149 Responses of bovine ovarian preantral follicles to FSH in vitro and in vivo

In this study, we hypothesized that preantral follicles in cattle respond to FSH via cAMP accumulation and that the hormone has a positive effect on preantral follicle growth. In experiment 1, preantral follicles at the primary stage of development were isolated from slaughterhouse-derived ovaries and cultured in groups of 20 for 15 min in medium containing vehicle, 200 ng mL⁻¹ human recombinant FSH, or 20 µM forskolin (positive control). In seven replicates, follicles were harvested after the 15-min culture and snap-frozen before being subjected to measurement of cAMP with a high-sensitivity (0.006 pmol mL⁻¹) ELISA (Enzo Life Sciences). Preantral follicles exposed to FSH and forskolin had greater concentrations of intracellular cAMP compared with vehicle (1.05, 1.13, and 0.86 ± 0.06 pmol mL⁻¹, respectively; P = 0.02). In experiment 2, five 14-mo-old cycling beef heifers were used in a crossover experiment with fixed treatment order. On Day 0 (0 h), each heifer had all visible follicles aspirated from both ovaries via transvaginal ultrasound-guided ovum pickup. Immediately after follicle ablation, animals received 25 mg of dinoprost (Lutalyze, Zoetis Animal Health) and a progesterone insert (CIDR, Zoetis Animal Health). In the first round of the experiment, all heifers received i.m. injections of saline (vehicle; 10 mL/injection) at 24, 36, 48, 60, 72, and 84 h after follicle ablation. In the second round (21 days later), i.m. injections of 50 mg of FSH (10 mL/injection for a total of 300 mg/animal; Folltropin V, Vetoquinol) were administered at 24, 36, 48, 60, 72, and 84 h after follicle ablation. Blood samples were obtained immediately before injections at 24, 36, 48, 72, and 96 h for analysis of plasma FSH via radioimmunoassay (sensitivity 0.05 ng mL⁻¹). Twelve hours after the last injection (96 h), ovarian cortex biopsies were obtained from both ovaries via modified transvaginal ovum pickup using a 16 G notch-biopsy needle and processed for histological analysis of follicle numbers after hematoxylin-eosin staining. Data were analyzed via two-way ANOVA (cAMP levels), one-way ANOVA (histological data), or repeated measures ANOVA (FSH levels). Circulating FSH increased between 0 and 72 h in both treatments (P = 0.001). As expected, administration of exogenous FSH caused an overall increase in plasma FSH (P = 0.03), which was detectable at 48 h (first injection at 24 h) in FSH-treated compared with vehicle-treated animals and lasted until the time of ovarian biopsy at 96 h. Histological analysis of cortical biopsies revealed that the proportion of primary follicles was greater in FSH-treated samples compared with control (31 ± 0.04% vs. 26 ± 0.04%, respectively; P = 0.05); the proportion of primordial and secondary follicles was similar. In conclusion, exposure of primary-stage preantral follicles to FSH in vitro increased intracellular cAMP levels. Moreover, administration of FSH to heifers increased the proportion of primary follicles. These results indicate that short-term ovarian stimulation with FSH promotes the growth of preantral follicles, and that these follicles respond to FSH via the canonical cAMP pathway when stimulated in vitro, likely mediated by the FSH receptor.

Funding for this study was provided by USDA-NIFA 2023-67015-40941 and a UC-Davis Jastro Shields award to AM.