EXPRESSION OF TUMOR-NECROSIS-FACTOR-ALPHA IN MOUSE SPERMATOGENIC CELLS

Enriched fractions of spermatogenic cells were isolated by unit gravit y sedimentation and analyzed both for the presence of secreted tumor n ecrosis factor-alpha (TNFalpha) in vitro by bioassay and for the prese nce of TNFalpha mRNA by Northern blot analysis. Small quantities of bi oactive TNFalpha were consistently detected in medium conditioned by r ound spermatid fractions. Both pachytene spermatocyte and round sperma tid fractions contained RNA that hybridized with murine cDNA probes fo r TNFalpha, with pachytene spermatocytes containing a normal 1.9-kilob ase (kb) transcript, while round spermatids contained principally an a pproximately 2.8-kb transcript. Both the normal size transcript and th e larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed throu gh an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids-appeared uninduced by LPS treatment. In situ h ybridization for the TNFalpha message by using digoxigenin label antis ense TNFalpha riboprobe labeled pachytene spermatocytes, round spermat ids, and presumptive interstitial macrophages. Spermatogonia and elong ating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis r esulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes o r round spermatids, hybridized with human TNFalpha receptor p60 probe in Northern blot analysis. These results are consistent with the worki ng hypothesis that spermatids release TNFalpha, which is detected by S ertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.