Effects Of Melatonin-containing Water On In Vitro Development Potential Of Oocytes In Mice

Melatonin is a powerful antioxidant that plays an important role on the reproduction of female animals.The current study was designed to investigate the effects of melatonin on the development potential of oocytes on female mice by adding melatonin to the drinking water,in order to provide references for further improving livestock breeding potential.At first instance,the mice were given free access to drinking water containing melatonin with different concentrations（0μg/mL,3μg/mL,30μg/mL and 300μg/mL）for 21days.Then,we examined the serum levels of melatonin,estradiol（E₂）and follicle-stimulating hormone（FSH）.Further,other parameters such as,the number of follicles in ovary,development potential of embryo following in vitro fertilization（IVF）,as well as the relative expression levels of apoptosis and estrogen receptor-related genes（BAX,BCL2,GPR30）in 2-cell embryos and blastocysts were examined.The summary of results is given as follows:（1）After melatonin supplementation with different concentrations for 21 days,the serum melatonin level shows the regular circadian rhythm which the initial rise at 21:00（230.74±4.94～256.2575±0.22pg/mL）,peaked at 23:00（336.63±23.43～321.25±33.99pg/mL）and then declined to regular levels at 5:00 daytime（241.14±16.03～268±20.42pg/mL）.The values of melatonin levels were significantly higher（P<0.05）in group supplemented with 30μg/mL melatonin during the day 9:00-17:00 and 5:00 am（269.80±17.75～274.89±17.08,275.21±2.88μg/mL）as compared to the control group（210.64±19.28～240.51±15.08,241.14±16.03μg/mL）,whereas,no significant（P>0.05）changes in E₂and FSH levels were observed among all other melatonin supplemented groups and control group.（2）The prepared H&E stained sections of ovaries were observed through light microscope.Our histological observations revealed that the number of follicles was significantly higher（P<0.05）in melatonin group supplemented with 30μg/mL（15.65±3.29）as compared to the control group（7.78±5.52）.（3）Our experiment on developmental potential of embryos demonstrated that there was no significant difference（P>0.05）in rates of 2-cell embryo development as compared to the control following IVF.However,when developed into hatching blastocysts,the rates of 2-cell embryo development were significantly higher in group supplemented with30μg/mL melatonin（85.70%±8.10）as compared to that of 0μg/mL（72.10%±8.13）and300μg/mL（60.32%±17.24）melatonin groups（P<0.05）.（4）The studies on relative expression of apoptosis and estrogen receptor related genes in 2-cell embryos and blastocysts demonstrated that the expressions of BAX,BCL2,and GPR30 genes were significantly lower in the 2-cell embryos in groups supplemented with medium and low concentrations of melatonin（3μg/mL,30μg/mL）as compared to the control group（P<0.05）.The relative gene expression of GPR30 and BCL2/BAX in 2-cell embryos in group supplemented with 300μg/mL melatonin was also significantly lower than that in the control group（P<0.05）.However,in blastocysts,the expression of BAX was significantly lower than that of control group（P<0.05）;the expression of BCL2 was the lowest in the group supplemented with 3μg/mL melatonin,moreover,no significant difference was observed in the expression of this gene among the four treated groups（P>0.05）.The ratio of BCL2/BAX in the blastocysts of group supplemented with 30μg/mL melatonin was significantly higher than the control group（P<0.05）.The expression of GPR30 gene in groups with different concentrations of melatonin was significantly lower than that of the control group（P<0.05）.Taken together,melatonin（0μg/mL、3μg/mL、30μg/mL and 300μg/mL）was added to the drinking water of female mice for 21 days,the serum melatonin levels was increased in group with 30μg/mL melatonin during the daytime,however,there were no effects on E₂and FSH serum levels.The blood melatonin levels still showed the regular rhythmic changes.Moreover,It obvious that the supplementation of melatonin can maintain the number of dominant follicles in the mouse ovary and inhibit the expression of apoptotic genes in the blastocyst,which in turn may increase the development rates of hatching blastocysts via IVF technology in oocytes.