The Effect Of Copper On In Vitro Maturation Of Porcine Oocytes And Subsequent Developmental Competence After In Vitro Fertilization

In mammal,early embryo development depends on the gene in oocyte before zygotic genome activation,at the same time,oocyte cytoplasm contains abundant nutrients for cleavage.Oocyte maturation as the previous link is the basis of embryonic development.Normal development of embryo,including nucleuar mature of meiosis,cytoplasm mature of related proteins and mRNA accumulation.Nutrition environment change in oocyte maturation affectsthe development ability,enabling each period of embryo development change.Cu²⁺with REDOX activity,and as related enzymes cofactors of cellular respiration,reducing the toxicity of free radicals and iron metabolism.Copper in the study of in vitro maturation of oocytes are mainly concentrated in cattle,but copper on porcine oocytes in vitro maturation（IVM）and in vitro fertilization（IVF）has been rarely reported the influence of subsequent embryonic development in detail,Therefore,this study aimed to investigate the effects of copper on the porcine oocytes IVM and embryo development after IVF,so as to provide a basis for determining the development competence of copper and oocyte as well as the rational addition of copper during IVM.During porcine oocyte IVM,the maturation medium was supplemented with different concentration of Cu²⁺（0μg/mL,1μg/mL,2μg/mL and 3μg/mL）,and then analyzed oocyte nuclear maturation,intracellular glutathione（GSH）and reactive oxygen species（ROS）level,gene mRNA expression level of cumulus expansion,maturation,redox and copper transportation and embryos viability after IVF.Results showed that after 44 h of IVM,1)compared with the control group,the 1μg/mL Cu²⁺group showed significantly increased the number of oocytes in the metaphase II stage（P<0.05）;The cumulus cell expansion area was higher in the 1μg/mL and 2μg/mL Cu²⁺groups（P<0.05）;2μg/mL Cu²⁺group of intracellular GSH level is significantly higher（P<0.05）;In each Cu supplementation groups,the mRNA expression level of PCNA,which is related developmental ability,was relatively higher significantly（P<0.05）,the relative mRNA expression level of GDF9,associated with maturation,was significantly increased in 2μg/mL Cu²⁺group（P<0.05）;2)Cu supplementation decreased intracellular ROS level(1μg/mL and 2μg/mL Cu²⁺groups)（P<0.05）;In addition,the relative mRNA expression level of SOD1 was significantly increased in each Cu supplementation groups（P<0.05）,and NRF2 mRNA transcription level was increased in 3μg/mL Cu²⁺group（P<0.05）,and decreased in 1μg/mL and 2μg/mL Cu²⁺groups,both of which were related to antioxidant respondse.3)The decrease level of copper concentration in culture medium increased significantly after IVM,and there was significant difference among all groups（P<0.05）;In each Cu²⁺treatment,the relative mRNA expression of CTR1 was significantly increased（P<0.05）,and the relative mRNA expression of ATP7A was significantly decreased（P<0.05）,which regulated the absorption and transport of copper in cells.4)The cleavage rate of 1μg/mL and 2μg/mL Cu²⁺groups was significantly higher（P<0.05）.The blastocyst formation rate of 1μg/mL Cu²⁺group was significantly higher（P<0.05）.The number of blastocyst cells in all Cu²⁺treatment groups had an upward trend.In conclusion,in the IVM test,our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves the relative mRNA expression of CTR1 and ATP7A of copper transport in oocytes and the uptake of copper in cells,1μg/mL and 2μg/mL Cu²⁺treatment promote the nuclear and cytoplasmic maturation of porcine oocytes that reduces oxidative stress mainly by regulating the mRNA expression of SOD1 and NRF2,and improve embryonic development competence of porcine embryos.