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

Abstract

BACKGROUND: Spermatogonial stem cells (SSCs) are infrequentself-renewing cells among the type A spermatogoniawithin the seminiferous tubules and are the basis of spermatogenesisin mammalian testis. An adequate number of SSCs is aprimary requirement for the study of their behavior, regulation, andfurther biomanipulation. OBJECTIVES: In this paper, we studiedthe development of the primary co-cultures of type A spermatogoniaand prepubertal bovine sertoli cells in the presence of ColonyStimulating Factor 1 (CSF1), a potential contributor in the SSCniche. METHODS: The effect of different concentrations of CSF1(0, 10, 50 and 100 ng/mL) on the colonization activity of spermatogonialcells was assessed 4, 7 and 11 days after the beginning of theculture by counting the total number of colonies and measuring theirarea in each group of the present experiment. Immunofluorescentstaining against OCT4 and vimentin led to the confirmation of thenature of both the SSCs and sertoli cells. RESULTS: Results showedthat the total number of colonies from day 4 to 11 increasedsignificantly in all groups, independent of CSF1 concentration. Inaddition, the total number and total area of colonies were higher (notsignificant) in 10 and 50 ng/mL CSF1 treatments than the controland 100 ng/mL CSF1 groups in all the three evaluations during theexperiment. However, this difference was only significant (p<0.05)between the total area of colonies in the control and 10 ng/mLCSF1groups at day 4 of co-culture. CONCLUSIONS:It was concluded thatCSF1 can be a suitable growth factor for improving SSCs colonizationin vitro, particularly during the first days of culture whereaccompanying sertoli cells still have not proliferated sufficiently tosupport the propagating spermatogonial cells.