Isolation and Culture of Immature Rat Type A Spermatogonial Stem Cells

Abstract

Spermatogenesis is a complex process of cellular renewal and differentiation that begins with the divisions of the type A spermatogonial stem cells and ends as late spermatids are released into the seminiferous tubule lumen as spermatozoa (Dym 1983). In vivo spermatogonial stem cells can be directed to one of three fates: (1) renew themselves into other stem cells; (2) differentiate into type B spermatogonia and eventually spermatocytes and more mature germ cells; or (3) degenerate. Despite abundant studies on the morphology and kinetics of spermatogonial cell renewal and differentiation (Clermont 1966, 1969; Dym and Clermont 1970; Huckins 1971; Oakberg 1971), very little is known about the regulation of spermatogonial cell proliferation or degeneration in mammals. Attempts to study spermatogonial proliferation in organ cultures have been made by several investigators (Martinovitch 1937, 1939; Steinberger et al. 1964; Ghatnekar et al. 1974; Aizawa and Nishimune 1979; Curtis 1981; Boitani et al. 1993). In all these studies, differentiation stopped at the pachytene spermatocyte stage. Similarly, spermatogonial proliferation and differentiation up to pachytene spermatocytes have been observed in cocultures of spermatogenic cells with Sertoli cells in serum-free defined medium supplemented with hormone and growth factors (Tres and Kierszenbaum 1983; Hadley et al. 1985). However, differentiation beyond pachytene spermatocytes into young spermatids was observed in seminiferous tubule segments cultured for 4–6 days (Parvinen et al. 1983). Thus, it is likely that cellular interactions are important for spermatogonial proliferation and differentiation. However, the Sertoli cell factors which are responsible for regulating this process are not known. It has been observed that up to 75% of spermatogonia undergo spontaneous degeneration before maturation (Abe 1987; Huckins 1978). Although the withdrawal of gonadotropins by hypophysectomy or immunoneutralization enhances the degeneration of germ cells (Raj and Dym 1976; Russell and Clermont 1977), other factors promoting degeneration of germ cells in intact animals have not been identified. In order to examine the regulation of spermatogonial renewal, differentiation, and degeneration under controlled culture conditions, it is important to develop an in vitro model system. In this direction, we have isolated rat type A spermatogonial cells from 9-day-old rats (Dym et al. 1995) and purified them to greater than 95% purity by sedimentation velocity at unit gravity followed by differential plating in fetal bovine serum (FBS)-supplemented medium. The present study was undertaken to standardize culture conditions which would allow the type A spermatogonia to survive for longer periods of time.