Effects of inducers and maturation duration on in vitro maturation and fertilization of mouse oocytes, and preimplantation embryo development

Abstract

In vitro fertilization and development of embryos from oocytes matured in vitro are still by far much lower than those matured in vivo, suggesting that oocytes and/or procedures for the oocyte maturation being used are of suboptimum. This study was carried out on the purpose of developing improved procedures for IVM, IVF, and cultivation of embryos in vitro, concentrating on the effects of oocyte maturation inducers (PMSG and hCG), oocyte maturation durations in vitro (3-24 hours), and intraoocyte cyclic adenosine monophosphate (cAMP), on in vitro oocyte maturation, fertilization, and preimplantation embryo development. In the first experiment, the effect of oocyte maturation inducers was explored. Oocytes at germinal vesicle (GV) stage obtained from various groups of immature female mice 48 hours after injection of various concentrations (5, 7.5, 10 IU) of either PMSG or hCG were cultured in a maturation medium for 24 hours under an atmosphere of 5% CO2 in air at 37 °C. Oocytes developed to metaphase II (Mil) stage were subsequently tested for their efficiencies for fertilization and development to blastocysts in T6 medium. In the second experiment, the effect of in vitro maturation durations was explored. GV stage oocytes obtained 48 hours after 7.5 IU PMSG or hCG injection were cultured in maturation medium for 3-24 hours. At various intervals, oocytes developed to Mil stage were subsequently tested for their efficiencies for fertilization and development to blastocysts as in experiment I. The level of intraoocyte cAMP, zona thickness, and zona hardness, were also measured to elucidate the effect of oocyte maturation duration in vitro on these factors, which accordingly affect their fertilization and development. Results from this study suggested that the suitable concentration of gonadotropins for the stimulation of immature female mice to obtain immature oocytes for IVM/IVF studies in the mouse model is 7.5 IU/mouse. However, hCG, more effective than PMSG, plays a major role in nuclear maturation (increase oocyte maturation: 87.6 ± 3.4% versus 76.4 ± 3.2%, p< 0.05), cytoplasmic maturation (increase the fertilization rate: 65.5 ± 3.4% versus 51.0 ± 4.7%, p<0.05 ; decrease the level of intra oocyte cAMP : 0.03 fmol/oocyte versus 0.08 fmol/oocyte, p<0.05 ; and decrease the zona digestion time : 1,882 ± 147 seconds versus 1,668 ± 103 seconds, p<0.05), and preimplantation embryo development (increase blastocyst development : 64.4 ± 2.5% versus 57.6 ± 3.1%, p<0.05) respectively. The optimum maturation duration of the GV stage oocytes for the highest rate of IVM/IVF studies in this mouse model is 15 hours. In conclusion, hCG is superior to PMSG, and 15 hours is the optimum duration for in vitro oocyte maturation yielding the highest rate of fertilization and preimplantation development of embryo in vitro. In addition, decreasing the level of intra oocyte cAMP increase the percentage of oocyte maturation, fertilization, and preimplantation embryo development, and decrease the zona hardness