Effect Of Exogenous Melatonin On Development Of Cryopreserved Mâ…¡ Oocytes In Mouse

This study was conducted to investigate the effect of different concentrations of exogenous melatolin (MT) on the development of mouse mature oocytes after cryopreservation. Mouse mature oocytes(MII) were cryopreserved with Open Pulled Straw(OPS) first, and cultured in M2 with varied concerntration(0 mol/L,10-9 mol/L, 10-7 mol/L,10"5 mol/L,10"3 mol/L) of MT for 1 hour, following that the level of ROS and GSH from oocytes, the viability of embryos after parthenogenetic activation, as well as the cell numbers in blastocyst were detected, expression level of mRNA from five genes (Hsp90aal, Hsfl, Hspa1b, Nrf2 and Bcl-xl) in oocytes treated with three different concentrations (0 mol/L,10-7 mol/L,10-3 mol/L) of MT were tested. The results are shown below.(1) ROS level from frozen-thawed oocytes treated with 10"3 mol/L MT for 1h after vitrification was remarkably lower than that in MT-untreated group (P<0.05) Significant difference on GSH level was not found between MT treated groups and untreated ones (P>0.05)(2) Frozen-thawed oocytes after vitrification were cultured in MT-treated M2 for 1 h. The ability of parthenogenetic activation embryos which developed to 2-cell weakened as the concerntration of additional MT increased, while there was no significant difference between 10-9～10-5 mol/L MT-treated groups and untreated ones (P>0.05), moreover, the cleavage rate of oocytes from 10-3 mol/L MT treated group was significantly lower than that from untreated ones (P<0.05). The ability of parthenogenetic activation embryos which developed to blastocyst didn’t be changed with the increased MT concerntration (P>0.05). Cell numbers of blastocyst decreased following the concentration of additional MT increased, though the significant difference was not found (P<0.05)(3) Results from real-time fluorescence quantification PCR showed that, the expression of Hsp90aal, Hsfl, Hspa1b, Nrf2 and Bcl-xl was detected in 10-3 mol/L,10-7 mol/L and 0 MT-treated groups, while the expression amount of these five genes in 0 MT group was higher than that in 10"3 mol/L MT group which the expression of Hsfl、 Hsp90aal and Bcl-xl was significantly lower than that in 0 MT group (P<0.05) Compared to the 10-7 mol/L MT group, the difference of expression of Hsp90aal and Bcl-xl was significant in 10’3 mol/L MT group (P<0.05). The expression amount of Hspa1b and Hsfl was slightly lower in 10-7 mol/L MT group than that in 0 mol/L MT group, no significant difference was found (P>0.05). the expression amount of Nrf2, Hsp90aal and Bcl-xl was up-regulated, whereas significant difference was only found in Hsp90aal (P<0.05)(4) In vitro fertilization experiments, the oocyte cleavage rate and blastocyst rate of freezing group was significantly lower than that in the fresh group (fresh) (P<0.05), it has no effect of 10-7 mol/L melatonin on oocyte cleavage rate and blastocyst rate of no effect (P>0.05)Under the experiment conditions, addition of different concerntration of MT (10-9,10-7, 10"5,10-3 mol/L) to the frozen-thawed mouse MIl oocytes cultured for 1h after cryopreservation makes no prominent improvement on the following development of the oocytes.