Immortalization of germ cells and somatic testicular cells using the SV40 large T antigen

We report the immortalization, using the SV40 large T antigen, of all the cell types contributing to a developing seminiferous tubule in the mouse testis. Sixteen peritubular, 22 Leydig, 8 Sertoli, and 1 germ cell line have been established and cultured successfully for 90 generations in a period of 2.5 years. Immortalized peritubular cells were identified by their spindle-like appearance, their high expression of alkaline phosphatase, and their expression of the intermediary filament desmin. They also produce high amounts of collagen. Immortalized Leydig cells are easily identifiable by the accumulation of lipid droplets in their cytoplasm and the production of the enzyme 3-β-hydroxysteroid dehydrogenase. Some Leydig cell lines also express LH receptors. The immortalized Sertoli cells are able to adopt their typical in vivo columnar appearance when cultured at high density. They exhibit a typical indented nucleus and cytoplasmic phagosomes. Some Sertoli cell lines also express FSH receptors. A germ cell line (GC-1spg) was established that corresponds to a stage between spermatogonia type B and primary spermatocyte, based on its characteristics in phase contrast and electron microscopy. This cell line expresses the testicular cytochrome c_t and lactate dehydrogenase-C₄ isozyme. These four immortalized cell types, when plated together, are able to reaggregate and form structures resembling two-dimensional spermatogenic tubules in vitro. When only the immortalized somatic cells are cocultured, the peritubular and Sertoli cells form cord-like structures in the presence of Leydig cells. Fresh pachytene spermatocytes cocultured with the immortalized somatic cells integrate within the cords and are able to survive for at least 7 days. The ability to perform coculture experiments with immortalized testicular cell lines represents an important advancement in our ability to study the nature of cell-cell and cell-matrix interactions during spermatogenesis and testis morphogenesis.