The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2B were specifically expressed in PS whereas those encoding the transition 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 Sertoli cell monolayers in bicameral chambers, both the number and the viability of the cells decreased during the coculture. However, both parameters 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 decrease in the levels of p19 and TH2B mRNAs but also to an enhancement in the relative amounts of TP1 and TP2 as compared to the amounts present on the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the levels of the four mRNAs studied during the coculture period. DNA flow cytometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decrease in the tetraploid cell population (4C). No such changes were observed in Sertoli cell-only cultures. By contrast, the haploid population decreased 3-fold during the first week in RS-Sertoli cell cocultures. Immunocytochemical studies demonstrated further that 5-bromo-2'-deoxyuridine-labeled PS of stages V-VIII were able to differentiate into RS under the present coculture conditions. Hence, although clearly imperfect, the present coculture system should help to clarify the local regulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.

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-Initial studies to establish an in vitro system allowing survival and multiplication of porcine spermatogonia are described. Purified spermatogonia from 3-week-old pigs were cultured for 9 days alone or in the presence of Sertoli cells in either control medium or in medium supplemented with 5% fetal calf serum (FCS). Under either condition the number and the viability of the cells decreased with time, but both parameters were positively influenced by the presence of FCS. However, very few, if any, spermatogonia were able to take up BrdU under either condition. In another series of experiments, small fragments of seminiferous tubules from 3-week-old pigs were cultured in the presence of FCS, or seeded on an extracellular matrix. Under these conditions the number of cells decreased between day 0 and day 2 or day 5, then it remained roughly constant until the end of the culture. The number of spermatogonia decreased 2.5 fold during the two-week culture period. Spermatogonia were able to incorporate BrdU until the end of the experiment. The number of BrdUlabeled spermatogonia was higher when tubule-segments were seeded on an extracellular matrix. Then, the effects of the association of FCS and extracellular matrix were tested. The number of spermatogonia, during the whole culture period, was higher in serum-containing cultures than in serumfree cultures. As for the number of spermatogonia able to incorporate BrdU at different days, is decreased 3 fold between day 2 and 14 irrespective of the culture conditions. By contrast, the number of spermatogonia, labeled with BrdU between day 1 and 2, measured on days 5 to 14 of culture, was higher in serum-containing cultures. Finally, the number of spermatogonia labeled between day 1 and 2 was higher from day 5 onward than the number of spermatogonia able to take up BrdU between days 4 and 13. Taken together, these results indicate that intercellular communication and extracellular matrix are important for spermatogonia multiplication and that FCS promotes the survival of spermatogonia under in vitro conditions. spermatogenesis / stem cells / in vitro Reprod. Nutr. Dev. 40 (2000) 305-319 305