INHIBIN AND INHIBIN ALPHA-CHAIN PRECURSORS ARE PRODUCED BY IMMATURE RAT SERTOLI CELLS IN CULTURE

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.