Effects of Gonadotropin Treatment and Withdrawal on Follicular Growth, Cell Proliferation, and Atresia in Ewes1

Abstract

To determine the effects of FSH-P treatment and subsequent withdrawal on follicular growth, cell proliferation, and atresia, ewes (n = 4 ewes/treatment group) received twice daily injections of saline or FSH-P beginning on Day 13 of the estrous cycle (length of the estrous cycle = 16.5 days) and were slaughtered after 0, 48, or 72 h of treatment (i.e., on Days 13, 15, or 16). Some treatment groups received FSH-P from Day 13 until slaughter (FSH-P-treated), whereas some received FSH-P for 24-48 h followed by saline for 24-48 h (FSH-P withdrawal). All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analogue) 1 h before slaughter. For both ovaries from each ewe, the number and surface diameter of all visible antral follicles were determined, and antral follicles were classified as small (< or = 3 mm), medium (> 3 mm to < or = 6 mm), or large (> 6 mm). As an index of the rate of cell proliferation, BrdU was immunolocalized in paraffin-embedded tissue sections, and the labeling index (LI; BrdU-labeled nuclei as a percentage of total nuclei) was determined for granulosa and thecal cells of nonatretic and early atretic antral follicles of known diameter. Follicular status (atretic vs. nonatretic) was evaluated morphologically. Moreover, the presence of apoptosis was detected in situ by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method. For untreated and saline-treated ewes, the number of small follicles per ewe increased (p < 0.01) from Day 13 to Day 15, then decreased again on Day 16, whereas numbers of medium and large follicles did not differ across days. Compared with saline-treated ewes, ewes receiving FSH-P from Day 13 until slaughter had fewer (p < 0.05) small but more (p < 0.05) medium and large follicles. Compared with FSH-P-treated ewes, FSH-P withdrawal resulted in fewer (p < 0.05) medium and large but more (p < 0.05) small follicles. Across all follicular size classes, granulosa and thecal cell LI of nonatretic follicles was decreased (p < 0.05) by FSH-P withdrawal compared with FSH-P treatment. Additionally, across all follicular size classes, FSH-P withdrawal increased (p < 0.01) the percentage of follicles that were atretic compared with saline or FSH-P treatment. Histochemical staining of early and advanced atretic follicles showed that granulosa cells are the predominant site of cell death (apoptosis) during follicular atresia. Thus, compared with continuous FSH-P treatment, withdrawal of FSH-P resulted in decreased numbers of medium and large follicles, decreased proliferation of follicular cells, and an increased incidence of atresia associated with granulosa cell death. This model should prove useful for studying the mechanisms regulating follicular growth and atresia in ewes.