Flow cytometric analysis of granulosa cells from developing rat follicles

in Reproduction

Summary. Immature rats were treated with diethylstilboestrol (DES) or pregnant mares' serum gonadotropin (PMSG) and forward angle light-scatter (FALS) and 90° light-scatter (90° LS) signals were used to measure the size and the granularity (internal organization) of the granulosa cells, respectively. The results confirmed the presence of two major populations of granulosa cells in the ovaries of both groups of rats, with the same percentage of larger cells in both treatments (52·3% in DES, 49·5% in PMSG). Since DES treatment brings about granulosa cell growth while PMSG treatment causes growth and differentiation, it is evident that there is heterogeneity in granulosa cell sizes during different states of growth and differentiation. There was also heterogeneity in sizes of granulosa cells harvested from follicles of small (< 210 μm), medium (210–420 μm) and large (> 420 μm) diameter. Quadrant analysis of granulosa cells in various fractions collected from Percoll gradients suggested an increase in granularity in the small and large granulosa cell populations. Cell cycle analysis of small and large granulosa cell populations collected from large follicles of rats treated with PMSG indicated that each population was distributed in G0/G1, S and G2/M phases. These results demonstrate that populations of small and large granulosa cells exist in rat ovarian follicles during various stages of growth and differentiation.

Keywords: flow cytometry; granulosa cells; heterogeneity; follicle isolation; DNA analysis; rat

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