The Role Of G_s A In Regulation Of Mouse Oocyte Development

Background and research purposeIn mammals,the functional oocytes undergo a complex growth process commonly referred to as oocyte maturation,which includes meiotic recovery and extensive cytoplasmic and nuclear modifications.This process is crucial in forming a high-quality oocyte.Various genetic and environmental factors regulate oocyte maturatioin,thereby affecting the quality of oocytes and developmental conpetence.Oocyte quality,a key limiting factor in female fertility,affects monospermic fertilization early embryonic survival and implantation,pregnancy maintenance,fetal development,and even disease occurrence.During oogenesis,oocytes are regulated by various genes that assemble into a molecular program todetermine the morphology and quality of oocytes.Abnormal gene expression generally results in dysfunction or dislocation of the oocyte component,such as mitochondria,spindle,or cortical granules(CGs),which consequently reduces the potential of oocytes and adversely affects the quality of the subsequently generated embryo.The stimulatory heterotrimeric Gs protein alpha subunit(Gsa)is a ubiquitous guanine nucleotide-binding protein that regulates the intracellular cAMP signaling pathway by coupling multiple receptors,and consequently participates in a wide range ofbiological events.In mouse ovaries,Gsa is present at a high level in oocytes instead of in follicular somatic cells and is located in both plasma membrane and cytoplasm Gsa reportedly synthesizes cAMP in the oocytes and inhibits the meiotic resumption in immature mammalian oocytes.Injection with an inhibitory Gs? antibody or a dominant-negative form of Gs? into the cytoplasm causes meiosis to resume in mouse oocytes within the follicles.The Gs-linked receptor GPR3 activates Gs? in oocytes.GPR3 knockout mice allow meiosis in the ovaries to precociously resume and reduce female fertility in aging mice.Adenylylcyclase 3,the primary isoform activated by Gsa in mouse oocytes,generates cAMP to maintain meiotic arrest.AC3 null mouse also exhibits premature resumption of meiosis in oocytes within follicles and phenotype of fertility reduction Thus,Gs? signaling is not only required to maintain meiosis arrest in mammalian oocytes but is also important for female fertility.In vitro experiment showed that Gs? maintains meiotic arrest in mammalian oocytes.However,the function of Gs? in oogenesis and developmental capacity in vivo remains unknown.Gs? null mutation(-/-)in mice causes embryonic lethality.To investigate the function of Gs? in oocyte development,we generated oocyte-specific Gsa knockout mice by using Zp3-Cre/LoxP systemThe scientific issues that should be solvedThis thesis was discussed mainly focuses on the following questions.(1)The effect of Gs? on female fertility(2)The function of Gs? in regulation of oogenesis and oocyte development ability(3)The possible molecular mechanism of Gs? in affecting of oocyte qualityResearch methods and results obtainedIn order to solve scientific problems mentioned above,we takes mice as experimental material in this thesis,by using the Cre-LoxP system,we obtained the oocyte-specific knockout of Gs? mice model firstly,namely Zp3-Gs? conditional knockout mice.Zp3-cre is expressed in oocytes of primary follicles and in oocytes of subsequent stage of follicular development.we can use the Zp3-Gs? conditional knockout mice to study the function of Gs? in follicular development and oocyte meiosis maturation Zp3-Gsa conditional knockout mice were viable,exhibiting no apparent growth,activity,and developmental abnormalities.For investigating the effect of oocyte-specific knockout of Gs? on female fertility,a breeding assay was conducted by mating the control and Zp3-Gs? conditional knockout female mice with males of proven fertility for 6 months.The result showed that female Zp3-Gs? conditional knockout mice were completely infertile.The number of oocytes in mutant mice collected from natural ovulation was comparable with that in the control(wildtype mice).Bilateral ovarian weight of the control and Zp3-Gs? conditional knockout mice showed no significant difference.Meanwhile,ovarian histomorphometric analyses were performed on the adult Zp3-Gs? conditional knockout mice,there were follicles of all developmental stages presenting in these ovaries.To further study the effect of Gs? on follicular development,follicle counts were performed in mice of different ages.At 2 months of age,the number of follicles in each stage was comparable between the control and Zp3-Gs? conditional knockout mice.However,at 6 months of age,the umber of activated follicles in the ovaries of the Zp3-Gs? conditional knockout mice was considerably decreased.Since Zp3-cre is not expressed in primordial follicles,we did not observe abnormalities in the number of primary follicles in aging knockout mice.However,the significantly reduced number of growing follicles in aging Zp3-Gs? conditional knockout mice indicated that Gs? may play a role in follicular development and contribute in preserving the functional ovarian reserve in aging females.Although deletion of Gsa in oocytes resulted in abnormal follicular development,it was not the primary cause of infertility in Zp3-Gsa conditional knockout mice.We detected the embryos from Zp3-Gs? conditional knockout mice,we found.that embryonic implantation in the uterus of Zp3-Gs? conditional knockout mice at E5.0 was unsuccessful,further work-up revealed that severe defects occurred in preimplantation embryos.Compared with the control,the fertilization ability of oocytes from Zp3-Gs? conditional knockout mice considerably decreased.Some of the oocytes from Zp3-Gs? conditional knockout mice were fertilized,but few visible normal pronuclei were noted;immunofluorescence analysis showed that these zygotes displayed defects in efficient pronuclei formation Subsequent development of embryos from Zp3-Gs? conditional knockout mice was also defected;these embryos were arrested at the two-cell stage or degenerated all along preimplantation development.No embryo from Zp3-Gsa conditional knockout females developed to the blastocyst stage,resulting in infertility of Zp3-Gs? conditional knockout females.To further understand the defects in oocyte development capacity caused by Gsa deletion,we focused on oocyte maturation and quality.Oocytes retrieved from antral follicles of mice were analyzed and cultured in vitro.When isolated from ovaries,some of the oocytes in Zp3-Gs? conditional knockout mice were prermturely resumed meiosis,the percentage of GV-stage oocytes in Zp3-Gs? conditionml knockout mice notably decreased compared with the control.The function of Gsa on regulation of oocyte meiotic arrest was verified in vivo.Moreover,degenerated oocytes in Zp3-Gs?conditional knockout mice notably increased compared with those in control mice.After in vitro culture,only part of Gs?fl/fl,Zp3-Cre oocytes resumed meiosis,the rate was obviously decreased compared with the control oocytes.We observed a progressive increase rate of degenerated oocytes in Zp3-Gs? conditional knockout mice during culture,which was responsible for the decrease in meiotic resumption rate.These results suggest that deletion of Gsa in oocyte not only leads to dyssynchrony of oocyte meiosis state,but also impairs the meiotic competence of oocyte.We assessed the morphology of MII oocytes derived from each group.The number of oocytes in Zp3-Gs? conditional knockout mice collected from natural ovulation was comparable with that in the control;but oocytes of Zp3-Gs? conditional knockout mice displayed a high rate of abnormal morphology.Superovulation induced the increased rate of morphological abnormal oocytes in Zp3-Gs? conditional knockout mice compared with spontaneous ovulation.Therefore,Gs? deletion in oocytes substantially affects oocyte quality.To further analyze the poor quality of oocytes caused by Gsa deletion,we examined the ovulatedMII oocytes by immunocytochemical staining to observe spindle organization and CG distribution.Compared with the control mice,oocytes from Zp3-Gs? conditional knockout mice displayed high rates of abnormal spindle and misaligned chromosomes.In Zp3-Gs?conditional knockout mice,high proportion of MII oocytes showed partial premature CG exocytosis compared with the control.These abnormal cell structures also proved that Gsa deletion carused the deterioration of mouse oocyte quality.To determine whether the decline of oocyte quality was caused by oxidative stress,we further measured the level of intracellular ROS and the le vel of antioxidant GSH.We found that levels ofROS in GV and MII oocytes from Zp3-Gs? conditional knockout mice markedly increased cotrpared with the control.Zp3-Gs? conditional knockout MII oocytes considerably reduced GSH level and the ratio of GSH/GSSG conpared with the control.The results indicated that there was oxidative stress in Gs?-deleted oocytes.By detecting mitochondrial parameters,we found abnormal distribution of mitochondria in oocytes of Zp3-Gs? conditional knockout mice,mtDNA copy numbers and mitochondrial membrane potential in oocytes of knockout mice were significantly reduced in comparison with the control We evaluated the ATP level in MII oocytes of both genotypes.Oocytes from Zp3-Gs? conditional knockout mice exhibited considerably lower ATP content than the control.We found that early apoptosis occurred in Gs?-deleted oocytes examined by Annexin-V staining.Then we treated the oocytes of Zp3-Gsa conditional knockout mice with antioxidant NAC,we found that NAC supplement improved the developmental competence of Gsa-deleted oocytes substantially,the fertilization and cleavage rates in Zp3-Gs? conditional knockout mice increased,and some of these embryos developed to blastocysts.The defects in oocyte quality and developmental competence induced by Gs? deletion were partially rescued.Therefore,we speculate that oxidative stress may play an important role in deteriorates oocyte quality and female infertility in Zp3-Gsa conditional knockout mice.To sum up,Gs? plays an important role in development of mouse oocytes,which is involved in the regulation of oocyte meiotic state,oocyte maturation,maintenance of oocyte quality and early embryonic development capacity.Gsa deletion results in increased level of ROS,mitochondrial dysfunction and apoptosis in oocytes.Targeting Gs alpha signaling pathway may help prevent and treat female infertility in clinical practice.Targeting the Gs? pathway may be helpful to prevent and treat female infertility in clinic.Research significanceDespite in vitro studies have documented Gsa is required to maintain meiotic prophase arrest in mammalian oocytes,the exact role of Gs? in female fertility in vivo remains largely unknown.In this study,we investigated the function of Gs? in oogenesis,fertilization,and early embryo development by deleting Gsa in oocytes in mice.Here,we firstly revealed that oocyte-specific deletionof Gs? results in complete female infertility,and oxidative stress plays an important role in this event.Deletion of Gs? induced deterioration of oocyte quality and impaired early embryo development.Meanwhile,antioxidant supplementationpartially rescued the defects of Gsa-delected oocytes.In this thesis,the functibn of Gsa during the development of mouse oocytes in vivo was studied.We believe that this study could have a great interest to the field of gametogenesis and fertilization The Gsa conditional knockout mice generated in this paper may provide a ideal animal model of female infertility in humans,it would provide theoretical basis and direction of the treatment for female infertility.