Culture of porcine spermatogonia: effects of purification of the germ cells, extracellular matrix and fetal calf serum on their survival and multiplication.

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 BrdU-labeled 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 serum-free 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.

Localization and quantitative expression of mRNAs encoding the testis-specific histone TH2B, the phosphoprotein p19, the transition proteins 1 and 2 during pubertal development and throughout the spermatogenic cycle of the rat.

Expression of the testis-specific histone TH2B, the phosphoprotein p19, and the transition proteins TP1 and TP2, was localized in the rat testis and quantified, using in situ hybridization of their mRNAs with radiolabeled probes and image analysis. In a first study, expression was assessed during testicular development between day 2 and day 65 postpartum. TH2B mRNAs appeared first in preleptotene spermatocytes (PL) on day 12 and in pachytene spermatocytes (PS) on day 18; p19 mRNAs were present in PS from day 18 onward, and TP1 and TP2 mRNAs were detected in round spermatids (RS) from day 32 onward. In the second trial, the expression of these four genes was studied throughout the cycle of spermatogenic epithelium in mature animals. TH2B mRNAs were localized in B spermatogonia at stage V, and in PL at stages VII and VIII but no longer in leptotene and zygotene spermatocytes. Thereafter, TH2B mRNAs were observed in PS from stages III-IV to XIII. The steady-state mRNA level per cell was high in PS with a maximum at stages IX-X. p19 mRNAs were present in PS from stages III-IV onward and in RS up to stages 1-2 of spermiogenesis. The maximum mRNA level per cell was observed in PS between stages VII and XIII. The presence of TP1 mRNAs was restricted to spermatids from steps 6 to 15-16 of spermiogenesis while TP2 mRNAs were detected in spermatids only between step 7 and step 13. The highest steady-state amounts of mRNAs were observed between step 7 and step 14 for TP1 and between step 10 and step 12 for TP2.

Pre- 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 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.