| In animal husbandry,ovarian dysfunction,especially ovulatory dysfunction,is one of the main causes of infertility and reduced conception rate during artificial insemination in livestock,but the external inducers of ovulatory dysfunction remain unclear.It has been shown that external factors such as feed amino acids and heat stress can affect the synthesis of milk protein by affecting the phosphorylation level of downstream target proteins of m TOR signaling pathway during livestock breeding,but whether these external factors can regulate mammalian ovulation and subsequent embryonic development through m TOR signaling pathway has not been reported.In order to explore a new method to induce ovulation and improve embryo survival in mammals,this study assessed the species conservation of m TOR signaling activation during ovulation in four mammalian species: Bos taurus,Homo sapiens,Macaca mulatta and Mus musculus by analyzing a transcriptome sequencing database associated with mammalian ovulation,and further analyzed the effects of the m TOR signaling inhibitor rapamycin and the activator MHY1485 on ovulation process and embryo development using mice as model animals.The main study contents and results are as follows:(1)Differentially expressed genes(DEGs)were detected in the transcriptome database related to ovulation in four mammals including buffalo,human,macaque and mouse,and KEGG analysis was performed to successfully screen the m TOR signaling pathway,indicating that the activation of m TOR signaling was species conserved in mammals.(2)The activation of m TOR signal in the ovulatory period of mouse ovaries after superovulation treatment was detected,and it was found that the m TOR signal of all types of cells in follicles was extensively activated.(3)Rapamycin was injected into the ovulatory phase of mice during superovulation treatment.Transcriptome sequencing was performed on mouse ovaries,and then KEGG analysis was performed on DEGs to successfully obtain ovulation related signaling pathways.(4)Two groups of mice were injected with rapamycin and MHY1485 during ovulation treated with superovulation,and then the effect of m TOR signaling on ovulation process was examined,and it was found that activators and inhibitors had no significant effect on ovulation number and oocyte nuclear maturation.(5)Mice in estrus were taken,respectively,after injection of rapamycin and MHY1485 breeding,then collected blood samples,ovary and embryo,found that rapamycin treatment on corpus luteum number,serum progesterone,and cholesterol levels,corpus luteum function gene expression level and the number of early embryos and pregnancy had no significant influence,but significantly reduce the number of the middle of a pregnancy embryo;MHY1485 treatment had no significant effect on the number of embryos.(6)Rapamycin was injected into the ovulation stage of mice during superovulation treatment,and the oocytes were collected for in vitro fertilization.It was found that rapamycin treatment reduced the total number of cells in blastocysts,but had no significant effect on the development efficiency of early embryos.(7)Mice were treated with rapamycin and MHY1485 at the ovulation stage of superovulation,and then the structure and function of oocytes related to energy were detected.The results showed that the volume of oocytes treated with rapamycin increased significantly,the mitochondrial membrane potential increased significantly,and the content of lipid droplets decreased significantly.It was speculated that rapamycin accelerated the consumption of oocyte energy reserve,resulting in the loss of embryo.However,the mitochondrial membrane potential of oocytes treated with MHY1485 decreased significantly,and the content of lipid droplets increased significantly.In conclusion,rapamycin impaired embryo development by affecting oocyte energy reserve.Although MHY1485 has an effect on oocyte energy reserve,it does not affect embryo development. |
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