Effects Of Astaxanthin On Mouse Oocyte In Vitro Maturation And Early Embryo Development

In vitro maturation(IVM)of oocytes has been used in assisted human reproduction,animal embryo production,animal cloning and transgenesis.However,the maturation rate,blastocyst rate,pregnancy rate and live birth rate of in vitro matured oocytes are significantly lower than those of in vivo matured oocytes,and the rate of abnormal embryo development and embryonic arrest is significantly higher.The main reason for the decrease in quality of in vitro matured oocytes is that oocytes are separated from the relatively stable maternal hypoxic environment and the redox balance is disrupted,resulting in excessive accumulation of intracellular reactive oxygen species(ROS)and subsequent oxidative stress.Therefore,it is important to alleviate the oxidative stress of in vitro matured oocytes,improve the quality of oocytes and enhance the efficiency of in vitro oocyte maturation to promote the wide application of in vitro oocyte maturation technology.Astaxanthin is a natural carotenoid,mainly found in marine organisms,and is one of the most powerful antioxidants in nature.In recent years,studies on astaxanthin have focused on tumor,diabetes,inflammation and immunomodulation,while in the field of reproduction,astaxanthin has been reported to enhance the developmental potential of aging oocytes after ovulation in cattle,but the effect of astaxanthin on mouse oocyte maturation in vitro has not been reported.In order to investigate the effect of astaxanthin on mouse oocyte maturation in vitro,we added different concentrations of astaxanthin to the culture medium and found that the addition of 2.5 μM astaxanthin significantly increased the maturation rate of mouse oocytes.The spindle is the main organelle involved in the nuclear maturation of oocytes.In this study,we found that astaxanthin had no significant effect on the spindle morphology of oocytes during MⅠ and MⅡ periods by immunofluorescence staining.To further investigate the mechanism by which astaxanthin promotes the maturation of mouse oocytes in vitro,the present study revealed that 273 genes were down-regulated and 245 genes were up-regulated in the oocytes of the astaxanthin-added group by single-cell transcriptome sequencing,and the differential genes were associated with mitochondrial homeostasis and protein homeostasis.Subsequently,mitochondrial activity was measured in in vitro matured oocytes,and a significant increase in mitochondrial activity was found in the oocytes of the astaxanthinadded group;ROS levels were measured in vitro matured oocytes,and a significant decrease in ROS levels was found in the oocytes of the astaxanthin-added group.Next,to investigate the effect of astaxanthin on the protein homeostasis of in vitro matured oocytes,this study showed that 24 proteins were up-regulated and 69 proteins were down-regulated in the oocytes of the astaxanthin-added group,and several important biological processes,including DNA methylation,chromosome assembly,protein deacetylation and metabolic processes,were involved.The quality of oocytes determines the potential of early embryonic development.In this study,we innovatively applied laser perforation of the zona pellucida of in vitro matured oocytes to successfully fertilize in vitro matured oocytes,and found that the fertilization rate and 2-cell rate of oocytes in the astaxanthin-added group were significantly increased.Androgenetic embryos could be obtained by removing the female pronucleus through micromanipulation during the zygote period,but the developmental efficiency of androgenetic embryos was low due to oxidative stress damage during the in vitro manipulation.In this study,we found that the addition of astaxanthin significantly increased the 4-cell rate of androgenetic male embryos during the in vitro culture.In conclusion,the addition of astaxanthin to the culture medium can significantly increase the in vitro maturation rate of mouse oocytes,enhance the quality and developmental potential of oocytes,and promote early embryonic development in mice.In this study,we investigated the effects of astaxanthin on in vitro maturation of oocytes and improved the in vitro maturation technique of mouse oocytes,which is of great significance for the wide application of in vitro maturation of oocytes.