immunological (I) activities in vitro. To establish the basis for this decrease, culture medium from Sertoli cells stimulated with dbcAMP was fractionated by dye-affinity chromatography, reverse-phase HPLC, and preparative PAGE. Two peaks of inhibin activity were identified: a predominantly bioactive 29-kDa peak I material (B:I ratio = 5.0) and a bio-inactive, immunoactive 27-kDa peak II material (B:I ratio = 0.1). Evidence of a subunit structure was established by iodination and immunopurification using an inhibin alpha-subunit antiserum. On reduction, peak I (29-kDa) material showed bands of 19 kDa and 14 kDa, whereas peak II (27-kDa) material showed a single 20-kDa band. On the basis of HPLC retention position, molecular mass, evidence of subunit structures and their molecular masses, and inhibin in vitro bio- and immunoactivities, peak I and II materials were most likely inhibin and the alpha-subunit precursor protein pro-alpha C. Western blotting of Sertoli cell culture medium using antiserum directed against the NH2 terminal region (alpha N) of the alpha-subunit precursor also indicated the presence of 24-kDa alpha N. It is concluded that after dbcAMP stimulation, Sertoli cells produce 29-kDa inhibin and the alpha-subunit precursor proteins pro-alpha C and alpha N. The production of the alpha- subunit precursor in addition to inhibin provides an explanation for the decrease in the inhibin B:I ratio following dbcAMP stimulation of Sertoli cells in culture.


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