Size, number, cellular proliferation, and atresia of gonadotropin-induced follicles in ewes

Abstract

To determine the effect of exogenous gonadotropin on size, number, cellular proliferation, and atresia of follicles, ewes (n = 3- 5/treatment/day) received an injection of vehicle or FSH-P (a pituitary extract) twice daily on Days 13, 14, and 15 (5, 4, and 3 mg FSH-P/injection, respectively; Day 0 = estrus) and were slaughtered on Days 14, 15, or 16 (i.e., after 24, 48, or 72 h of FSH-P treatment, respectively). An additional group of ewes (Day 13 control) received no treatment and were slaughtered on Day 13. All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analog; 5 mg/kg-1 BW) 1 h before slaughter. For both ovaries from each ewe, number and surface diameter of all visible follicles were recorded, and antral follicles were classified as small (≤ 3 mm), medium (> 3 mm to ≤ 6 mm), or large (> 6 mm). To evaluate rate of proliferation of follicular cells, ovaries were fixed by perfusion with Carnoy's solution, and BrdU was immunolocalized in paraffin-embedded sections by use of a specific primary antibody and indirect immunoperoxidase detection. As an index of the rate of cellular proliferation. Labeling index (LI: number of BrdU-labeled nuclei expressed as a percentage of total nuclei) of granulosa and thecal cells was determined by image analysis of antral follicles of known diameter. Follicular status (atretic vs. nonatretic) also was evaluated morphologically by using the histological sections. After 24 h of treatment (i.e., on Day 14), FSH-P-treated ewes had an increased (p < 0.01) number of medium follicles compared with vehicle-treated ewes. After 48 and 72 h of treatment (i.e., on Days 15 and 16). FSH-P-treated ewes had more (p < 0.01) medium and large follicles than vehicle-treated ewes. In addition, on Day 16, FSH-P- treated ewes had fewer (p < 0.01) small follicles than vehicle-treated ewes. Overall, granulosa cell LI was greater (p < 0.01) than thecal cell LI. In addition, across all groups, granulosa cell LI decreased (p < 0.01) as follicular size increased, whereas thecal cell LI was similar for small and medium follicles but decreased (p < 0.05) for large follicles. Across all follicular size classes, granulosa cell LI was greater (p < 0.01) for FSH-P- treated ewes than for vehicle-treated ewes after 24 h of treatment (Day 14), but not after 48 or 72 h of treatment (Day 15 or 16). Across all days and size classes of follicles, incidence of atresia was less (p < 0.01) for FSH- P-treated than for vehicle-treated ewes. Thus, FSH-P treatment decreased the incidence of atresia and induced recruitment and growth of ovine follicles as evidenced by 1) the increased number of medium and large antral follicles, 2) the increased LI of follicles after 24 h of FSH-P administration, and 3) the decreased incidence of atresia on Days 14 to 16.