| ForewordMammalian cell will initiate a complex protein'network in response to DNA double-stranded breaks (DSBs), which includes cell cycle checkpoint, DNA damage repair and apoptosis. DNA damage signal transduction pathway is a highly conserved one, which could be devided into three parts: sensors, transducers and effectors. Phosphoinositide 3-kinaserelated protein kinases (PI-3K) family are activated according to different DNA damage sources and quickly recruited to the damage site.It phosphorylates downstream kinases, which work cooperatively in order to carry out the concordant cellular responses to DNA damage, including H2AX, which is rapidly phosphorylated at Ser139 to formγH2AX once DSBs occurs and is characterized as the marker of DNA damage. Ataxia telangiectasia mutated (ATM) can be activated by the chromatin modification associated with DNA damage, making it the most match kinase for H2AX phosphorylation of PI-3K family. ATM also regulates cell cycle checkpoint and apoptosis: Activated ATM phosphorylates and activates cell cycle checkpoint kinases Chk1 and Chk2, mediates cascade reaction and amplifys damage signal.Chk1/Chk2 phosphorylates its effectors to activate cell cycle checkpoints (G1/S, S, and G2/M) by different signaling pathways according to different DNA sources, so as to stop the cell cycle and provide time for DNA repair.Semen cryopreservation is an very important technology in assisted reproductive technology (ART) field. But it could produces excessive reactive oxygen species (ROS) during frozen-thawed process and induces oxidative damge on sperm.With the application of ART, especially intra-cytoplasmic sperm injection(ICSI),which avoids multiple natural barriers for sperm screening, the opportunity raises for DNA-damge sperm to fertilize and develop into embryo. DNA-damage sperm still can fertilize, while it is unable to repair damaged DNA for mature sperm, and depends on the repair of zygotes after fertilization. However, the research on repair in zygote fertilized with oxidative stress-induced DNA-damage sperm is limited.Because of the association between oxidative stress-induced DNA-damage and ROS, it may be beneficial for ART outcomes by adding antioxidant into the ART system to reduce the DNA damage caused by ROS. Epigallocatechin-3-gallate (EGCG) is the best candidate due to its strongly anti-oxidation activity and protective effect on cell and DNA. It was added to the ART system and found it could promote embryo development, but the mechanism remains unknown.Objective1. To explore repair mechanism in zygote fertilized with oxidative stress-induced DNA-damage sperm.2. To explore protective mechanism of EGCG on zygote fertilized with oxidative stress-induced DNA-damage sperm.Methods1. KM mouse sperm was added to capacitation agent of fresh or 1mM H2O2 for capacity in 5%CO2,37℃incubator for 1.5 hours,so as to get fresh and oxidative DNA-damage sperm for IVF, adjusting the concentration of sperm to 1-2.5×106/ml. It was devided into two groups: the control group without H2O2 in capacitation agent and the oxidative DNA -damage group with 1mM H2O2 in capacitation agent, pSer1981-ATM ,pSer345-Chk1 and pThr68-Chk2 signaling in zygotes of pronucleus stage was detected by immunofluorescence and analyzed.2. The zygotes fertilized with oxidative DNA-damage sperm were got from the first part and divided into two groups: the blank group without EGCG neither in insemination agent nor development agent; the experimental group with 17.5μg/ml EGCG both in insemination agent and development agent. Zogotes of above two groups were observed every one hour from 16.5 hours post-insemination(hpi) to 23.5 hpi and analyzed their fertilization rate,one- and two-cell cleavage rate. PSer1981-ATM signaling in zygotes of above the observation time between the blank group and the experimental group were detected by immunofluorescence and analyzed. Results1. PSer1981-ATM and pSer345-Chk1 signaling were found in zygote with treated group while no positive result was found in the control group. No pThr68-Chk2 signaling was found in neither the control group nor the oxidative DNA-damage group.2. The fertilization rate, the cleavage rate of one- and two-cell embryos was 44.5%,97.8% and 71.3% respectively of the experimental group, 45.2%,100% and 61.5% respectively of the blank group,there was no statistical significance for fertilization rate, one- and two-cell cleavage rate between them(P>0.05).The cleavage rate of one-cell embryos of the experimental group was 32%,70%,83%,87%,89%,90%,95%,96% respectively from 16.5 hpi to 23.5 hpi,while the cleavage rate of one-cell embryos of the blank group was 21%,34%,68%,86%,89%,91%,96%,98% respectively. The cleavage rate of one-cell embryos at the same observation time between the blank group and the experimental group was analyzed and found that there was statistical significance for them when at 17.5hpi and 18.5hpi(P<0.05),while there was no statistical significance at the other observation time (P>0.05).The zygotes in the experimental group entered M phase about one hour earlier when determined as the time at which 50% of the embyros had cleaved. PSer1981-ATM signaling was found in the experimental group and it was stronger than that in the blank group, fluorescence analysis showed the intensity was 0.0196±0.010347 and 0.008327±0.006603 for the experimental group and the blank group respectively,there was statistical significance(P<0.05).Conclusions1. It relied on the mRNA and proteins stored in oocytes for the repair of oxidative DNA-damage sperm post fertilization. It might be ATM that initiated the DNA damage repair mechanism in zygotes; meanwhile, ATM might activate Chk1, initiate the cell cycle checkpoint and delay cell division, so as to gain time for repair.2. It was possible that EGCG mediated the DNA damage repair mechanism by accelerating the speed for entering M phase and prolonging the duration of M phase; EGCG might promote the repair of zygote fertilized with oxidative DNA-damage sperm which mediated by ATM... |
PDF Full Text Request