| During male gametogenesis, the spermatogonia became primary spermatozoa through mitosis, and then became fourth equivalent cytoplasm, round sperm through meiosis. After maturation, they participated in embryo development with the mature oocyte through fertilization. But in female gametogenesis, the primary oocyte formed a matured egg and 1~3 polar bodies (PB). In the past, it was thought that polar bodis are a non-functional, small cell with the chromosome and few cytoplasms.The objective of this study was whether the reconstituted embryo with the second polar body (PB2) can develop. Firstly, female mouse was superovulated with PMSG + hCG and keep in the same cage with male mouse, the pronuclear embryo was obtained at the different time after hCG injection with different motheds to investigate the existing time of pronuclear embryo. Then pronuclear embryo was cultured with three different mediums to select the best to culture in vitro. Meanwhile, the polar body and pronuclear's behavior was observed. After obtaining the pronuclear embryo with PB2, enucleating the female pronuclear and aspirating the PB2, PB2 was injected into the enucleated pronuclear embryo cytoplasm. The reconstituted embryo was cultured in vitro. The results as follows:Studies on the time of obtaining the pronuclear embryo. The superovulated mouse was killed atthe different time after hCG injection with two different methods (in vivo development and in vitroculture) to obtain the pronuclear embryo. The results demonstrated that the rate of the pronuclearembryo increased with the hCG injection time. From 18h to 24h after hCG injection with in vivodevelopment methods, the rate of the pronuclear embryo increased from 14.93% (10/67) to 79.66%(47/59) and the results was significantly different (p<0.01); the rate decreased slightly to 73.81%(62/84) at 26h after hCG injection. So these results showed that at 24h after hCG injection the rateof the pronuclear embryo is highest with in vivo development methods. From 18h to 26h after hCGinjection with the in vitro culture methods, the rate of the pronuclear embryo increased from 2.82% (8/248) to 92.74%(230/248), and the results was significantly different (p<0.01). The rate of the pronuclear embryo was higher (79.66% VS 77.02%) at 24h with in vivo development methods comparing with in vitro culture methods, but the pike value appeared (92.74%) at 26h with IVC methods and the difference is significant comparing with the data at 24h (p<0.01). The results showed that the pronuclear embryo can be obtained at 24h after hCG injection, and the embryo developmental speed in vivo was higher than that in vitro.The pronuclear embryo was cultured with M16, mM16 (M16+taurine+EDTA.NA2+glutamine) and mCZB. The results were that the cleavage of the pronuclear embryo was 69.90% (72/103), 94.91% (317/334) and 63.16% (60/95) respectively. The results of mM16 were different comparing with that of M16 and mCZB (p<0.05), but there was no difference between M16 and mCZB (p>0.05). That showed the taurine and EDTA.Na2 and glutamine was useful to the pronuclear embryo development in vitro.During in vitro culture, we observed the pronuclear and polar body can exist for a long time, and some of them can reach to 32h after hCG injection. The male pronuclear and female pronuclear was clear in the cytoplasm, and the polar body in perivitalline space. In the early stage of pronuclear embryo, the distance between the male and femal pronuclear was large, and the protruding formed in the oocyte membrane surface. With the development of the embryo, the male and femal pronuclear moved toward to one another, and at last overlaped. But we didn't observe the pronuclear fusion or association.At the 24h after hCG injection obtaining the embryo, the pronuclear embryo with or without PB2 was analyzed. The pronuclear embryo with PB2 was 217 in all 431 embryos (50.35%), the pronuclear embryo without PB was 104 (24.13%) and the abnormal embryo was 32 (7.42%), and the rest was the pronuclear embryo with PB1 or...
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