Study On The Effect And Molecular Mechanism Of C-type Natriuretic Peptide On The Morphology Of Meiotic Spindle In Mouse Oocyte

During the ordinary in vitro maturation of mammalian oocytes,the concentration of 3',5'-cyclic adenylate monophosphate(cAMP)in oocytes rapidly decrease due to the separation from the follicular environment under in vivo conditions.,causing early resumption of meiosis and completion of nuclear maturation when the cytoplasm is not ready for maturity,eventually leading to the asynchrony of nuclear and cytoplasmic maturation,thus affecting the subsequent developmental capacity of oocyte.During maturation of oocytes,normal spindle morphology is required for proper chromosome segregation and organelle distribution,which are directly related to whether the fertilization and embryo cleavage can proceed successful.A variety of substances have been used to induce the simultaneous nuclear and cytoplasmic maturation of in vitro matured oocytes,C-type natriuretic peptide(CNP)is one of them.Although scientists generally believe that CNP enhanced oocyte maturation rate and subsequent developmental capacity by maintaining meiotic arrest by gaining more time for cytoplasmic maturation of oocytes.However,whether CNP can directly or indirectly regulate cytoplasmic maturation of oocytes(especially spindle formation)the maturation has not been well studied.In this study,we studied the regulation of CNP on the assembly of MII spindles and its pathways,as well as the expression level of genes involved in spindle assembly in oocyte maturation.The effect of CNP on the cytoplasmic maturation of mouse COCs in the process of IVM was explored,and we suppose to reveal its regulation mechanism further.The research content and results are as follows:(1)In order to detect the effect of CNP on nuclear maturation and morphology of the MII spindle of mouse oocytes matured in vitro,the COCs with GV from mouse ovary treated with PMSG for 24 h were cultured in 50 nM CNP for 24 h and then cultured in mature medium containing 10 ng/mL EGF for 16 h(CNP treatment group)or directly cultured in mature medium containing 10 ng/mL EGF(in vitro control)for 16 h.And in vivo matured oocytes obtained form PMSG 48 h + hCG 16 h treated mouse(in vivo control).The matured oocytes were collected and stained with Hoechst33342.The chromatin morphology of oocytes was observed to judge the nuclear maturation and the maturity rate of each group was counted in combination with the discharge of polar bodies.The results showe that the maturation rate(78.47 ± 5.24%)of the CNP treatment group was significantly higher than that of the in vitro control(57.53 ± 3.20%)(P<0.05).This indicated that meiosis arrested in 50 nM CNP for 24 h significantly increased the maturation rate of mouse oocytes treated with PMSG for 24 h.And then all three groups' matured oocytes were used to observe the situation of spindle assembly by immunofluorescence,and the proportion of oocytes with normal MII spindle morphology in each group was counted.The results showed that the ratio of mature oocytes with normal spindles in CNP treatment group was 76.03 ± 2.46%,which was significantly higher than that of the in vitro control(54.73 ± 2.37%)(P<0.05)and was not significantly different from the in vivo control(fully mature in vivo)(82 ± 3.48%).This indicates that the in vitro 50 nM CNP arrested culture for 24 h significantly promoted the correct assembly of the spindle during mouse oocyte maturation by some aspects.(2)The most important pathway downstream of CNP is cAMP-PKA pathway.In order to test whether CNP promotes the correct assembly of spindle during mouse oocyte maturation through this pathway.First,ELISA was used to detect cAMP levels in oocytes after incubation of mouse COCs for 2 h with or without CNP.The results showed that the cAMP level was 0.155 ± 0.037 pmol/mL after COCs culture for 2 h without the addition of CNP,while the cAMP level was 0.582 ± 0.047 pmol/mL in the CNP group,which was significantly higher than the former(P<0.05).It is indicated that CNP significantly increased the level of cAMP in oocytes during in vitro arrested culture.And then the PDE3 A inhibitor Milrinone,which can also increase cAMP levels,and PKA inhibitor H89,were used in tests.In addition to the previous test group and control group,the mouse COCs were pre-incubated for 24 h in culture medium supplemented with 2.5 ?M Milrinone(Milrinone group)or 25 ?M H89+ 50 nM CNP(CNP + H89 group),respectively,and then matured in mature medium.After maturation.The oocytes were subjected to immunofluorescence staining,and the ratio of matured oocytes with a normal MII spindle morphology was counted.The results showed that the ratio of mature oocytes with normal MII spindle morphology in Milrinone group was 64.4 ± 2.17%,which was significantly higher than the in vitro control group(54.73 ± 2.367%)and CNP + H89 group(15.89 ± 1.78%)(P<0.05),but was significantly lower than the results of in vivo control(82 ± 3.477%)and CNP treatment group(76.03 ± 2.46 %)(P<0.05).The ratio of mature oocytes with normal MII spindle morphology in CNP + H89 group(15.89 ± 1.80%)was significantly lower than the other groups(P<0.05).These results indicate that CNP passes cAMP-PKA pathway promoted the proper assembly of spindle in the process of mouse oocytes maturation.(3)Aim to further verify that CNP promoted the expression of genes related to spindle assembly,such as TPX2 and KIF4,through the cAMP-PKA pathway,qRT-PCR was used to detect the relative expression level of TPX2 and KIF4 mRNA in oocytes that treatment with 50 nM CNP,2.5 ?M Milrinone or CNP+25 ?M H89 for 24 h in vitro and with PMSG for 48 h in vivo.The results showed that 50 nM CNP arrested 24 h significantly increased the expression of TPX2 and KIF4 mRNA(P<0.05)compared with which treated with PMSG for 24 h,and there was no significant difference from which treated with PMSG for 48 h.The expression of TPX2 and KIF4 mRNA was significantly increased by treatment with 2.5 ?M Milrinone for 24 h compared with the result from which treated with PMSG for 24 h(P<0.05).The treatment with 25 ?M H89 and 50 nM CNP for 24 h significantly reduced the expression of TPX2 and KIF4 mRNA relative to other groups(P<0.05).These results show that CNP promotes the expression of TPX2 and KIF4 mRNA in mouse oocyte by cAMP-PKA pathway.In summary,CNP promotes the expression of spindle assembly-related genes,TPX2 and KIF4,in mouse oocytes through cAMP-PKA pathway,which promotes the correct assembly of oocyte MII spindle and improves its maturation effect.