Abnormal distribution of cortical granules in cynomolgus monkey one-day old MII oocytes which were immature at oocyte retrival after controlled ovarian hyperstimulation

Abstract

OBJECTIVE: Developmental competence of cynomolgus monkey (macaca fascicularis) oocytes, which were immature at oocyte retrieval after controlled ovarian hyperstimulation (COH) and reached to metaphase II stage (MII, 1-day old MII oocytes) after overnight culture, were low compared with mature oocytes at oocyte retrieval (fresh MII oocytes). In this study, ultrastructures of 1-day old and fresh MII oocytes were assessed. DESIGN: An experimental study using a monkey model was performed. MATERIALS AND METHODS: Female cynomolgus monkey (5 years old) was used for COH and oocyte retrieval was carried out using a laparoscope. Cumulus cells were removed from oocyte using hyaluronidase. Some of immature (germinal vesicle (GV) and metaphase I (MI)) and mature (fresh MII) oocytes were fixed for control as below. Immature oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for 24h. Control and 1-day old MII oocytes were prefixed by glutaraldehyde, postfixed by osmium, dehydrated and embedded in EPON blocks. Ultrathin sections were observed by transmission electron microscope (TEM) after staining. RESULTS: Fifty oocytes (14 GV, 21 MI, 9 MII and 6 degenerated oocytes, respectively) were retrieved. Immature oocytes (12 GV and 19 MI) were cultured. Seven mature oocytes were obtained. By observation using TEM, the morphological change of mitochondria during meiotic maturation was assessed. Mitochondria were oval at GV stage and some of them extended at MI stage. Flattened and crescent-shaped mitochondria which encircled smooth endoplasmic reticulum were observed in both fresh and 1-day old MII oocytes. However, in 1-day old MII oocytes, cortical granules were observed not only in the surface but also inside of oocyte. In addition, large fragments were observed in the perivitelline space. Microvilli were observed in all oocytes. CONCLUSIONS: These results suggest that cytoplasmic maturation is incomplete in 1-day old MII oocyte because of improper localization of cortical granules. The unbalance of maturation between nucleus and cytoplasm may impair the developmental competence of 1-day old MII oocyte.