The effect of follicular fluid on pregnenolone accumulation and 3B-hydroxysteroid dehydrogenase activity in porcine granulosa cells from small ovarian porcine antral follicles in vitro
Girmus, Ronald L.
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Progesterone (P₄) secretion by porcine granulosa cells (GC) from small (1-2 mm) ovarian follicles is enhanced by incubation with charcoal treated and filtered fluid from large (6-12 mm) porcine, ovarian follicles (LFF1). This study examines the enzymatic steps in the conversion of cholesterol to progesterone at which LFF1 acts to enhance P₄ secretion. The effect of nafoxidine, an anti-estrogen, on LFF1's enhancement of P₄ secretion and LFF1's effects on cellular DNA content were also studied. GC from small, antral follicles were incubated in monolayer for 3 days using an initial inoculum of 5 x 10⁵ cells. P₄ and progesterone (P₅) concentrations were determined by specific radioimmunoassay and cellular DNA content determined by a fluorometric assay. To study LFF1's effect on P₅ production, 0 to 5 x 10⁻⁵ M P₅ were added for the 3 day incubation with LFF1 or serum. P₄ secretion by LFF1 treated cells was not increased until 10 M P₅ was present, whereas 10 M P₅ enhanced P₄ secretion by controls. When 5 x 10 M P₅ was present, treated cells produced more P₄ than controls. This implies that LFF1 increased P₅ production and utilization. When 5 x 10⁴ viable GC were inoculated, 10⁻⁶ M P₅ saturated the enzymes of both treated and control cells. LFF1 treated cells secreted more P4 than controls at saturating concentrations of P₅. During a 3 hour incubation with 10⁻⁶ M P₅ following a 3 day incubation with LFF1 or serum, the rate of P₄ secretion by LFF1 treated cells and controls was similar over the first 1/2 hour. P₄ secretion by LFF1 treated cells remained linear for 2 hours while controls stopped secreting P₄ after 1 hour. This may be due to a difference in the subcellular compartment containing the enzyme. 10⁻⁶ M nafoxidine blocked LFF1's enhancement of P₄ secretion in 2 of 4 experiments. LFF1 increased cellular DNA content by 60+7% in 5 of 9 experiments. The interpretation of LFF1's effect on P₄ secretion is the same if data are expressed per culture, cell #, or ug DNA. 3B-hydroxysteroid dehydrogenase (3B-HSD) activity was measured in 10,000 x g supernatant fractions of GC after 3 day incubations with LFF1 or serum. There was no difference in Km but Vmax was increased 60% in LFF1 treated cells. These results imply that LFF1 increases P₅ production and the number of 3B-HSD molecules.