Analyzing Nuclear And Cytoplasmic Quality Of Oocytes Matured In Vitro Or Derived From Follicle Culture In Vitro

Objective:In vitro maturation（IVM）of oocytes and in vitro culture of follicles are both promising assisted reproductive techniques（ART）for infertility treatment,although they are still not routine manipulations for human ART due to reduced embryo development.This study had two purposes,one was to clarify the possible reasons for reduced capacity of in vitro matured oocytes,and the other was to explore a safe and effective follicular serum-free culture system,to understand the influence of this culture system on oocyte quality,and to improve culture conditions for follicular development and oocyte maturation.Methods:1.For the detection of mitochondrial function,we designed four experiments.The first experiment was to show the distribution of mitochondria by fluorescence staining;the second was to analyze the mitochondrial membrane potential;the third was to determine the intra-cellular ROS level;and the last one was to calculate the mtDNA copy numbers by qPCR.2.For the detection of nucleus function,we investigated spindle assembly,and chromosome congression.3.We aimed to establish an effective serum-free secondary follicle culture system.4.We performed experiments to analyze calcium oscillations and embryo development after parthenogenetic activation of oocytes from IVM and follicular culture.Results:1.We found that oocytes with cumulus cells showed better performance in comparison with denuded oocytes in pronuclear formation rate and production of calcium waves.In vitro matured oocytes had increased mitochondria clusters,decreased mitochondrial membrane potential,increased ROS levels,and increase mitochondrial DNA copy number.These results were not different in oocytes matured in vitro with or without cumulus cells.In vitro matured oocytes showed significantly lower activation and embryonic development rates,and their ability to produce Ca²⁺oscillations was much lower in response to parthenogenetic activation,especially in oocytes matured in vitro without cumulus cells with nearly half of them failing to produce calcium waves upon strontium chloride stimulation.2.We established a 2-dimensional follicular serum-free culture system with 3-dimensional effect that can make secondary follicles develop into antral follicles（78.52%）,generating developmentally mature oocytes in vitro（66.45%）.The oocytes in this serum-free system completed the first meiosis;spindle assembly and chromosome congression in most oocytes matured from follicular culture were normal.However,these oocytes showed significantly lower activation and embryonic development rates,and their ability to produce Ca²⁺oscillations was also lower in response to parthenogenetic activation,after which a 2-cell embryonic developmental block occurred.Oocytes matured from follicular culture displayed increased abnormal mitochondrial distribution and increased ROS levels when compared to in vivo matured oocytes.Conclusion:1.We concluded that in vitro matured oocytes had increased mitochondria clusters,decreased mitochondrial membrane potential,and increased ROS levels,indicating a decrease in mitochondrial function.To this end,the mitochondrial DNA copy number increased,perhaps to compensate for function.These results also indicated that in vitro maturation conditions did not support complete acquisition of oocyte developmental competence.Low rate of embryonic development and Ca²⁺oscillations were severely disturbed in oocytes matured in vitro,especially in the denuded oocyte group,which may be correlated with impaired mitochondrial function.These data are important for understanding the reason for reduced capacity of in vitro matured oocytes and the importance of cumulus cells for oocyte quality.2.We established a serum-free follicle culture system that effectively supports 3-dimensional structure of follicles and oocyte meiotic maturation,which can serve as a biological model to study the mechanisms underlying follicular development,and to assess harmful effects of specific compounds or conditions on follicular development.However,although MII spindles and mtDNA numbers in most oocytes derived from this system are basically normal,they showed decreased developmental competence,which needs to be improved by further investigations.