Inhibin in immature rat Sertoli cell conditioned medium: a 32 kDa CY./?-B dimer

Conditioned medium of cultured Sertoli cells from 21-day-old rats was used as starting material for the isolation of inhibin. Inhibin activity was monitored by the dose dependent suppression of the folliclestimulating hormone release of cultured rat pituitary cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the highly purified inhibin preparation revealed a 32 kDa protein after silver staining, which could be separated in subunits of 18 kDa and 12 kDa after reduction. Western blot analysis with an antibody recognizing the 22 N-terminal amino acids of the a-subunit of 32 kDa bovine inhibin confirmed the presence of a 32 kDa inhibin molecule under non-reducing conditions, whereas an 18 kDa cY-subunit was found after reduction. An antibody recognizing the /3-A subunit of inhibin did not yield a signal after Western blotting. N-terminal amino acid sequence analysis of two highly purified preparations of inhibin obtained using different methods yielded the sequence predicted for a 32 kDa @-B dimer on basis of cDNA nucleotide sequence. This result is in agreement with the large excess of P-B over /3-A mRNA in the rat testis.