| This study investigated the effects of epictechin (EC) on the maturation quality of oocytes from mice which accepted repeated superovulation, via detecting the mtDNA copy number in both oocytes and embryos and exploring the antioxidant capacity of ovary, so as to explain the relationship between mtDNA copy number and developmental potential in oocyte, as well as the mechanism that EC impact of oocyte maturation.Firstly, the effects of EC on mtDNA copy number and developmental competence of mouse oocytes after in vitro maturation (IVM) was investigated. Cumulus-oocyte complexes were respectively cultured in the maturation medium supplemented with different concentrations of EC (Oμmol/L,5μmol/L, 10μmol/L,15μmol/L and 20μmol/L) for 16 h, and found that the mtDNA copy number in each EC treatment group were increased than that of control group, and there was remarkably increase in the both groups of 10μmol/L (618223±220584) and 15μmol/L (777851±120487) compared with the control group (304380±60963, P<0.05), but the mtDNA copy number of 10μmol/L group was more closed to that of the oocytes from the nature ovulation (618223±220584 vs 587688±204035, P>0.05). Furthermore, the result of oocytes IVM showed that adding with 10μmol/L EC into the maturation medium, there were no significant effect in the maturation rate of oocytes, but the developmental rate of blastocyst after PA was significantly improved than that of control group (55.71±2.84%vs 48.04 ±3.54%, P<0.05).Secondly, the treatment process of epicatechin in mice were evaluated. The results showed that the mtDNA copy numbers of oocytes from intraperitoneal injection group had no significant difference than that of the gavage group (495758 ± 76910 vs 523196±89292, P>0.05), but both of them had a significant increase compared with their control groups respectively (495758 ±76910 vs 281266 ±42266,523196 ±89292 vs 275836 ±33959, P>0.05). In contrast to the control group, the expression of PGC-1α and Tfam gene in ovarian tissue from injected EC group significantly increased (P<0.05). Moreover, there was no significant difference in oocyte mtDNA copy numbers between injected EC 2 d group and injected EC 7 d group (489723 ± 148982 vs 495758±203486, P>0.05), and the oocyte numbers after superovulation between injected EC 2 d group and control group (22.80 ± 5.84 vs 21.35 ± 5.12) also had no significant difference (P>0.05).Lastly, the effects of epicatechin on the oocyte developmental potential in mice during repeated superovulation were investigated. The results showed that the numbers of 2-cell stage embryos obtained from control groups significantly reduced with the increase of ovarian stimulation cycles, while did not change significantly in the EC-treated groups. Furthermore, it also showed no significant difference in the numbers of harvested embryos between superovulation one time group and the EC-treated groups with different stimulation cycles (20.25 ± 1.06 vs 19.25 ± 1.95vs20.33 ± 1.61vs 21.00 ± 0.71, P> 0.05). While 2-cell stage embryos were cultured in vitro, the rate of blastocyst formation and hatched blastocyst presented a downward trend with the increase of ovarian stimulation cycles. Especially, the rates of hatched blastocyst in the control groups significantly decreased (62.65 ± 0.21 vs 53.52 ± 3.13 vs 42.81 ± 4.83, P<0.05). But it decreased more slowly in the EC-treated groups. When superovulation cycles got to five time, the apoptosis rates of blastocyst in the both of EC-treated group and control group were significantly increased (P<0.05), and the control group was significantly higher than EC-treated group (3.74 ± 0.86 vs 3.10 ± 0.74, P<0.05). Results of qRT-PCR analysis showed that the mtDNA copy number in EC-treated group was significantly higher than that of the control group (489723 ± 148982 vs 298333 ± 139487, P<0.05) in the first stimulation cycles. However, there was no significant difference between each groups (769066 ± 389862 vs 811676 ± 473759 vs 743958 ± 224432 vs 771691 ± 341214, P> 0.05) when stimulation cycles got to 3 and 5. In ovarian stimulation 5th cycle, the expressions of GPX1, PRDX3, SOD and GLS2, and the content of GSH (46.68 ± 2.67 vs 37.74±3.80, P<0.05) in ovary from EC-treated group were significantly higher compared with the control group.These results indicated that:(1) Adding 10μmol/L EC into the maturation medium could increase the mtDNA copy number of oocytes, and contribute to improve its subsequent developmental competence. (2) The supplement of EC via the intraperitoneal injection or intragastric treatment can increase the oocyte mtDNA copy number in mice. (3) Treatting with 10 mg/kg of EC for once daily continuing 2 days during mice superovulation cycles can increase the oocyte mtDNA copy number, but has no effect on the quantity of harvested oocytes. (4) Repeated ovarian stimulation can induce the reduction of harvested embryos number and quality. But EC supplement can significantly improve the abnormality all of the above. |
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