Effects Of Melatonin On Heat Stress Porcine Oocytes In Vitro Maturation

The objective of this study was to research the effect of heat stress on the porcine oocytes at GV stage as well as the effect of melatonin supplemented in the in vitro maturation media on the oocyte under the condition of heat stress. The oocytes which collected form slaughtered sows were exposed to a physiological condition of heat stress i.e. under a in vitro circumstance remain a temperature of 41℃, the treatment last for 4 hours then the oocytes maturated to MII stage spontaneously. The Mitochondrial copy number in the oocyte, the distribution of Mitochondrial in cytoplasm, the fluorescence intensity of mitochondria stain, the express of specific genes correlated with the function of mitochondria which encoded by nuclear and mitochondria itself were detected, the embryo developmental ability originated from parthenogenetic activation of oocytes were also assessed in our study.The porcine oocytes were cultured in maturation medium supplemented with different concentrations of melatonin(0, 10-9M, 10-8M) under normal(38.5℃) and heat sock(41℃) conditions as group of normal control(C), C+M(-9), C+M(-8) and heat cock control(H), H+M(-9),H+M(-8). The oocytes in each group were maturated to the stage of MII, embryos were obtained though the method of parthenogenetic activation, the developmental of embryos were observed. The experimental results showed that, compared with the control group of normal condition i.e. group C, the blastocyst rate were significantly improved in the group of C+M(-8)(P<0.05); in the heat sock group(H), the developmental capacity of oocyte to the blastocyst stage were significantly compromised(P<0.05), in group H+M(-9) and H+M(-8) the blastocyst rate were both improved with the application of melatonin(P<0.05). The H+M(-8) group can significantly improve the blastocyst rate(P<0.05). While H+M(-9) group just have a trend of increase the capacity of oocyte development to the blastocyst stage, but the difference of this group was not significant(P>0.05). The cleavage rate was not influenced with the treatment of melatonin, and there is no significant difference between each group of cleavage(P>0.05).Mitochondrial copy number can be used as an important parameter to assess the state or degree of oocyte maturation. This study further researched the effect of heat stress on the mitochondrial copy number of mt DNA in porcine oocytes. In this experiment, porcine oocytes were divided in to 6 groups as described above and maturated to the MII stage. The samples were collected to quantify the copy number of mitochondrial DNA used the method of RT-PCR. The experimental results showed that the copy number of mt DNA in group C+M(-8) had improved significantly compared to the control group(C)(P<0.05); when the oocytes exposed to heat stress,the copy number of mt DNA decreased significantly in the group(H)(P<0.05), but improved significantly when melatonin supplied in the maturation medium of group H+M(-9) and H+M(-8)(P<0.05). All above coincide with the data obtained from the development of embryo derived from parthenogenetic activation, demonstrated that oocyte developmental competence compromised by heat stress may correlated with the phenomenon that reduced copy number of mt DNA in cytoplasm could influence the maturation of oocyte cytoplasm.During the process of oocyte maturation the distribution of mitochondrial could been shifted, the Mito-Tracker could be used to visualize the distribution of mitochondrial in the porcine oocyte at the MII stage. Compared with the normal control group(group C),The mitochondrial distributed inhomogeneously in the oocyte cytoplasm and appeared abnormal empty cavities; in the group H+M(-8) the distribution of mitochondrial back to normal status. This demonstrated the fact that melatonin could also improve the quality of oocyte maturation through influence the distribution of mitochondrial in the cytoplasm. After assessing the quantified fluorescence signal intensity of Mito-Tracker stain, it was showed that melatonin can enhance the fluorescence signal intensity of group C+M(-8)(P<0.05). The fluorescence signal intensity of Mito-Tracker stain was significantly compromised when oocytes exposed to heat stress in group H(P<0.05), but improved when melatonin added in the maturation medium in the group H+M(-8) significantly(P<0.05). All the results evidenced the fact that melatonin enhanced the activity of mitochondrial in the cytoplasm in porcine oocyte and maintained mitochondrial membrane potential in normal level simultaneously.After detecting the expression of genes which associated with the replication of mt DNA encoded by nuclear in porcine oocyte, the results showed that C+M(-8) could improve the expression of the specify gene Tfb1 m compared with control(P<0.05). In another case, the expression of Tfb1m、POLG2 was down regulated in group H(P<0.05), the expression of Tfb1m、POLG2、POLG and Ssbp1 was up regulated in the melatonin treated group H+M(-8) than group H significantly(P<0.05). It is shows that melatonin may be regulate the gene expression of nuclear encoded mitochondrial related genes to influence the copy number of mt DNA, such a process could have further influence on oocyte development to improve the oocyte quality and relieve the injury effect of heat stress to the porcine oocyte.To study further the relationship between nuclear encoded genes and mitochondrial encoded genes, ND2 and ND6 were analyzed. The test shows that in group C+M(-8) the expression level of ND2 and ND6 were significantly improved(P<0.05), but compromised in the heat stress groups significantly compared with the normal control group C(P<0.05), there were no significant difference between group H and H+M(-8)(P>0.05). From what has been discussed above, we concluded that in normal conditions melatonin could upregulate the expression of mitochondrial genes then improve the process of oxidative phosphorylation to produce enough ATP to support oocyte development.