| Assisted reproductive technology and its derivative technology has been developed rapidly in recent years.From the initial in vitro fertilization and embryo transfer(IVF- ET), to gradually matured single follicle intracytoplasmic sperm injection(ICSI), frozen embryo transfer(FET), and the pre-implantation genetic diagnosis /screening(PGD/PGS)which is popularized recently, more and more reproductive related technical challenges have been overcome and more and more infertile couples benefit from it.Gamete and embryo freezing technology is an important part of assisted reproduction and fertility preservation areas, where the slow freezing and vitrification of embryos has been routinely used in assisted reproduction centers around the world, but because of oocytes' large volume, including more water, with no nuclear membrane surrounding the nucleus,even low membrane permeability, all these features make it the most difficult to successfully freeze-thaw among all the cell types. A number of previous studies have already shown vitrification technology is more efficient than slow freezing. Therefore, the successful application ofoocyte vitrification technology is not only important for the improvement and development of human assisted reproductive technology, but also play a significant role in the nuclear transfer engineering and reproductive resources' preservation of rare species.Vitrification technology is an ultra-rapid freezing cooling process of using high concentrations of cryoprotectants to replace cytosol of water, thus avoid the formation of intracellular ice crystals, therefore prevent the freezing injury. However, ultra-fast freezing of oocytes vitrification process and the cryoprotectants itself will do harm to the internal ultrastructure and accessory structures of oocytes(such as the zona pellucida, spindle, cytoskeleton, etc.),hence affecting the recovery rate of vitrificated oocytes?fertilization rate after IVF and the development potential of embryoes. Researches in the field of oocyte freezing techniques are mostly concentrated in comparing two freezing methods(slow freezing and vitrification),frozen efficiency of different carriers, establish and improvement of freezing and stem, etc. However,researches on specific mechanism of freezing technology affecting oocytes(such as gene expression, epigenetic modifications, etc) are very rare. Oocyte-specific genes are a class of genes associated with the development of oocytes, involved in the generation and maturation of follicles?fertilization and early embryonic development. But very few studies are combining oocyte vitrification with oocyte-specific genesnow.In this study, mature(MII) oocytes were treated with vitrification-thaw fluids or went through vitrification-thaw procedure,fresh oocytes as control group. measure the m RNA level of five major oocytes-specific genes(H1foo?Bmp15?Gdf9?Sohlh2?Nobox) and early embryo development situation,to explore the effact of vitrification-recovery solution toxicity and vitrification technique on mature oocyte oocyte-specific genes expression level and the development of early embryo development potential after in vitro fertilization. By comparing methylation levels of H1foo-? gene in fresh and vitrified- MII oocytes,preliminary explore the mechanism of oocyte vitrification affectting the expression of specific histone H1 foo.First, mature oocytes of C57 BL / 6 mice were randomly divided into:(1) fresh group,(2)exposed to vitrification and thaw solution without being plunged into liquid nitrogen,(3) vitrified by regular method using cryotop.40 in each group were taken to measure oocyte-specific gene( H1 foo, Bmp15, Gdf9, Sohlh2, Nobox) m RNA expression levels.We found one of H1 foo subtypes--H1foo-?,which m RNA expression in vitrification-thawed group was significantly lower than the solution treated group and the control group of fresh(P <0.05), while the other subtype H1foo-? and the other four oocyte-specific gene Bmp15, Gdf9,Sohlh2 and Nobox m RNA expression has no significant differenceamong 3 groups(P> 0.05). Western blot is used to test the protein expression levels of two H1 foo subtypes in the fresh group and vitrification group, the results showed coincidence with Real-time PCR results---- H1foo-? protein levels in the vitrification recovery group was significantly lower than fresh group.Further study was taken to measure the H1 foo gene promoter sequence methylation pattern.Vitrification group(100 MII oocytes)methylation degree(87.5%) is higher than the fresh group(75%), but the difference was not statistically significant(P> 0.05).In the second part of the experiment, 197 mature oocytes were randomly divided into three groups, 64 fresh oocytes in the control group,65 oocytes in vitrification solution treatment group, 68 in vitrification recovery group.After in vitro fertilization,Compared early 2-cell embryos rate and blastocysts rate obtained from each group. In vitrification solution treated group 2-cell rate(cleavage) and blastocyst rates were77.42% and 56.25%, respectively, with control group 2-cell rate81.25% and blastocyst rate 57.7%, there's no significant difference(P>0.05).However, in frozen-thawed group, the 2-cell rate(63.93%) after in vitro fertilization were significantly decreased(P <0.05) compared with the control group and vitrification solution treated group, while the blastocyst rate(53.84%) decreased compared with other two groups,only the difference was not statistically significant(P> 0.05).In conclusion, vitrification and thaw solutions have no obvious effect on the expression of oocyte-specific genes and the potential of early embryonic development after in-vitro fertilization. Whereas the vitrified-thawed process would significantly decrease the developmental rate to 2-cell embryos and oocyte-specific histone H1foo-? expression level both in m RNA and protein.To find out the mechanism of this effect,BSP was used to measure the methylation level of promoter sequence in H1 foo gene, it turns out that the methylation level in vitrification group was higher than control group,but the difference has no statistically significance.It still need further reaserch to prove. |
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