Morphometric analysis of the effects of follicular fluid on granulosa cells from immature porcine antral follicles
McLean, Mark Philip
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The purpose of this study was to examine the influence of in vitro incubation with fluid from large follicles on the ultrastructure of granulosa cells from small porcine follicles and to correlate morphological changes with the cell's steroidogenic activity. Follicular fluid's actions in preventing granulosa cell atresia were also examined. Granulosa cells from small (1-2 mm diameter) porcine ovarian follicles were cultured in either charcoal extracted follicular fluid from large (6-12 mm diameter) follicles or serum with equal parts of TC 199. 5 Initially 5 X 10⁵ viable cells were inoculated into Falcon multiwells in 0.5 ml TC 199 containing 10% porcine serum. After 48 hours the media were removed and replaced with 50% fluid from large follicles or 50% serum. Cultures were maintained at 5% CO₂:95% air, 37C and high humidity. Representative samples of granulosa cells were examined using Nomarski (interference contrast) optics each day for 5 days. Cells were prepared for electron microscopy after 2 and 5 days in the media containing 50% fluids and compared to freshly collected cells and to cells incubated in 10% serum for 2 days. Quantitative data obtained by morphometric analysis of micrographs from each treatment group were analysed statistically. The total cell, nuclear, cytoplasmic and several organelle volumes were determined. Average size, surface area and number of organelles within a treatment group were quantified. Results were analysed using Analysis of variance and Student-Newman-Keuls multiple comparison tests. Ultrastructural features of the granulosa cells were correlated with data obtained from radioimmunoassay of the culture media's progesterone content. Petroleum ether extracts of the culture media were assayed in triplicate for progesterone content using a rabbit antiserum against 11-ahydroxy- progesterone hemisuccinate-BSA. Background progesterone contents of unincubated media were determined and subtracted from progesterone content of incubated media. Data were analysed statistically using Student's t test. Granulosa cells incubated with follicular fluid appeared epithelioidlike with ultrastructural features (microvilli, and densely staining bodies) which are related to the cells steroidogenic capacity. Serum treated cells appeared fibroblast-like and contained numerous lysosomal profiles suggesting regressive or atretic cellular changes. Follicular fluid treated cells secreted more progesterone than serum treated cells. This study has confirmed that charcoal extracted follicular fluid from large follicles can stimulate progesterone secretion and modify the ultrastructure of granulosa cells from small antral follicles. It is hypothesized that the actions of follicular fluid on the cells's ultrastructure may be related to follicular fluid's enhancement of steroidogenesis and inhibition of atresia.