Effects Of Chemokine CXCL12 On Oocyte Maturation And Embryo Development In Bovine And Porcine

In vitro maturation(IVM)and in vitro production is widely used in biological breeding to efficiently expand and disseminate superior germplasm resources,while the developmental potential of the derived oocytes and embryos is inferior to that of in vivo-derived ones.Oocyte maturation is a complicated but delicate process involving nuclear and cytoplasmic maturation.In most cases,cytoplasmic maturation and the full acquisition of developmental capacity of oocytes cannot be automatically completed along with nuclear maturation.Many studies have shown that IVM can result in cytoplasmic maturation defects,such as abnormal cortical granule(CG)distribution,mitochondrial dysfunction,and abnormal cytoskeleton assembly,which seriously affects the ability of oocytes to support the embryonic development after fertilization.The maternal environment provides a dynamic developmental program for oocytes and embryos.The secretion of signaling molecules,such as hormones,growth factors,and cytokines,represents a key mechanism underlying how reproductive systems(mainly the ovary,oviduct,and endometrium)control oocyte and embryo development.These molecules regulate the complex biological events occurring during development by binding to membrane receptors on oocytes and embryos.Studies have shown that chemokines maintain high expression levels in the reproductive system tissues of mammals during the estrous cycle.Apart from playing an immune role in the reproductive system,whether these chemokines are involved in the regulation of oocyte maturation and embryonic development is a question worthy of attention.By analyzing published transcriptome data,this study screened out CXCL12 and CXCR4,which had potential functions on oocyte and embryo development of bovine and porcine.We explored the effect and mechanism of CXCL12 on oocyte maturation and embryo development of bovine and porcine,and preliminarily explored whether this mechanism is conserved in multiple species.The main research contents and results are as follows:(1)Heatmap of chemokine and their receptor genes were performed by analyzing public transcriptome data from bovine and porcine reproductive system tissues,oocytes,and embryos derived from in vivo.The results showed that CXCR4 was the highly expressed chemokine receptor gene in oocytes and early embryos of bovine and porcine.During the estrous cycle,the expression of chemokine genes in the reproductive system tissues of bovine and porcine showed a kind of “dominance”.The expression level of CXCL12 was the highest in oviduct and CXCL17 in bovine endometrium.The expression level of CCL21 was the highest in ovary and CXCL16 in porcine oviduct.Their expression levels maintained at least two times higher than other chemokine genes during the estrous cycle.(2)According to the expression level of chemokine genes in the reproductive system tissues,combined with the expression level of corresponding chemokine receptor genes in oocytes and embryos,CXCL12-CXCR4 was selected as potential functional chemokinereceptor for oocytes and embryo development in bovine and porcine.By analyzing amino acid sequences and protein domains,it was found that the structure of CXCL12 and CXCR4 were highly conserved in 9 mammal species including human,mouse,pig,cow,sheep,horse,rabbit,dog,and camel.(3)By measuring CXCL12 content in preovulation follicles,immature follicles and IVM medium,it was found that the content of CXCL12 in the follicular fluid of preovulation follicles was significantly higher than that in the follicular fluid of immature follicles and IVM medium,indicating that the oocyte developed in the physiological environment containing CXCL12.CXCL12 supplementation did not affect bovine and porcine oocyte nuclear maturation,but significantly increased blastocyst formation efficiency and cell number after parthenogenesis,fertilization,and cloning.(4)Transcriptome analysis of bovine and porcine MII oocytes derived from in vivo revealed that PTPN11,encoding SHP2 protein,was the highly expressed signaling molecule gene in CXCL12-invovled signaling pathways.Western blotting of bovine and porcine MII oocytes showed that CXCL12 activated SHP2 and enhanced its tyrosine phosphorylated protein level,promoting the transition of SHP2 from autoinhibition to an open conformation.(5)The effect of CXCL12 on bovine and porcine oocyte cytoplasmic maturation was evaluated through organelle redistribution,cytoskeletal reorganization,and molecular maturation.The results showed that CXCL12 promoted cumulus expansion,peripheral distribution of cortical granules,mitochondrial function,and protein synthesis by activating SHP2.In addition,Western blotting of bovine and porcine MII oocytes showed that CXCL12 promoted the activation of MAPK1/3 signaling pathway by activating SHP2.In summary,this thesis demonstrated that CXCL12-CXCR4 had common effects on bovine and porcine oocyte maturation and embryo development.CXCL12-CXCR4 activated SHP2 and enhanced its tyrosine phosphorylated protein level to promote the transition of SHP2 from autoinhibition to an open conformation and activation of MAPK1/3 signaling pathway,and finally improved the developmental potential of oocytes and subsequent embryonic development.This study can provide high-quality oocytes and embryos for animal cloning and livestock breeding,and provide new ideas for further improving the efficiency of in vitro production.