The Study of Repair in Zygote Fertilized with Oxidative DNA Damaged Sperm and the Antioxidant Effect of Epigallocatechin-3-gallate(EGCG)
|Keywords||ROS Oxidative stress-induced DNA damage Fertilized egg ATM EGCG|
Introduction of DNA double-strand breaks (DNA double-stranded breaks, DSBs) occur in mammalian cells will start a complex protein network to monitor and repair: cell cycle checkpoint, DNA damage repair and apoptosis. DNA damage response is highly conserved signal induction process, damage the sensor, signal transduction and signal effects can be divided into three parts. Damage the sensor molecule 3 - phosphatidylinositol kinase (Phosphoinositide 3-kinaserelated protein kinase, PI-3K) family members for different sources of injury is activated quickly gathered to the injury site, the rapid phosphorylation of downstream effector kinases, through a synergistic The role of highly coordinated response to DNA damage. The H2AX Preparation as family members of the PI-3K molecule, is formed after the 139th serine residue is rapidly phosphorylated gammaH2AX. The latter is considered to be DNA damage repair markers, while the ability of the ATM may be DNA damage associated chromatin modification activated, making it a family of PI-3K procure H2AX phosphorylated kinases most closely matches. , ATM is also involved in the regulation of the cell cycle checkpoint: of activated phosphorylated ATM cell cycle checkpoint kinase Chk1 or Chk2 the active Chk1/Chk2 thus sequential phosphorylation of different effectors, and according to the type of DNA damage, the formation of different The signaling pathway activated cell cycle checkpoint (G1 / S, s, G2 / M), cell cycle arrest, DNA damage repair time. Semen cryopreservation assisted reproductive technology (Assisted Reproductive Technology, ART) in the field of technology, the freezing and thawing process of producing excessive quantities of reactive oxygen species (Reactive oxygen species, ROS) can cause oxidative stress, DNA damage sperm. The ART application of the DNA damage in sperm develop into embryos opportunities, especially intracytoplasmic sperm injection (Intra-cytoplasmic sperm injection, ICSI), due to a variety of natural barriers to avoid the natural process of fertilization screening of sperm , increasing the risk of DNA damage sperm directly into the egg. DNA damage in sperm can still be fertilized, but the loss of mature sperm ability to self-repair DNA damage repair after fertilization, the fertilized egg to rely on. At present, research on oxidative stress-induced DNA damage in sperm fertilized egg to repair is very limited, for the direction of research for the perfect DNA damage repair sperm fertilized egg theory is undoubtedly of great significance. It is because of oxidative stress in sperm DNA damage and ROS is closely related to the antioxidant protection agent is added to the ART system to reduce ROS-induced oxidative stress injury may be an effective way to improve ART treatment outcomes. Epigallocatechin gallate (Epigallocatechin-3-gallate, EGCG) has a very strong antioxidant activity, can protect cells and DNA from damage as an ideal in many antioxidant protection agent, try Add to in vitro fertilization culture system, showing the role to promote embryonic development, but the the thawed sperm fertilized egg protection mechanism is not clear. Clarify EGCG provides a new method of oxidative stress-induced DNA damage in sperm fertilized egg protection mechanism, not only to provide a theoretical basis for the study the frozen sperm oxidative stress, DNA damage control measures, but also to find ways to reduce the risk of DNA damage in sperm genetic strategy ideas. The purpose of a preliminary study oxidative stress-induced DNA damage in sperm the fertilized egg DNA damage repair mechanisms. Explore the protection mechanism of EGCG on oxidative stress-induced DNA damage in sperm fertilized egg. Method 1. Kunming (Kunming, KM) mice sperm into the fresh sperm capacitation liquid plus 1mMH 2 O 2 of sperm capacitation solution, 5% CO 2 , 37 ℃ incubator incubated for about 1.5 hours to get fresh sperm and oxidative stress DNA damage in sperm, adjust the sperm concentration of 1-2.5 × 10 6 sup> a / ml, respectively, in vitro fertilization normal control group, DNA damage and oxidative stress in group fertilized egg. By immunofluorescence to detect the formation of pronuclear stage embryos of the normal control group and oxidative stress-induced DNA damage group pSer1981-ATM, pSer345-Chk1, and pThr68-the CHK2 focus of comparison. 2 based on the semen and embryo culture solution is added with EGCG, oxidative stress-induced DNA damage group fertilized egg divided into two groups: untreated groups by semen and embryos Add EGCG culture medium; Add Group by semen and embryo culture medium Add a 17.5μg/ml EGCG. From 16.5 to 23.5 hours after fertilization, the fertilization rate of the untreated groups and add a group, the two-cell formation and cell formation was observed 1 hour observation, calculate the fertilization rate, cleavage rate of a cell and cell cleavage rate and compared; choose Add Group and add group each observation point in time the fertilized egg, the immunofluorescence assay pSer1981-ATM focus formation and compared. Results 1 immunofluorescence the groups fertilized egg pSer1981-ATM signal found in the normal control group fertilized egg pSer1981-ATM, pSer345-Chk1 and pThr68-Chk2 no signal; oxidative stress-induced DNA damage in the group visible strong In of pSer1981-ATM fluorescence signal and pSer345-Chk1 fluorescence signal, pThr68-Chk2 no clear signal. Not add a group, the fertilization rate, a cell cleavage rate and cell cleavage rate was 45.2%, 100% and 61.5%, respectively, the Add Group fertilization rate, cleavage rate of a cell and cell cleavage rate were 44.5%, 97.8% and 71.3%, add a group compared with the untreated groups, fertilization rate, cleavage rate of a cell and cell cleavage rate differences were not statistically significant (P gt; 0.05) 23.5 hours from 16.5 hours after fertilization until after fertilization, did not add the group of cell cleavage rates were 21%, 34%, 68%, 86%, 89%, 91%, 96%, 98%; Add a The cell cleavage rates were 32%, 70%, 83%, 87%, 89%, 90%, 95%, 96%; add a group and add a set of cell cleavage rate was compared with that for each point in time, 17.5 hours after fertilization and fertilization after 18.5 hours (equivalent to the first mitosis late G2 phase of the cell cycle) cell cleavage rate difference was statistically significant (P lt; 0.05), the other point in time a cell cleavage rate the difference was not statistically significant (P gt; 0.05) Time after fertilization as the independent variable, a cell cleavage was established function of the dependent variable, as observation points to cell cleavage rate of 50% of the time point, when the one-cell embryo cleavage rate of 50%, add a group is 18.28598 hours after fertilization, add a group is 17.10280 hours after fertilization, add into the separatist group than in the untreated groups ahead of time about 1 hour. Did not add the group and add a group pSer1981-ATM fluorescence signal strength 0.008327 ± 0.006603 and 0.0196 ± 0.010347, the difference was statistically significant (P lt; 0.05). Conclusions. Oxidative stress DNA damage sperm DNA damage repair depends on the stored oocytes mRNA and protein, may be provided by the ATM start of the fertilized egg DNA damage repair mechanisms at the same time, ATM is also possible to activate Chk1 activated cell cycle checkpoint, cell division is delayed, so as to gain time to repair. 2. EGCG role in the first cell cycle after fertilization, to accelerate the speed of the fertilized egg into the cleavage, extend the duration of the first cleavage; EGCG may contribute to oxidative stress-induced DNA damage in sperm fertilized egg ATM mediated damage repair.