Isolation of bovine spermatogonial cells and co-culture with prepubertal sertoli cells in the presence of colony stimulating factor-1

ABSTRACT

Spermatogonial stem cells [SSCs] are infrequent self-renewing cells among the type A spermatogonia within the seminiferous tubules and are the basis of spermatogenesis in mammalian testis. An adequate number of SSCs is a primary requirement for the study of their behavior, regulation, and further biomanipulation. In this paper, we studied the development of the primary co-cultures of type A spermatogonia and prepubertal bovine sertoli cells in the presence of Colony Stimulating Factor 1 [CSF1], a potential contributor in the SSC niche. The effect of different concentrations of CSF1 [0, 10, 50 and 100 ng/mL] on the colonization activity of spermatogonial cells was assessed 4, 7 and 11 days after the beginning of the culture by counting the total number of colonies and measuring their area in each group of the present experiment. Immunofluorescent staining against OCT4 and vimentin led to the confirmation of the nature of both the SSCs and sertoli cells. Results showed that the total number of colonies from day 4 to 11 increased significantly in all groups, independent of CSF1 concentration. In addition, the total number and total area of colonies were higher [not significant] in 10 and 50 ng/mL CSF1 treatments than the control and 100 ng/mL CSF1 groups in all the three evaluations during the experiment. However, this difference was only significant [p<0.05] between the total area of colonies in the control and 10 ng/mLCSF1 groups at day 4 of co-culture. It was concluded that CSF1 can be a suitable growth factor for improving SSCs colonization in vitro, particularly during the first days of culture where accompanying sertoli cells still have not proliferated sufficiently to support the propagating spermatogonial cells