254 DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES AFTER IN VITRO GROWTH, NUCLEAR TRANSFER, AND IN VITRO FERTILIZATION

Abstract

Long-term effects of in vitro maturation of oocytes and in vitro culture of fertilized eggs have been reported in ruminants, mice, and humans. However, effects of in vitro oocyte growth are unknown. Although a large number of non-growing oocytes can be a gamete resource, very few oocytes ever acquire competence to support full-term development after in vitro growth. The objective of the study was to evaluate different culture conditions and the long-term effects of in vitro oocyte growth on the production of offspring. Oocytes of newborn, 10-day-old, and adult BDF1 (C57BL/6N × DBA2) mice were cultured for 22, 11, and 1 day(s), respectively. The results showed that alpha-MEM medium was superior to Waymouth medium in oocyte growth (68.6 ± 3.87 µm vs. 61.7 ± 3.26 µm, respectively; P < 0.001), and in maintenance of follicular integrity (69% vs. 30%; P < 0.001) when non-growing oocytes from newborn mice were cultured. However, oocytes grown in vitro were incompetent to support meiotic maturation by themselves in the case of either the 22-day culture of oocytes from newborn mice (1/59 in alpha-MEM vs. 1/65 in Waymouth) or the 11-day culture of oocytes from 10-day-old mice (51/140 in alpha-MEM vs. 2/157 in Waymouth), and none of them developed to the blastocyst stage. Subsequently, to examine the nucleic competence of oocytes grown in vitro, serial nuclear transfers were carried out. Karyoplasts from oocytes grown in vitro using alpha-MEM were fused with the GV oocytes grown in vivo after enucleation. The reconstituted oocytes were cultured in alpha-MEM. After 14 h, MII chromosomes of the reconstituted oocytes were transferred into the enucleated and ovulated MII oocytes in order to provide cytoplasmic competency. The results showed that when the donor oocytes attained a diameter of e60 µm, the reconstituted oocytes could develop into pups at extremely high rates (30-41%) after in vitro fertilization (IVF) and embryo transfer in the case of either the 22-day culture of oocytes from newborn mice (7/17) or the 11-day culture of oocytes from 10-day-old mice (25/77). A significant difference was not observed in the competence to develop to term of the reconstituted oocytes when compared with that of the oocytes reconstituted from the control GV (25/52; P > 0.05). When the donor oocytes attained a diameter of 50–60 µm, the reconstituted oocytes also could develop into pups (7/33); however, their efficiency was significantly reduced when compared with that of the reconstituted oocytes from the control GV (P < 0.05). On the other hand, the weight of the offspring depended on the duration of culture, and offspring from non-growing oocytes (1.48 ± 0.17 g) were heavier than those of the IVF control (1.25 ± 0.14 g; P < 0.05). In conclusion, we have demonstrated that using a nuclear transfer technique combined with in vitro growth of oocytes was sufficient to produce functional oocytes, and long-term culture for oocyte growth did not affect the nucleic ability of oocytes to develop to term; however, fetal growth may be susceptible to the duration of culture.