In vitro growth of preantral follicles in cats

Abstract

EXP. 1 The objective was to determine effects of thyroxin (T4)and activinA 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 d in 20-µL microdrops of basic culture medium supplemented with various concentrations of T4 (0.5, 1.0 or 2.0 µg/mL) or activin A (10, 100 or 200 ng/mL). 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 controls, 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 and 200 ng/mL activin A were larger than those of the controls (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 a14-d culture, whereas T4 supplementation had no beneficial effects. EXP. 2 The objective was to optimize alginate gel concentrations for feline preantral follicle culture. Preantral follicles with round or oval shape were mechanically isolated from ovaries of domestic cats, and individually encapsulated with 0.25% (n = 15), 1.0% (n = 31) or 2.0% alginate gel (n = 22), respectively. Each encapsulated follicle was cultured in a 96-well plate containing 100 µL medium comprised of M199 supplemented with 0.23 mM sodium pyruvate, 2 mM L-glutamine, 12.5 mMHepes, 0.3% BSA, 1% ITS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 1.0 mIU/ml growth hormone, 2.3 µg/ml FSH, 10 ng/ml IGF-I and 10 ng/ml activin A. Follicle morphology and diameter were determined on Days 0, 3, 7 and 14. The follicles encapsulated in 0.25% and 2.0% alginate gel reached their maximal diameters on Day 7 and maintained their sizes till Day 14. In contrast, diameters of the follicles encapsulated in 1.0% alginate increased continuously from Day 0 to 14 and finally reached greater size than follicles in the other alginate concentrations (P<0.05). Most cultured follicles exhibited intact basement membrane throughout culture. Our findings suggested that 1% alginate gel is optimal supporting cat preantral follicle growth in the 3-D culture system. EXP. 3This study aimed to investigate freezing effects of ovarian tissues on survival of preantral follicles and observe in vitro growing of preantral follicles retrieved from cryopreserved ovarian cortical tissues of a cheetah post-mortem. After 29 hours cold storage, ovarian cortices were cut into small pieces (2.0 x 2.0 x 1.0 mm3) and allocated to be frozen using a passive cooling container (n=3 pieces) or vitrification (n=3 pieces). After one year of storage, 23 and 58 preantral follicles were mechanically isolated from ovarian tissues cryopreserved using a passive cooling container and vitrification, respectively. Of 23 and 58 isolated follicles, 10 and 12 morphologically intact and viable were selected to be in vitro grown in a culture medium for 7 days. Diameters and diameter gains were examined on Days 0, 3 and 7. Follicle viability was assessed on Day 7. Diameters of follicles retrieved from the slow freezing ovarian tissues decreased gradually from 53.5 ± 14.2 µm on Day 0 to 50.9 ± 17.1 µm with 2 out of 10 viable on Day 7 whereas those frozen using vitrification maintained their diameters between 50.7 ± 15.6 µm and 50.5 ± 17.9 µm on Days 0 and 7, respectively, with 2 of 12 viable. In conclusion, preantral follicles obtained from cryopreserved cheetah ovarian tissues can be grown in vitro for 7 days. However, optimization of freezing protocol and culture medium are required to improve the viability and growing rate.