The Study Of Development And Cell Cycle Checkpoint Response Of Early Embryos In Vitro Fertilized With DNA-damaged Sperm Induced By Oxidative Stress

Background Cryopreservation of human semen is of great importance in assisted reproductive technology (ART) and has been widely used as a vital method for the fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. The major defect of frozen-thawed spermatozoa is that it may cause a decrease in the viability and motility of sperm, and destruction of DNA integrity, which is especially prominent in infertile men. Mechanisms underlying the cryodamage to spermatozoa are not yet known, but recently the excessive generation of reactive oxygen species (ROS) has been considered as a major contributing factor. ROS can cause several forms of sperm DNA damage, e.g. chromatin cross-linking, chromosome deletion, base oxidation and DNA strand damage. H2O2 has the strong oxidative activity and can generate (O2 ? -) and hydroxyl radical (? OH) persistently when react with O2. Thus different concentrations of it in this experiment were used as reagents inducing DNA damage to establish the DNA-damaged model of oxidative stress of cryopreserved spermatozoa to investigate its mechanisms of repair.In eukaryotes, in order to maintain the integrity of the genome, DNA damage activates the system of DNA repair and cell cycle checkpoint, which monitors DNA damage, regulates DNA repair and controls cell cycle process and programmed cell death. Activation of cell cycle checkpoint signal pathways can slow down the process of cell cycle, open up a time window for transcription and translation of genes either involved in repair of DNA damage or in cell apoptosis. Sperm can not repair DNA damage by itself, however the DNA-damaged sperm is still capable of fertilizing oocyte and has potentiality of development and DNA damage can be repaired after fertilization. But so far there have been few studies investigating the role of DNA damage and repair system or cell cycle checkpoint signal pathways in embryonic development. In this study, we first explored a reasonable oxidative stress DNA-damaged model of cryopreserved spermatozoa. Based on this model, we studied the activation of cell cycle checkpoint signal pathway which triggered mitotic cell cycle arrest or delay in early mouse embryos in vitro fertilized with DNA-damaged sperm induced by oxidative stress.Objective (1) To study the development of mouse embryos in vitro fertilized with DNA-damaged sperm induced by oxidative stress. (2) To research the activation of cell cycle checkpoint response during the development of mouse early embryos in vitro fertilized with DNA-damaged sperm induced by oxidative stress.Methods (1) Observe the fertilization rate of the fresh sperm group and the 1.0mM H2O2 treated sperm group, the cleavage rate and following development of 1-cell embryos and 2-cell embryos in vitro fertilized with the two groups by the inverted microscope. (2) Indirect immunofluorescent detection of Bromodeoxyuridine(BrdU) incorporation to determine the onset and duration of S-phase of the mitotic cell cycles of 1-cell embryos. Access the onset of G1-phase by the time when zygotes of each group emitted the second polar body. Assess the mean length of the first cell cycle after observations of first cleavage rate for embryos fertilized with fresh semen and semen with H2O2 treatment. And then calculate the duration of different phases(G1-phase,S-phase,G2/M-phase)of the first mitotic cell cycle according to the above-mentioned.Results (1) The fertilization rates the fresh sperm group and the 1.0mM H2O2 treated sperm group were 71.4±16.1% and 62.6±12.3%,respectively, and the difference was not statistically significant (P>0.05). After 24 hours post-insemination, the cleavage rates of 1-cell embryos in vitro fertilized with the fresh sperm and the 1.0mM H2O2 treated sperm were 98.3±0.5% and 97.2±1.6%, and there was no significant difference between the two groups (P>0.05). And after 48 hours post-insemination, 2-cell embryonic cleavage rates were 95.3±0.7% and 87.3±6.5%, respectively, and the difference was also not statistically significant (P>0.05).The time when the cleavage rate of 1-cells embryos reached≥95% was 20±0.9 hours post-insemination in fresh sperm group and 22.5±1.1 hours post-insemination in 1.0mM H2O2 treated sperm group, and the difference was statistically significant (P<0.05). Therefore, the cleavage time of 1-cells embryos in vitro fertilized with 1.0mM H2O2 treated sperm delayed for about 2.5 hours on average. The time when the cleavage rate of 2-cells embryos reached≥95% was 48.8±1.5 hours post-insemination in fresh sperm group and 51±2.7 hours post-insemination in 1.0mM H2O2 treated sperm group, and there was no statistically significant difference (P>0.05). Compared with fresh sperm, 1-cell embryos of 1.0mM H2O2 treated sperm group came through the delayed mitotic division, and the 2-cell embryos had a similar cleavage time with the normal embryos. (2) The time point of initiation of G1-phase of 1-cell embryo in fresh sperm group and 1.0mM H2O2 treated sperm group were 1.8±1.0 hours post-insemination and 1.7±1.1 hours post-insemination respectively, and there was no statistically significant difference (P>0.05). The onset of S-phase in fresh sperm group and 1.0mM H2O2 treated sperm group were 9.8±0.6 hours post-insemination and 10.3±1.0 hours post-insemination, and there was no statistically significant difference (P>0.05). The end of S-phase in fresh sperm group and 1.0mM H2O2 treated sperm group were 17.0±0.8 hours post-insemination and 17.7±0.6 hours post-insemination, and there was also no statistically significant difference between the two groups (P>0.05). So, the duration of G1 phase in fresh sperm group and 1.0mM H2O2 treated sperm group were 7.2 hours and 7.4 hours respectively. The G2/M phase in 1.0Mm H2O2 group lasts nearly for 4.8 hours, which is about 1.8 hours longer than the fresh sperm group which lasted for about 3 hours. And this indicated that 1-cell embryo in vitro fertilized with 1.0Mm H2O2 treatment had a prolonged G2/M-phase.Conclusions (1) The fertilizition rate and cleavage rate of mouse early embryos in vitro fertilized with DNA-damaged sperm induced by oxidative stress are not changed, but 1-cell embryos come through a delayed mitotic division, while 2-cell embryos had a similar cleavage time with the normal embryos. (2)1-cell mouse embryos in vitro fertilized with DNA-damaged sperm induced by oxidative stress activates the of G2/M cell cycle checkpoint to delay the progress of the first mitotic cell cycle.