Study On Roles Of PGC-1? And NRF-1 In The Process Of Goat Follicular Atresia,Oocyte Aging And Cloned Embryonic Development

In the process of ovarian follicular growth and development,only a few folliclescould mature and ovulate,while most follicles undergo atresia.If the oocytes could notfertilize in time,unfertilized oocytes will be aging,which severely affects the subsequentdevelopment potential.Durig this period,mitochondria is crucial in animal folliculardevelopment,oocyte maturation and subsequent embryo development,which is coined asmitochondrial biogenesis.Underlying this biological process,PGC-1? and NRF-1,ascorroborated by a large body of recent research,proved to play foundamental roles.However,the mechanism of the regulation of the two genes in follicular atresia,oocyteaging and embryo development is unveiled.In addition,oocyte quality severely limits thedevelopment of assisted reproductive technology and transgenic cloned technology.Therefore,understanding the mechanisms of follicular development,atresia and oocytesaging,exploring the molecular mechanisms of oocyte aging and identifying potentialpredictive markers of embryonic development,are significant to improve the efficiency ofthe assisted reproductive technology,and enrich our understanding about the reproductivephysiology and endocrinology.This study contained the following 4 parts:1.Study on roles of PGC-la and NRF-1 in follicular development and atresia of goatovariesThe aim of this study was to investigate whether PGC-1? and NRF-1 played roles ingoat follicular atresia.The localization of four proteins(PGC-la,NRF-1,BCL-2 andBAX)in the various developmental stages of goat follicle was investigated byimmunohistochemical staining.Antral follicles with different sizes(? 2 mm,2-5 mm and>5 mm)and states(healthy and atresia)were chosen to study the expression pattern ofthese four genes by qRT-PCR and western blotting.In addition,the levels of E2 and P4during follicular development were studied by radioimmunoassay.The immunostaining results showed that the two mitochondria-associated proteins were mainly stained in thegranulosa cells in all types of goat follicles.Levels of E2 and the ratio of E2 to P4 inhealthy follicles were significantly higher than those in atretic follicles(P<0.05).Transcription and translation products of PGC-la and NRF-1 in granulosa cells were moreabundant in large healthy follicles(P<0.05).There was a trend toward reduced expressionof PGC-1? and NRF-1 in granulosa cells of atretic follicles compared with that of healthyfollicles.Furthermore,relative expression of BAX and the ratio of BAX to BCL-2 ingranulosa cells were higher in atretic follicles than in healthy follicles(P<0.05).Theseresults suggest that changes of the mitochondria-associated gene expression patterns ingranulosa cells may lead to follicular atresia during goat follicle development.2.Study on the roles of PGC-la and NRF-1 on apoptosis of goat granulosa cellscultured in vitroWhether PGC-1? and NRF-1 could regulate granulosa cell apoptosis wereinvestigated by interference or overexpression test.The results showed that the PGC-laand NRF-1 mainly localized in the cytoplasm of granulosa cells.Compared with controlgroup,interference of PGC-1? or NRF-1 expression by shRNA in the granulosa cellssignificantly reduced the mtDNA copy number(P<0.05),decreased the expression levelsof anti-oxidative related genes SOD 2,GPx and CAT(P<0.05),increased the expressionlevels of apoptosis-related genes caspase 3,caspase 9 and BAX(P<0.05),and the ratio ofBAX/BCL-2(P<0.05),enhanced intracellular caspase 3 and caspase 9 activity(P<0.05),and significantly increased the apoptosis of granulosa cells(P<0.05).On the other hand,overexpression of PGC-1? or NRF-1 genes could alleviate these effects.In addition,cellcycle analysis showed that interference and overexpression of PGC-1? or NRF-1 had nosignificant effect on cell cycle distribution of the granulosa cells.In summary,aberrantexpression of PGC-1? and NRF-1 may lead to the apoptosis of granulosa cells,thus resultin follicular atresia.3.Study on the roles of PGC-la and NRF-1 in goat oocytes agingIn the present study,the age-dependent molecular changes in the goat oocytes wereinvestigated.Firstly,the quality of the goat oocytes with different in vitro culture time(24,30,36,48 and 60 h)was evaluated by the developmental rates of parthenogeneticactivation embryo and cumulus cells(CCs)apoptosis.Secondly,the relative geneexpression levels of six genes(PGC-1?,NRF-1,HAT 1,SNRPN,HAS 3 and SMAD 2)were analyzed during the goat oocyte aging process.Thirdly,the changes of seven genes(PGC-1?,NRF-1,BAX,BCL-2,HAS 2,STAR and SOD 1)in CCs during the oocyte agingprocess were further studied.The results showed that the blastocyst rate of the 30 h groupwas significantly lower in comparison with the 24 h group(P<0.05),but there was nodifference in cleavage rate between the two groups.Additionally,the number of apoptoticcells significantly increased with the culture time prolonged(P<0.05,respectively).Moreover,the relative gene expressions of PGC-1?,NRF-1 and SMAD 2 significantlydecreased from 24 to 36 h(P<0.05).However,the levels of HAT 1 and HAS 3 exhibited aslowly increasing with the cultivation extended.These genes in the aged immature oocyteshad the same expression patterns in the aged mature oocytes.Furthermore,the levels ofPGC-1?,BCL-2,HAS 2 and SOD 1 quickly reduced and BAX significantly increased from24 to 36 h in aged CCs(P<0.05).These genes may be used as markers to predict theoocyte aging process.In conclusion,goat oocytes started to age at 30 h in vitro culture,and the gene expression patterns of oocytes and CCs significantly changed along with thegoat oocyte aging.In addition,PGC-la gene maybe as a marker to predict the oocyte-quality.4.Study on roles of PGC-1? and NRF-1 in goat cloned embryos in vitro developmentThis study further analyzed whether metabolomics could be used to evaluate theeffect of mitochondrial related genes on embryo development potential.In this study,gaschromatography-mass spectroscopy(GC-MS)based metabolomics was used to assess theculture media of goat cloned embryos collected from A(high quality)and B(low quality)groups.The results showed that A group presented higher percentages of blastocystscompared with their counterparts(P<0.05).The metabolic differences presented amongdifferent groups.Moreover,the relative gene expressions of PGC-1? and NRF-1 weresignificantly lower in group B than their counterparts A group(P<0.05,respectively).Dueto differences in metabolic substances were closely associated with energy metabolism,and PGC-1? and NRF-1 were the center regulater of mitochondrial energy metabolism.These results suggested that abnormal expression of PGC-1? and NRF-1 may lead tomitochondrial dysfunction and further decrease the quality of goat transgenic clonedembryos.This method may be helpful to select high quality embryo and improve cloningefficiency.