Studies on follicular fluid stimulation of progesterone secretion by porcine granulosa cells from immature antral follicles time course and influence of an inhibitor of cholesterol synthesis

Includes bibliograhpical references.Follicular fluid from preovulatory porcine follicles stimulates progesterone secretion by granulosa cells from small antral follicles. This study was undertaken to examine the time course of this stimulation and whether or not de novo cholesterol synthesis is required. Granulosa cells were aspirated from small (1-2 mm) porcine follicles. Follicular fluid was collected from large (6-12 mm) follicles and charcoal-extracted to remove 99% of endogenous steroids. Granulosa cells were cultured in Medium 199 supplemented with either equal parts of fluid from large follicles (50% LFF1) or adult porcine serum (50% serum). Charcoal-extracted serum was used in some of the experiments. Between 2-5 X 10⁵ viable cells were inoculated into Falcon multiwells containing 0.5 ml of Medium 199 containing 10% porcine serum (10% serum). Within 24 hours of culture the granulosa cells had attached, and the media were removed and replaced with 50% serum or 50% LFF1. All treatment groups were triplicated per experiment. Cultures were placed in an incubator maintained at 5% CO₂-95% air, 37°C, and high humidity. For the time course experiments the cultures were incubated for various time intervals between 2 and 48 hours. To terminate the cultures the media were removed and stored at -20°C until assayed for progesterone. The numbers of attached cells present per well were determined by Coulter counting. The cells were washed with Eagles medium and incubated with .25% Puck's trypsin until all the cells were detached. Prior to terminating the cultures observations were made of the cells utilizing an inverted stage microscope with Hoffman modulation and phase contrast optics. To examine whether LFF1 stimulation of progesterone secretion was due to an increase in de novo cholesterol synthesis, granulosa cells were incubated for 24-48 hours in 50% serum or 50% LFF1 with or without trans-1, 4-bis [2-chlorebenzylaminomethyl] cyclohexane dihydrochloride (AY-9944), an inhibitor of cholesterol synthesis. The concentrations of AY-9944 utilized were 5 X 10-⁴, 10-⁴, and 10⁻⁵M AY-9944. Petroleum ether extracts of the culture media were assayed in triplicate for progesterone content by radioimmunoassay. A rabbit antiserum against 11-α hydroxyprogesterone hemisuccinate-BSA was used. The progesterone content of unincubated media were determined in each experiment and subtracted from the progesterone content of the incubated media. Data were analyzed statistically by Student's t tests. In order to determine the effectiveness of AY-9944 in inhibiting cholesterol synthesis granulosa cells (4-5 X 10⁷ viable cells) were incubated in 5 ml of saline containing glucose, 5μCi of Acetic acid, sodium salt [1,2⁻¹⁴C] with or without 10⁻⁴M AY-9944. Cultures were gassed with 5% C0₂-95% air, sealed, and placed in a shaking water bath (37°C) for 4 hours. The media and the cells were homogenized and extracted with petroleum ether. The ether extracts were assayed for progesterone content. Both the unlabelled progesterone and ¹⁴C-progesterone content of the media were determined by scintillation counting of ³H and ¹⁴C bound to the antibody. These experiments demonstrated that LFF1 stimulation of progesterone secretion by cells from small follicles occurs consistently within 24 hours of culture. The earliest stimulation observed in these studies was between 2-5.5 hours of incubation. AY-9944 was effective in blocking the incorporation of ¹⁴C-acetate into progesterone. Progesterone secretion was significantly greater in the presence of LFF1 and AY-9944 than progesterone secretion in the presence of serum and AY-9944. Inhibition of progesterone secretion by AY-9944 was greater in the cultures containing serum than those containing LFF1. Thus LFF1 stimulation of progesterone secretion does not appear to require de novo cholesterol synthesis, and LFF1 may act by stimulating the conversion of preformed cholesterol into progesterone.M.S. (Master of Science