Effect of insulin supplementation on in vitro maturation of pre-antral follicles from adult and pre-pubertal mice.

Abstract

This study was aimed to determine the impact of insulin concentrations on in vitro pre-antral follicle growth, survival, antrum formation rate, and retrieval of mature oocytes in mice. Mice pre-antral follicle growth were recorded on days 2, 4, 6, 8, 10, and 12 in α-modified essential media (α-MEM) supplemented with insulin concentrations of 6, 8, and 10μg/ml along with 10% FBS, 100mIU/ml follicle stimulating hormone, 10mIU/ml luteinizing hormone, 100μg/ml penicillin, and 50μg/ml streptomycin. After 12d of growth in vitro, follicles were allowed to mature for 16-18h in α-MEM supplemented with 1.5IU/ml human chorionic gonadotrophin (hCG) and 5ng/ml epidermal growth factor (EGF). The initial diameter (54.86 ± 2.5μm) of mice oocyte progressively increased in all the three insulin concentration groups and attained a maximum size on day 12 (71.90 ± 2.8μm). Supplementation with higher concentrations of insulin (both 8 and 10μg/ml) significantly enhanced antrum formation without effecting the oocyte diameter and percent retrieval of mature oocyte in all the three concentration groups. Both in vitro cultured as well as in vivo collected follicles and oocytes showed similar localization and expression of oocyte maturation markers SAS1B and GDF9. Insulin concentration of 8μg/ml was found to be optimal for in vitro follicle culture of adult mice (42-49d). Optimized follicle culture conditions were also assessed successfully with pre-pubertal mice (12-14d); however, adult mice showed higher follicle survival, antrum formation, and more mature oocytes production in comparison to pre-pubertal mice.