| During mammalian oocytes cultured in vitro,the level of cyclic adenosine-3,5-adenosine monophosphate(c AMP)in mammalian oocytes decrease rapidly due to the absence of in vivo follicular environment,which leads to the recovery of the activity of maturation promoting factor(MPF)in mammalian oocytes and the initiation of meiosis prematurely.Due to the early initiation and completion of meiosis,oocytes do not have enough time to complete the material reserve needed for cytoplasmic maturation,so cytoplasmic maturation is not sufficient,and the nuclear maturation(meiosis)and cytoplasmic maturation of oocytes cultured in vitro are out of sync.Therefore,the quality of oocytes obtained is low and the ability of subsequent development is poor.As the main center of oocyte energy production,mitochondria provide energy for oocyte maturation and subsequent development through a series of oxidative phosphorylation processes.At the same time,mitochondria are closely related to the production and elimination of reactive oxygen species(ROS)in oocytes,so they play an important role in regulating oxidative stress in oocytes.It has been proved that C-type natriuretic peptide(CNP)can maintain the meiotic block of oocytes by acting on cumulus guanylate cyclase receptor(natriuretic peptide receptor 2(NPR2),which wins more time for cytoplasmic maturation,so that the oocyte cytoplasm can be fully matured,thus improving the quality of oocytes.Among them,whether CNP affects oocyte quality by regulating mitochondria function of oocytes and regulation mechanism need to be further analyzed.The purpose of this research was to investigate the effects of CNP on the morphology,distribution and oxidative metabolism of mitochondria and the expression of genes related to mitochondrial fusion,division and oxidative metabolism in mouse oocytes,and to explore the effects of CNP on the physiological status of mitochondria in mouse oocytes and its regulatory mechanism.The contents and results are as follows:(1)Select pre-estrus mice and inject intraperitoneal injection of pregnant mare serum gonadotropin(PMSG)to promote follicular development.Mice were sacrificed and ovaries were collected after PMSG treatment for 24 h to isolate mouse cumulus-oocytes for culture in vitro.At 1 h,2 h,3 h and 4 h of culture,cultured COCs were recovered for germinal vesicle break down(GVBD)detection,and it was found that oocytes occurred GVBD rapidly after culture for 2 h in basic culture medium,the rate of which was 53.42 ± 10.53%,and GVBD rate reached 85.86 ± 3.80% at 4 h,so 2 h of culture was selected as the detection time point for subsequent research on oxidative metabolism,number and distribution of oocyte mitochondria.(2)In order to detect the effects of CNP on mitochondrial oxidative metabolism of mouse oocytes in vitro,COCs were divided into two groups: control group(basic culture medium group)and CNP treatment group(basic culture medium with 50 n M CNP group).At the end of culture,cumulus cells were removed,and the levels of ROS,glutathione(GSH)and mitochondrial membrane potential in oocytes were detected by fluorescence probe and kit.The results showed that ROS content in CNP treatment group was significantly lower than that of control group(P < 0.05).GSH content and the level of mitochondrial membrane potential in CNP treatment group were both significantly higher than those in control group(P < 0.05).The results showed that CNP treatment decreased the level of ROS in mouse oocytes and increased the ability of antioxidant stress(increased level of GSH)and mitochondrial activity(increased level of membrane potential).(3)In order to detect the effects of CNP on the distribution and number of mitochondria in mouse oocytes in vitro,the mitochondria and nuclei of oocytes were stained to determine the morphology and distribution of mitochondria in oocytes,and the mitochondrial DNA,from two groups of mouse oocytes,was extracted and the copy number of mitochondria in oocytes was detected by q RT-PCR.The results showed that after 2 h of CNP treatment,the mitochondria of mouse oocytes were distributed around nucleus and there were small clusters around the nucleus,while the mitochondrial particles in control oocytes were small and uniformly distributed in cytoplasm,and the copy number of mitochondria in the oocytes treated with CNP was significantly higher than that in control group.It could be seen that the distribution of mitochondria in CNP-treated mouse oocytes was more reasonable,which was more conducive to the function of mitochondria;compared with control group,the copy number of mitochondrial DNA was also higher,which indicated that the number of mitochondria was more.The reasonable distribution of mitochondria and the increase of copy number may provide more sufficient energy supply and more reasonable energy distribution for cytoplasmic maturation and subsequent development of oocytes.(4)To further explore the mechanism of CNP increasing the activity of oocyte mitochondria,q RT-PCR and Western Blot techniques were used to detect the expression of genes related to mitochondrial fusion,division and metabolism in oocytes.The results showed that compared with control group,the m RNA expression of mitochondrial division related genes FIS1 and DRP1,the m RNA expression of mitochondrial fusion related genes OPA1 and MFN1/2,and the m RNA expression of mitochondrial metabolism related genes SIRT1,PGC-1?,TFAM and NRF1/2 increased significantly in CNP treated group.At the same time,protein expression of PGC-1?,the key regulator of mitochondrial activity,and phosphorylation level of CREB,the upstream regulator of PGC-1?,were both increased significantly in CNP treated oocytes.Further co-treatment of mouse oocytes with CNP and H89,which was the inhibitor of protein kinase A(PKA),an upstream regulatory molecule of CREB,showed that the expression of PGC-1? and the level of CREB phosphorylation in oocytes decreased significantly,but there was no significant difference compared with control group(P > 0.05).The results showed that CNP treatment promoted CREB phosphorylation,thus increased the expression level of PGC-1?.These results suggested that CNP promoted the expression of mitochondrial-related genes and proteins by activating PKA and then phosphorylating CREB,and finally improved the mitochondrial activity of oocytes.To sum up,CNP promoted the expression of mitochondrial related genes and PGC-1? protein in mouse oocytes through PKA-CREB pathway,and improved the activity and metabolic function of mitochondria in mouse oocytes. |
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