Apoptosis in equine granulosa cells and its relationship to cumulus expansion and oocyte chromatin configuration in ovarian follicles.

During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa cells, fractionated by agarose gel electrophoresis, stained with ethidium bromide and visualized with ultraviolet light or end-labelled with [32P]dideoxy-ATP, and autoradiography was performed after electrophoresis. In Expt 2, follicles (n=34) from four mares with at least one follicle >35 mm in diameter were processed. DNA was extracted from the granulosa cells; the cumulus oophorus was classified and the oocyte chromatin was stained with Hoechst 33,258 fluorescent stain. In Expt 1, apoptosis, as determined by the characteristic laddering of internucleosomal DNA fragments, was present in 45% of all follicles. Apoptosis was apparent primarily in follicles <20 mm in diameter and was present with greatest frequency in follicles 6-10 mm in diameter. Apoptosis was not detected in follicles >27 mm in diameter. The presence of sheared DNA of a wide range of different molecular masses, possibly indicative of necrotic cell death, was positively correlated with follicle size. In Expt 2, apoptosis was detected in 50% of follicles <20 mm in diameter but not in follicles > 30 mm in diameter. The oocytes had an expanded cumulus oophorus in 58% of follicles <20 mm in diameter, whereas 80% of follicles > 30 mm in diameter had a compact cumulus oophorus. In these mares, follicles <20 mm in diameter appeared to undergo apoptotic changes as well as cumulus expansion. These findings indicate that degeneration occurs in many follicles that are not destined for ovulation and that detection of apoptosis can be used as an indicator of follicular degeneration in mares. Apoptosis, as a marker of cell death, can be used to study the growth and selection of follicles, and to correlate the health of granulosa cells with oocyte quality.