Effects of thyroxin (T4) and activin A on in vitro growth of preantral follicles in domestic cats

Abstract

Preantral follicle culture is a promising technique for rescuing gametes from endangered animals that die abruptly. The objective was to determine effects of thyroxin (T4) and activin A on in vitro growth and morphology of preantral feline ovarian follicles. Preantral follicles (86.3 ± 18.7 μm) were isolated from fresh ovaries of domestic cats. Healthy follicles were cultured individually for 14 days in 20-μL microdrops of M199 supplemented with 0.23 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 12.5 mmol/L HEPES, 0.3% (wt/vol) BSA, 1% (vol/vol) insulin-transferrin-selenite solution, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 1.0 mIU/mL growth hormone, 2.13 μg/mL FSH, and 10 ng/mL insulin-like growth factor I. The effect of various concentrations of T4 (0.5, 1.0, or 2.0 μg/mL) or activin A (10, 100, or 200 ng/mL) on follicle growth and follicular integrity were assessed. Follicle diameter was measured on Days 0, 3, 7, and 14 of culture. Follicle morphology was characterized based on granulosa cell proliferation, dissociation of somatic cells, and detachment of oocytes from follicles. On Day 14, follicles were assessed for viability using ethidium homodimer-1 staining. In the control sample, diameters of follicles increased from initial sizes on Day 3, and peaked on Day 7. This pattern was also observed in both T4- and activin A-treated follicles. On Day 7, diameters and diameter gains of follicles treated with 10 ng/mL (mean ± SEM; 170.8 ± 7.6 and 35.9 ± 5.1 μm, respectively) and 200 ng/mL activin A (165.2 ± 10.4 and 32.8 ± 5.5 μm, respectively) were larger than those of the control follicles (P < 0.05). Furthermore, 10 ng/mL activin A increased percentage of viable follicles on Day 14 (46.9% viable; P < 0.05). Follicles treated with activin A had rapid granulosa cell proliferation until Day 7. In conclusion, activin A promoted growth of preantral feline follicles and supported follicle viability during a 14-day culture, whereas T4 supplementation had no beneficial effects.