Effect of Epigallocatechin-3-gallate (EGCG) on Fresh/post-thawed Sperm of Kunming Mouse in Vitro Fertilization and Embryonic Development
|Keywords||Table epigallocatechin gallate (EGCG) In vitro fertilization (IVF) Vitro Embryonic development Kunming (KM) mice|
Foreword assisted reproductive technology (assisted reproductive techniques, ART) has become infertile couples nurture the next generation of important therapeutic measures, however ART, such as semen freezing, IVF (in vitro fertilization, IVF) technology, the body can produce more higher levels of active oxygen species (reactive oxidative species, ROS), the gametes or embryos in oxidative stress (oxidative stress), induce the occurrence of mitochondrial changes, DNA damage, embryo developmental delay or block a series of oxidation stress damage. Currently, ROS has been considered the main reason for loss of embryos in vitro, and the application process ART embryonic cells against oxidative damage protection and repair after oxidative damage has attracted the attention of people. In vitro culture system is to increase the added antioxidants embryonic cells against oxidative injury in one of effective measures, unfortunately, has yet to find an ideal antioxidant. In recent years, tea attracted much attention because of its health benefits, researchers found that green tea extract the most active catechins - Table epigallocatechin gallate (epigallocatechin-3-gallate, EGCG) is a good anti-oxidants, not only suppression and scavenging oxygen free radicals, the role of chelating metal ions, but also presents a high degree of oxidation-reduction power; but also affect the repair system to promote DNA repair, anti-mutagenic, anti-tumor functions. Because animal experiments suggest that EGCG pregnant rats after administration, EGCG can be distributed to multiple fetal organs, and EGCG no obvious direct embryo - fetal toxicity and teratogenicity, which EGCG is considered to be an ideal antioxidant, EGCG The application of ART also begun to attract attention, but research is still very limited. Purpose 1. Analysis of EGCG on Kunming (KM) mice Fresh / thawed sperm IVF Embryos. (2) investigate the EGCG used in IVF embryo culture system security. Method 1. Were cultured in vitro fertilization system by semen, embryo culture medium supplemented with different concentrations of EGCG (0,5,10,15,25 μg / ml); or both by semen, embryo culture medium supplemented with different concentrations of EGCG (0,5,10,15,25 μg / ml), KM mice were observed and compared with fresh sperm IVF fertilization rate and embryo development (cleavage rate and blastocyst formation rate and very good 4 - cell embryos rate ). 2, while in vitro culture system by semen and embryo culture medium supplemented with different concentrations of EGCG (0,10,17.5,25,50 μg / ml), KM mice were observed and compared thawed sperm fertilization rate in vitro fertilization and embryonic developmental competence (cleavage rate, four - cell embryo formation rate, very good 4 - cell embryos and blastocyst formation rate ratio). Results 1 with the control group (EGCG 0μg/ml) compared to IVF culture system after adding 10μg/ml EGCG significantly improve the KM mice sperm IVF fresh blastocyst formation rate and very good 4 - cell embryos rate ( P lt; 0.05; P lt; 0.05). Three kinds of EGCG add: added separately by semen, embryo culture medium added individually or by semen and embryo culture medium at the same time added on in vitro fertilization outcome beneficial effects are present, in order to add a last way more obvious, but the difference was not statistically significant (P gt; 0.05). However, when added by EGCG concentration in semen increased to 25μg/ml, he could significantly reduce the rate of fertilization in mice (P lt; 0.05); embryo development medium supplemented with 25μg/ml EGCG, the cleavage rate decreased significantly (P lt; 0.01); And were found four - cell embryos and blastocyst formation. In addition, 5,15,20 μg / ml EGCG concentration added, no significant effect on in vitro fertilization in mice, as compared with the control group, the fertilization rate, cleavage rate, blastocyst formation rate and very good 4 - cell embryos observed rate of four index changes were not statistically significant (P gt; 0.05). 2. KM mouse sperm cryopreservation, the viability and vitality have undergone a decline; and thawed sperm IVF group and cleavage rates, blastocyst formation rate was lower than fresh sperm, the difference was statistically significant ( P lt; 0.05; P lt; 0.05; P lt; 0.05). With the control group (EGCG 0μg/ml) compared to when KM mice thawed sperm IVF culture system add 10,17.5 μg / ml EGCG after in vitro fertilization can improve fertilization rate, cleavage rate, four - cell embryo formation rate, very good 4 - cell embryos rate and blastocyst formation rate, where the fertilization rate and very good 4 - cell embryos rate increase was statistically significant (P lt; 0.05; P lt; 0.05), add to 17.5μg/ml concentration effect is more obvious. 25μg/ml EGCG added concentration on KM mice thawed sperm in vitro fertilization is not obvious, the observed indicators of change was not statistically significant (P gt; 0.05). When added to 50μg/ml concentration, the zygote is not observed. Conclusion 1. KM mice IVF culture system to add the suitable concentration of EGCG can improve sperm IVF fresh blastocyst formation rate and very good 4 - cell embryos rate, and promote embryo development. However, high concentrations of EGCG but it can lead to KM mice fresh IVF rates dropped significantly, and cause embryonic development block. IVF embryos that EGCG on concentration changes with added presence of beneficial and harmful effects. EGCG application must pay attention to their safety. (2) adopt by semen and embryo culture medium simultaneously with antioxidants, that is anti-oxidant protection measures to minimize damage throughout vitro fertilization, pre-implantation embryo culture process, embryonic cells may be more conducive to the protection and promotion of embryonic cell development. 3. KM mice in vitro culture system to add the suitable concentration of EGCG on IVF embryos thawed sperm present favorable impact; when EGCG concentration exceeds the appropriate range when added barred from IVF embryos thawed sperm. 4 compared with fresh sperm, even when thawed sperm in vitro fertilization should be noted that the use of anti oxidative damage protection.