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.