The gonadotropic regulation of granulosa cell steroidogenesis in vitro has been shown to be accompanied by cellular rounding. In this study, the possible relationship between cell shape, micro tubules, and granulosa cell steroidogenesis in vitro was further explored by culturing (24 h) granulosa cells obtained from antral follicles of
pregnant mare’s serum gonadotropin-treated rats in either Eagles’s Minimum Essential Medium alone (MEMcells) or in collagen gels (GEL-cells) in the absence or presence of coichicine, a micro tubule-depolymerizing agent previously shown to inhibit cell-spreading in vitro. Cellular morphology was assessed by electron microcopy
and compared with that seen in vivo. In addition, the influence of the various culture conditions on progesterone and 20a -hydroxy-pregn-4-en-3 -one (2Ocs -OH-progesterone) secretion was determined by specific radioimmunoassays.
Whereas the majority ofgranulosa cells in sections of antralfollicles appeared rounded in shape, cells cultured in MEM underwent considerable spreading and assumed a variety of shapes at the end of 24 h of culture. GELcells, on the other hand, remained rounded and had cellular diameters only slightly larger than those observed in
vivo. They also secreted more progesterone (almost 3-fold) and less 20n-OH-progesterone (0.6-fold) than MEMcells. Colchicine increased the secretion of progesterone (1.6-fold) and 20a-OH-progesterone (1.8-fold) comparably in MEM-cells but had no influence on the secretion of either progestin by GEL-cells. Hence, although coichicine-stimulated progestin secretion by granulosa cell monolayers appeared to reflect increased metabolism
of substrate-possibly due to a closer association between lipid droplets and mitochondria, the elevated secretion
of progesterone by GEL-cells may have been largely due to a shift in the equilibrium between progesterone
and its inactive 2 On-reduced metabolite. The high ratio of 20n-OH-progesterone to progesterone secretion seen
in MEM-cultured cells may be an adaptation of granulosa cell metabolism to culture as monolayers on plastic or
glass surfaces. The morphology of GEL-rather than MEM -cells resembled closely that seen in vivo. This culture
method may represent a more physiologic approach to the maintenance of granulosa and other steroidogenic
cells in vitro and provide a more appropriate means of assessing cytoskeletal function in the regulation of steroid
hormone production.
INTRODUCTION