Research On The Effects Of Melatonin And Growth Hormone On Human Oocyte Maturation And Early Embryo Development

Chapter Ⅰ Study on the effect of melatonin on human oocytematuration and early embryo developmentObjective:Oocyte maturation is an important determinant for oocyte quality,and fully mature oocyte is the core of the reproductive process.Inadequate oocyte maturation can lead to poor quality of oocytes,fertilization failure,abnormal fertilization,and aneuploid embryos.Embryos of poor quality can result in miscarriage,congenital anomalies,and other adverse clinical outcomes.Therefore,to promote human oocyte maturation and improve oocyte quality is not only beneficial for clinical outcomes of patients who pursue assisted reproductive technolgy,but also related to the offspring health of human.Some hormones and growth factors have been reported to promote human oocyte maturation,however,most studies are lack of in-depth mechanism research.Melatonin can promote oocyte maturation and early embryo development in non-primate species by its classical antioxidative function,but other mechanisms of melatonin on oocyte maturation have received little attention.The antioxidative effects of melatonin can help improve the fluidity of plasma membrane and reduce its rigidity,thus facilitating clathrin-mediated endocytosis(CME).However,few studies have reported whether melatonin can play a role in oocyte maturation by regulating CME.Therefore,the mechanism research of melatonin on human oocyte maturation centering on CME is the focus of this study.Methods:The beneficial effect of melatonin on human oocyte maturaion and early embryo development was studied by collecting GV oocytes discarded by clinical and donated for scientific research and adopting the methods of oocyte in vitro maturation and intracytoplasmic sperm injection(ICSI).Single oocyte transmission electronmicroscopy,real-time PCR and immunofluorescence were used to confirm the effect of melatonin on CME during human oocyte maturation at ultrastructure,RNA level and protein level,respectively.And dynasore,an inhibitor of CME,was utilized for further demonstration.In order to clarify how melatonin regulated CME,the expressions of melatonin membrane receptors in human oocytes were verified by real-time PCR,and then the changes in expression levels of CME marker proteins of human oocytes cultured in the presence of H2O2 with or without melatonin were determined by immunofluorescence.cAMP level in human oocytes was measured by enzyme-linked immunosorbent assay to investigate the mechanism of CME in regulating human oocyte maturation.Results:(1)Melatonin with its optimal concentration(10-11 M)significantly increased the maturation rate of human oocytes(71.9%vs.45.9%,P=0.03),the fertilization rate(81.4%vs.61.4%,P=0.017),and the blastocyst rate(32.2%vs.15.8%,P=0.039),which confirmed that 10-11 M melatonin could significantly promote human oocyte maturation and early embryo development.(2)During the process of human oocyte maturation,electron microscopy results showed that melatonin remarkably increased the numbers of clathrin-coated pits(2.0-fold,P=0.01)and vesicles(2.7-fold,P=0.029).The RNA levels of most CME marker genes in the melatonin group showed upward trends,and melatonin also significantly upregulated the expression levels of CME marker proteins clathrin(1.1-fold,P=0.015)and AP2(1.2-fold,P=0.016).Additionally,in the presence of the CME inhibitor dynasore,supplementation with melatonin at the same time could rescue its inhibitory effects on human oocyte maturation and CME.These confirmed that melatonin could enhance CME during human oocyte maturation.(3)There was no expression of melatonin membrane receptors in human oocytes;In the presence of H2O2,the marker proteins clathrin(0.6-fold,P=0.028)and AP2(0.8-fold,P=0.018)of CME were significantly decreased compared to the control;When melatonin was added at the same time,the expression levels of the marker proteins clathrin(1.7-fold,P=0.047)and AP2(1.3-fold,P=0.004)were significantlyupregulated compared to the H2O2 group,and reached the levels similar to the control group.This indicated that melatonin could regulate CME by its antioxidant and free radical scavenger effect,which is independent of melatonin receptors.(4)Compared to the cAMP levels(3.48 fmol/oocyte)and GVBD rates(66.7%)of oocytes from the control group,the highest cAMP level(4.10 fmol/oocyte)and the lowest GVBD rate(55.6%)were found in the oocytes of CME inhibitor dynasore group;While in the melatonin group,the cAMP level(3.15 fmol/oocyte)was the lowest,and the GVBD rate(75.0%)was the highest.This suggested that CME could influence human oocyte maturation by regulating cAMP levels in the oocytes.Conclusion:Melatonin promoted human oocyte maturation and early embryo developmnet.The mechanism research showed that melatonin enhanced CME by its antioxidative effects,and enhanced CME promoted human oocyte maturation by reducing intra-oocyte cAMP level.This study provided a strong scientific basis for melatonin to promote human oocyte maturation and improve oocyte quality.Chapter II Study on the effect of growth hormone on human oocyte maturation and early embryo developmentObjective:Growth hormone(GH)applied in vivo during ovulation induction in infertile patients can improve ovarian function,oocyte quality,and endometrium status,thereby benefiting clinical outcomes of patients who pursue assisted reproductive technology.In addition to the application in vivo,GH added into the in vitro culture medium has been reported to promote oocyte maturation and early embryo development in many animals.However,few systematic studies on the effects of GH on human oocyte maturation have been reported.Therefore,this study aimed to clarify the effect of GH on human oocyte maturation and early embryo development,and explore the corresponding mechanism of GH.Methods:The effect of GH on human oocyte maturaion and early embryo development was studied by collecting GV oocytes discarded by clinical and donated for scientific research and adopting the methods of oocyte in vitro maturation and ICSI.Single-cell RNA-seq was used to identify the changes of gene expression profiles in human oocytes after GH treatment,and real-time PCR was adopted to validate differentially expressed genes.Results:GH with its optimal concentration(200 ng/mL)significantly increased the maturation rate of human oocytes(70.0%vs.35.0%,P=0.004),and the fertilization rate(73.1%vs.60.3%,P=0.158)and blastocyst rate(25.0%vs.15.5%,P=0.214)in the GH group were with increased trends.Single-cell RNA-seq and real-time PCR data showed that GH remarkably upregulated genes related to meiotic progression,intracellular redox homeostasis,and oocyte developmental potential,such as AURKA(aurora kinase A,2.1-fold,P=0.007),PDIA6(protein disulfide isomerase family A member 6,2.5-fold,P=0.007),LING02(leucine rich repeat and Ig domain containing 2,5.5-fold,P=0.007),and CENPJ(centromere protein J,1.9-fold,P=0.039).Conclusion:GH promoted human oocyte maturation,probably by accelerating meiotic progression,balancing intracellular redox homeostasis,and promoting oocyte developmental potential.