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

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNFalpha) in vitro by bioassay and for the presence of TNFalpha mRNA by Northern blot analysis. Small quantities of bioactive TNFalpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNFalpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through 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 hybridization for the TNFalpha message by using digoxigenin label antisense TNFalpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNFalpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNFalpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.