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Evidence for Ovarian Granulosa Stem Cells: Telomerase Activity and Localization of the Telomerase Ribonucleic Acid Component in Bovine Ovarian Follicles¹

^a Flinders University of South Australia, Departments of Medicine and ^b Obstetrics-Gynaecology, Bedford Park, South Australia 5042, Australia ^c Louisiana State University, Medical Center, Department of Cellular Biology and Anatomy, Shreveport, Louisiana 71130 ^d Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60–100 µm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6–8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G₂/M phase. Telomerase activity (n = 3) was 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 carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

¹ This work was supported by the National Health and Medical Research Council of Australia, Flinders University of SA, and Flinders Medical Centre Research Foundation.

² Correspondence: Raymond J. Rodgers, Department of Medicine, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia. FAX: 618 8204 5450; ray.rodgers{at}flinders.edu.au

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