65 OVULATION OF IMMATURE OOCYTES WITH HIGH COMPETENCE RATES

Abstract

Collection of immature oocytes from antral follicles in superovulated mice is a widely established technique for retrieval of germinal vesicle (GV) and metaphase I (MI) oocytes. Investigators use their experience to select antral follicles under a microscope before puncturing the follicles. This is sometimes followed by screening of oocytes based on morphology and diameter, and usually mouse oocytes of small diameters or abnormal morphologies are excluded. Shortcomings with the current technique may include varied oocyte yields and collection of oocytes from primary and secondary follicles. Moreover, such immature oocytes were observed to have different chromatin configurations, cortical granule distributions, spindle-chromosome organizations, fertilization rates, and diameters. This study was designed to investigate the potential of ovulated immature oocytes, resultant from superovulated mice treated with an FDA approved phosphodiesterase 3A inhibitor named cilostazol (CLZ), to substitute for ovarian immature oocytes collected from antral follicles of superovulated mice. Swiss Webster mice were superovulated and gavaged with 7.5 mg of CLZ once, at the same time as hCG injection, or twice, at the same time as hCG plus 6 h post-hCG injection, to result in ovulation of MI or GV oocytes, respectively. Control ovarian GV or MI oocytes were collected from ovarian antral follicles of superovulated mice not treated with CLZ. Ten mice were used in each treatment or control group. Single or multiple administrations of CLZ resulted in mice ovulating 85.8 ± 3.9% MI oocytes or 95.2 ± 3% GV oocytes (mean ± SEM), respectively. Treated GV oocytes had significantly higher rates of advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had lower cAMP levels and higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P < 0.0001). Treated MI oocytes had significantly higher rates of normal spindle and chromosomes aligned at the metaphase plates and offspring than did control MI oocytes. Control or treated GV oocytes were found to have greater diameters than did control or treated MI oocytes, respectively (P < 0.007), indicating that initiation of meiotic maturation is associated with reduction in oocyte diameters and utilisation of cytoplasm proteins and cofactors. Moreover, control GV oocytes were found to have greater diameters than did treated GV oocytes (P = 0.007). This may refer to the readiness of treated GV oocytes to undergo germinal vesicle breakdown and transition into the MI stage, especially treated GV oocytes had high rates of meiotic development in comparison to control GV oocytes. Diameters of GV nuclei in treated GV oocytes were smaller than those in control GV oocytes (P = 0.006), which may also indicate a germinal vesicle that had started to undergo germinal vesicle breakdown. A similar significant difference was also noted with control and treated MI oocytes. In summary, we present a novel method for retrieval of immature oocytes at different stages of meiotic maturation. Treated ovulated immature oocytes had more uniform diameters and high developmental competence than did ovarian immature oocytes. Treated ovulated immature oocytes may substitute for ovarian immature oocytes and become an additional research resource.