| As a female reproductive cells, oocyte provides maternal genic materials to offspring, and also supports early embryo development before implantation. The quality of oocyte maturation determines if early embryo could developed normally. Establishment of oocyte quality evaluation standard is important to predict embryo development. In clinical, for cases with ovulation problem, the procedure of germinal vesicle (GV) stage oocyte maturation, fertilization and intracytoplasm sperm injection are the key steps for human assisted reproductive technology (ART). Thus, selection of oocytes with high developmental potential is the key to improve the results of human ART.There are three major morphology characters for evaluating oocytes quality:cumulus cells, zona pellucida (ZP), perivitelline space. The correlation between these three morphology characters and GV stage oocyte quality has not been widely investigated. In our experiments, based on the cumulus layers thickness, zona pellucid thickness as well as perivitelline space width, we classified mouse GV stage oocytes into several groups. The relationship between these three morphology characters and oocyte quality were studied. Further, by changing cumulus layers thickness, zona pellucid thickness as well as perivitelline space width, the reliability of these standard for evaluating oocyte quality was studied.Our data showed that:(1) oocytes with more than three cunmulus layers has higher ratio of maturation, fertilization and embryo development. Partial removing cumulus cells did not affect oocyte maturation, but impair oocyte fertilization and embryo development.(2) Oocytes with ZP between5μm to8μm (5μm≤Z<8μm) has higher maturation potential. On the contrary, those with ZP less than5μm(Z<5μm) could not be matured and fertilized. Although reducing zona pellucid from8μm (Z≥8μm) to the rage of5-8μm (5μm<Z<8μm) could improve maturation rate, neither the fertilization nor embryo development could be improved.(3) Wide perivitelline space (P>5μm) benefit first polar body extrusion. But this did not benefit fertilization as well as embryo development. Oocytes with perivitelline space less than1μm (P>5μm) could not matured. And this situation could not be conversed by widening the perivitelline space.Our results demonstrated that cumulus cells, ZP and perivitelline space can be used for predicting the potential of oocytes maturation, fertilization and embryo development. These results provide reference for human ART and have significance to improve the successful ratio. For most of mammals, the reproduction is the most important life events to their life cycle in addition to individual survival. Generations of reproduction start in females oocyte development and maturation, combined with fertilization and early embryo development. For the high economic value animals, such as cow, goat, often used (In Vitro Fertilization-Embryo transfer, IVF-ET) technology to expand the breeding, and the IVF-ET technology is one of the key steps in animal transgenic technology research. This technique is based on in vitro maturation of oocytes, therefore, mature oocytes quality directly affects the production of offspring.The signal molecules can also affect the normal development of one of the most important control way in oocyte maturation and early embryonic development in addition to the endogenous gene expression and regulation. This regulation is one of the most common and most direct way to play a role by paracrine factors. GDNF is a kind of neurotrophic factors, the earliest separated from the supernatant of rat glioma cell line. Studies have found that it is plays an important role in mice, pigs oocyte maturation, fertilization and early stage embryo development. Therefore, we use bovine oocytes as experimental material, studied the role of the GDNF on maturation, fertilization and embryo development.Western Blot and Immunostaining are used to sutudy GDNF location during matured follicle, GV oocyte, MII oocyte and early embryo. Right amount of GDNF factor was added to oocyte maturation and early embryonic development solution to observed effects on bovine oocyte maturation and early embryo. Specific siRNA was used to interference GFRA1and statistical bovine oocyte maturation rate.This study focused on finding cows in vitro maturation, fertilization and early embryo development must paracrine molecules, and find the best ratio, and ultimately improve the rate of oocyte maturation, fertilization and early embryo development rate. This research mainly studies in:(1) GDNF and its receptor(Glial cell line derived neurotrophic factor (GDNF) Family Receptor Alpha1, GFRA1) GFRA1located in bovine ovarian follicles, oocytes and early embryo.(2) oocyte maturation and early embryonic development culture medium was added the right amount of GDNF factor, observe the effect of GDNF on bovine oocyte maturation and early embryonic development.(3) with a specific si-RNA interference expression of GFRA1, statistics bovine oocyte maturation rate. |
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