Embryo Construction, Transfer And In Vitro Maturation Of Oocytes In The Cynomolgus Monkey

Research in Non-human primates (NHPs) has the potential to impact our understanding of neurophysiology, gerontics, psychology and behavior because of the similarities between human and their dissimilarities to rodents. It is clear that the current scientific needs can not be satisfied by transgenetic rodents. It is desirable to establish higher transgenetic animal model, therefore, alternative approaches must be considered and one of the most promising involves development of the assisted reproductive technologies (ARTs) on NHPs.In experiment 1,65 sexually mature cynomolgus monkey females were selected for ovarian hyperstimulation with different methods, oocytes were collected via laparoscopy. We screened out the best method, rFSH (Puregon) /hCG with GnRH agonist, which resulted 31.8 average tatal oocytes and 10.6 average oocytes in Metaphase II (MII). To construct embryos, collected MII oocytes were utilized for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The fertilized rate of ICSI was significant higher than that of IVF (43.5±11.6% vs 22.5±7.2%, P <0.01). 21 pronuclear embryos following IVF and 30 following ICSI were selected and tubal transferred to 25 recipients assisted by laparoscopy. Finally, four monkeys became pregnant resulting in birth of two healty infants, one pregnancy and one abortion. The pregnancy rate and implantation rate was 16% (4/25) and 7.8% (4/51). But when seperatting the groups of recipients according to blood E₂ assay , The pregnancy rate and implantation rate was 50% (3/6) and 25% (3/12) in the group 2 in which the recipients had E₂ surge two days before the day of embryo transfer.In experiment 2 of present study, we tested the effects of different factors on in vitro maturation (IVM) of oocytes in the stage of metaphase I (MI) and germinal vesicle (GV) which accounted for 2/3 oocytes drived from hyperstimulation. These work would help us to obtain more MII oocytes and disscuss the mechanism of oocytes IVM. First, we demonstrated that FBS significantly increased the ability of reaching MII of MI oocytes (67.2±24.3% vs 55.1±16.5%, P <0.05), but did not effect the ability of firtilization following ICSI. For GV oocytes, we disparted two groups with or without granulosa. Without granulosa, EGF, gonadotrophins (PMSG+hCG, short for Gn) and in combination did not effect the ability of GVBD, but Gn enhanced the rate of MII (37.5±8.9% vs 17.6±6.8%, P <0.05), and EGF inhibited the positive effect of gonadotrophins. With granulosa, more oocytes cultured in the medium with EGF and Gn reaching GVBD (75.0±0.0% vs 57.1±9.6%, P <0.05) and MII (58.3±7.2% vs 35.7±9.9%, P <0.05) compared with control. The result in co-culture group was further demonstrated by COCs. In conclusion, granulosa in synergy with EGF and gonadotrophins was significantly improve the maturation of GV oocytes (GVBD: 75.0±0.0% vs 51.6±9.6%, P <0.01; GVBD-MII: 58.3±7.2 % vs 22.6±8.4%, P <0.01). Immune analysis revealing the expression of EGF receptor (EGFR) on granulosa.