Cultivation of boar spermatogonia on Sertoli cells

We studied the in vitro effect of Sertoli cells on boar spermatogonia isolated from the testes of 60-day-old crossbred boars. In order to enrich the culture with spermatogonia, the cells were purified by density gradient centrifugation with the use of Percoll gradient followed by separation based on adhesive capacities of cells. We found lipid drops stained by Oil Red O in Sertoli cells. The experiments showed that the cultivation of boar spermatogonia in the presence of Sertoli cells (for up to 35 days) provide the same way of differentiation as in testes in natural conditions. After 10 days of cultivation, spermatogenic cells form groups, chains, and suspension clusters. By this time, spermatogenic colonies are formed; we analyzed the expression of Nanog and Plzf genes in these colonies by real-time PCR. The expression rate of Nanog gene in experimental cell clones obtained by the short-term cultivation of spermatogonia cells in the presence of Sertoli cells was 200 times higher than in freshly isolated spermatogonia cells. The product of Plzf gene expression was found both in freshly isolated spermatogenic cells and in cell clones obtained in vitro. After long-term cultivation of spermatogonia on Sertoli cells, we observed in vitro differentiation to the lineage of spermatogenesis and formation of separate motile sperm cells after 30–33 days. At this stage, the cell population was heterogeneous. In the absence of Sertoli cells, the differentiation of boar spermatogonia cells in culture stopped after 7 days of cultivation. The data show that the cultivation of boar spermatogonia cells on Sertoli cells contributes to their in vitro differentiation to the lineage of spermatogenesis and can help to obtain boar sperm cell culture.