A 29,000 M(R) PROTEIN DERIVED FROM ROUND SPERMATIDS REGULATES SERTOLI-CELL SECRETION

Within the last decade it has become accepted that germ cells can modulate Sertoli cell function in a paracrine interactive manner during the regulation of spermatogenesis. In this context, we undertook to identify a specific factor in round spermatid conditioned media that could stimulate Sertoli cell secretory function. Rat round spermatids isolated by centrifugal elutriation were cultured and the concentrated conditioned media were fractionated by Sephacryl S-200 gel filtration column chromatography. The biological activity of the fractionated round spermatid protein was assessed as stimulation of total protein and transferrin secretion from Sertoli cells that had been isolated from 18-day-old immature rat testes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the gel-filtration fractions showed two predominant proteins of 29,000 and 24,500 molecular weight which coexisted in the fractions containing the greatest biological activity. These two proteins were transferred to a nitrocellulose membrane and excised to raise polyclonal antibodies. Western blot analysis of the 29,000 M(r) protein demonstrated that it specifically occurred in round spermatid conditioned media, whereas no immunoreactive band was observed in either the conditioned media or cell lysates of other testicular cell types such as primary spermatocyes, Sertoli cells and peritubular myoid cells. Following subcellular fractionation of round spermatids by differential centrifugation, the 29,000 M(r) protein was detected by Western blots specifically in the cytosolic fraction of round spermatids, and was absent from the nuclear, mitochondrial, lysosomal and microsomal fractions. The antibody did recognize a few higher molecular bands in the cytosolic fraction which may represent precursor forms of the 29,000 M(r) protein. This suggests that the 29,000 M(r) protein in its mature form is secreted into the culture medium. Immunoprecipitation of the 29,000 M(r) protein from intact round spermatid protein, or from a gel filtration fraction of round spermatid protein which was highly enriched for the 29,000 M(r) protein, significantly reduced the stimulatory activity of these round spermatid protein preparations on Sertoli cell total protein and transferrin secretion. Sequence analysis of 32 amino acid residues from the NH2-terminus of the 29,000 M(r) protein indicates that this protein is not homologous with any known protein sequence or nucleotide sequence accessible from data banks. These results indicate that a potentially novel 29,000 M(r) round spermatid protein stimulates Sertoli cell total protein and transferrin secretion, and that the 29,000 M(r) round spermatid protein specifically localizes to round spermatids.