Study On In Vitro Maturation Of Porcine Oocytes And In Vitro Culture Of Parthenogenetic Embryos

Pork is the mainly meat of human daily life, the output of pork accounts for more than 40% of the total output of meat all over the world. In china, pig breeding is more than half of the world’s total breeding; hog industry occupies a very important position in agriculture and the whole national economy. It is extremely close to human in physiological and anatomical aspects can be a good human organ transplant donor. With the development of science and technology, in vitro fertilization, nuclear transfer, the embryo engineering technology such as organ transplantation is increasingly mature, the cultivation quality of oocyte maturation in vitro is one of the key factors to determine the success or failure, but up to now, we cannot control oocyte in vitro mature stablely. Parthenogenetic activation can provide a favourable experimental material to explore early embryonic developmental mechanism, analysis of related gene function in process of development. Currently, the studies of parthenogenetic activation method and the activation parameters have been relatively mature, but there are still some problems exist in the process of parthenogenetic embryos in vitro culture, and these problems are difficult to overcome, such as how to break the cell period in the future development of the block, how to increase the blastocyst rate, reduce the rate of deformation of cleavage, etc., these problems are the existing to be solved. So, the factors on in vitro maturation of porcine oocytes and in vitro development of porcine parthenogenetic embryos were observed, which can provide the reference basis for the related research in the future. These will have important significance for development of the embryo engineering and biomedicine including organ transplantation in the future.In the research, the porcine oocytes and embryos were exposed under different treatment in vitro maturation and parthenogenetic embryos culture, in order to optimize the porcine oocyte and embryo culture in vitro; tested the key genes related growth and development of porcine oocyte by real-time PCR, in order to further understand the molecular mechanisms of the oocyte and embryo growth. The results as follows:1. The embryos were treated with different concentrations (0 ng/ml,10 ng/ml,15 ng/ml,20 ng/ml,30 ng/ml, and 40 ng/ml) of EGF for 44 h; maturation rate, cleavage rate and blasiocyst raie of oocytes were compared. statistical analysis results showed that the cleavage rate and blastocyst rate of oocytes in maturation media with 10 ng/ml EGF were significantly higher than blank treatment groups (P<0.05). But there were no significant change in the maturation rate and cleavage rate of oocytes after the concentration of EGF more than 10 ng/ml. And there were no significant difference between the group of the 40 ng/ml EGF and the blank treatment group.2. There were four different concentration (0 ng/ml,0.5 ng/ml,1 ng/ml,2 ng/ml) of LIF in the test, the test is divided into three groups:the IVM group (only adding different concentrations of LIF to in vitro maturation medium), the IVC group (only adding different concentration of LIF in embryo culture medium), the IVM-IVC group (adding different concentration of LIF to IVM medium and IVC medium), after in vitro maturation culture and parthenogenetic activation, statistics for maturation rate, cleavage rate and blastocyst rate, the result showed:adding 0.5 ng/ml of LIF in IVM-IVC group(adding 0.5 ng/ml of LIF to IVM medium and IVC medium) is the most beneficial to improve in vitro maturation of porcine oocyte and embryonic development after parthenogenetic electricity activation.3. Removing cumulus cell in different time (18 h,24 h,38 h, and 44 h) after cultivation, statistics for maturation rate, cleavage rate and blastocyst rate, the results showed:maturation rate was no significant influence by the time of removing cumulus cell. The cleavage rate and blastocyst rate of oocytes of removing cumulus cell after cultivation of 18 h was significantly improved in the together culture group. There were no significant differences in the cleavage rate of removing cumulus cell after cultivation of 18 h and cultivation of 44 h, but the blastocyst rate of oocytes of removing cumulus cell after cultivation of 18 h were significantly higher than cultivation of 44 h in the alone culture group. So the 18 h may be a key point in vitro maturation of porcine oocyte. Destroying the structure of COCs at this time, may be give a stimulus signal to the cell connection between oocytes and cumulus cell by cutting off cell communication between cumulus cell and oocytes, which further affect the expression of some key genes in the oocytes, promote the karyoplasms mature synchronization.4. Examined the expression differences of four genes (GLUT1, CDX2, HSP70 and ploy A) in mature oocytes of porcine after treatment of different concentrations of LIF by real-time PCR. Researched expression patterns of the four genes, which in the different growth period of oocytes and parthenogenetic embryos. Real-time PCR results showed that:(1) GLUT1 gene expression in oocytes treated with 0 ng/ml and 1 ng/ml of LIF concentration is significantly higher than the 0.5 ng/ml treated group (P< 0.05); HSP70、CDX2 and ploy A gene expression are all highest in mature oocytes treated with 0 ng/ml of LIF concentration, and their expression quantity decreases with the increases of the LIF concentration. Show that LIF can inhibit HSP70, CDX2 and ploy A three gene expression.(2) GLUT1 gene expression quantity in oocytes is highest in 0 h of oocyte culture time (P< 0.05); CDX2 gene expression quantity is highest in blastocyst stage, its expression quantity is significantly higher than in other stages (P< 0.05); HSP70 and ploy A gene expression quantity is highest in oocyte culture to 44 h, its expression quantity is significantly higher than other groups (P< 0.05).The results showed the optimal adding concentration of EGF is 10 ng/ml in vitro maturation of porcine oocytes. Reasonable to add LIF can improve maturation rate of porcine oocytes and blastocyst rate of parthenogenetic embryos, it is conducive to improve and optimize the porcine oocytes and embryos in vitro culture system. And found two crucial points in time in vitro maturation of oocytes cultivate in the experiment, which is the 18 h and 44 h after cultivated, it can provide a new research direction for the further research about in vitro culture of porcine oocytes and other related research, it has great significance to explore the molecular regulatory mechanism in oocyte maturation. Detection of expression differences of key gene related growth and development, which can provide reference material for the future" studying gene expression differences of oocyte and embryo, that can lay a foundation for the further understand the molecular mechanism of growth and development of oocytes and embryos.