PREMEIOTIC AND POSTMEIOTIC EXPRESSION OF MALE GERM CELL-SPECIFIC GENES THROUGHOUT 2-WEEK COCULTURES OF RAT GERMINAL AND SERTOLI CELLS

The present study was aimed at examining, by reverse transcription pol ymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) o r round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2 B were specifically expressed in PS whereas those encoding the transit ion proteins TP1 and TP2 were specific to RS. This resulted in p19:TP1 and TH2B:TP2 ratios that were much higher in PS fractions than in RS fractions prepared by elutriation. When PS or RS were seeded on Sertol i cell monolayers in bicameral chambers, both the number and the viabi lity of the cells decreased during the coculture. However, both parame ters were equal to, or higher than, 60% after 2 wk. In PS-Sertoli cell cocultures, the ratios of p19:TP1 and TH2B:TP2 decreased dramatically during the second week of culture; this was due not only to a decreas e in the levels of p19 and TH2B mRNAs but also to an enhancement in th e relative amounts of TP1 and TP2 as compared to the amounts present o n the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the leve ls of the four mRNAs studied during the coculture period. DNA flow cyt ometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decr ease in the tetraploid cell population (4C). No such changes were obse rved in Sertoli cell-only cultures. By contrast, the haploid populatio n decreased 3-fold during the first week in RS-Sertoli cell cocultures . Immunocytochemical studies demonstrated further that 5-bromo-2'-deox yuridine-labeled PS of stages V-VIII were able to differentiate into R S under the present coculture conditions. Hence, although clearly impe rfect, the present coculture system should help to clarify the local r egulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.