Effects of follicle-stimulating hormone and androgen on proliferation of cultured testicular germ cells of embryonic chickens.

A germ-Sertoli cell coculture model was established to study effects of follicle-stimulating hormone (FSH) and testosterone (T) on testicular germ cell proliferation of the embryonic chickens. Germ and somatic cells were dispersed from 18-day-old embryonic testes and cultured in 96-well plates. Germ cells were characterized by expression of stem cell factor receptor c-kit. Germ cell proliferation was assessed by an increase in cell number and expression of proliferating cell nuclear antigen (PCNA). Results showed that the germ and Sertoli cells kept alive in serum-free McCoy's 5A medium supplemented with insulin, transferrin, and selenite (ITS medium). Germ cells adhered to the free surface of Sertoli cells that spread the filopodia and formed a monolayer in ITS medium. In the serum-containing medium, Sertoli cells displayed an increment with a flat squamous form and only a few very large germ cell masses were found in the free surface of Sertoli cells. Many germ cells showed apoptosis in the McCoy's 5A medium without ITS or serum. Only germ cells showed positive staining for c-kit in the coculture. Ovine FSH (0.25-1.0 IU/ml) significantly increased the number of germ cells, and PCNA-labeling index (P < 0.05). FSH also induced stronger c-kit expression compared with the control. In the FSH-treated groups, germ cells were manifested distinct knob-like form. Similar stimulating effect was found in the germ cell number by T treatments (10(-7)-10(-6)M). Furthermore, FSH (0.5 IU/ml) combined with T significantly promoted higher testicular germ cell proliferation (P < 0.05) compared with either FSH or T alone, which indicated that interaction of FSH and T might be additive. The above results showed that the serum-free germ-Sertoli cell coculture model allowed evaluating hormonal regulation of testicular germ cell proliferation. FSH and T promoted testicular germ cell proliferation probably through indirect effects on Sertoli cells.