Evidence for ovarian granulosa stem cells: Telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divi de indefinitely partly because they express telomerase. Telomerase is a rib onucleoprotein enzyme that repairs the ends of telomeres that otherwise sho rten progressively upon each successive cell division. In this study we car ried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 mu m) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and targe (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-re lated protein, were undertaken on follicles of each size (n = 3) by flow cy tometry; 12% to 16% of granulosa cells in all follicles were in the S phase , and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) wa s highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was c arried out on bovine ovaries, and telomerase RNA was detected in the granul osa cells of growing follicles but not primordial follicles. Two major patt erns of staining were observed in the membrana granulosa of antral follicle s: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly su pport our hypothesis that granulosa cells arise from a population of stem c ells.