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Granulosa cells were isolated from 15-day-old, 25-day-old or PMSG-primed rats and were separated by Percoll gradient (20 to 60%) into five fractions. The cells in fraction 2 were mostly small cells (6.96-9.57 microns) and fractions 3 and 4 had a relatively high population of large cells (10.96-13.05 microns) which were sorted to collect a pure population of large cells. Aliquots of small or large cells were cultured separately in serum-free defined DMEM/F-12 medium containing 50 ng FSH ml-1, or 10 ng transforming growth factor alpha (TGF-alpha) ml-1 for 3 days. In PMSG-primed rats, the large cells produced 3.2-fold more progesterone than did small cells (with FSH/TGF alpha: 255 +/- 35.0 versus 77.32 +/- 14.5 x 10(-6) ng, day 1). Large and small cells from 25-day-old rats produced similar amounts of progesterone (FSH/TGF alpha: 65.68 +/- 9.6 versus 78.25 +/- 12.3 x 10(-6) ng, day 1). In 15-day-old rats, large and small cells produced very low concentrations of progesterone (FSH/TGF alpha: 4.69 +/- 1.2 versus 2.66 +/- 1.0 x 10(-6) ng, day 1). Large cells from PMSG-primed rats had characteristics of steroidogenic cells, i.e. smooth endoplasmic reticulum and well-developed mitochondria with tubular cristae compared with small cells, whereas small and large cells from 25- and 15-day-old rats contained the regularly occurring organelles without the endoplasmic reticulum of the smooth variety and mitochondria with lamellar cristae. This study shows that the heterogeneity of granulosa cells is related to size, metabolic response to FSH, TGF-alpha or to both factors and morphological features, all of which may be associated with the transition from preantral to preovulatory stages of follicle differentiation.

reproduction

Functional and ultrastructural characteristics of two types of rat granulosa cell cultured in the presence of FSH or transforming growth factor (TGF- )