Mitochondria Localization Of ASZ1 Link Mfn-mediated Mitofusion To Germ Cell Maturation

Germ cell is the sole carrier of genetic material passed in sexual organisms,and can produce sexual gametes and offspring.In mammals,germ cells first appear at the proximal epiblast of the developing embryo,named primordial germ cells(PGCs).Then PGCs migrate to the gonads to become germline stem cells(GSCs).After mitosis and meiosis,GSCs form mature gametes(sperm or eggs).Mouse spermatogonial stem cells first arrest in the G1/G0 phase,then recover at postnatal day7-10,finally reach sexual maturity at 6-8 weeks after entering meiosis.The phase contains primary spermatocytes(leptotene,zygotene,pachytene,diplotene,diakinesis),secondary spermatocytes,round spermatids,and functional sperm.The number,size and shape,and distribution of mitochondria is very different during each germ cell development stage.In PGCs,spermatogonial stem cells and spermatocytes,mitochondria aggregate around the nuclear,where the ribosomal protein is rich and was named Nuage,also known as Intermitochondria cement(IMC).However,the role of mitochondria in the germ cell development is not yet clear.In the article,we have found that germ-specific genes Aszl located in the outer mitochondrial membrane,with a mitochondrial localization signal(MLS);as a mitochondrial targeting protein,Aszl combines with Mili and recruits Mili to mitochondria;Mutation of MLS in Aszl result in failure of Aszl to promote embryonic stem cells to differentiate into primordial germ cells in vitro.In addition,we also found that Asz1 forms homodimers,and interacts with Mfnl/2,which were the key protein in mitochondrial fusion regulatory machinery.Aszl can regulate mitochondrial morphology and promote mitochondrial fusion in a Mfnl/2-dependent manner.Through CRISPR-Cas System,we constructed an Aszl knockout mice,target mitochondrial localization signal.We discovered that male homozygous mice were infertile.In mice,the expression and localization of Mili/Vasa are abnormal.Nuages are missing,the transposon is up-regulated,indicating that Aszl's mitochondrial localization is essential for its function.These results also suggest that the action of Aszl may depend on Mfn1/2 through mitochondrial fusion.To determine the function of Mfh1/2 in germ cells,Vasa-cre/Stra8-cre lines were crossed with the original knockout line(Mfn null)to generate double heterozygotes.We found that Mfnl knockout male mice were infertile(Vasa-cre)or their reproductive capacity was decreased(Stra8-cre).At P7 Mfnl CKO Vasa-cre mice,SEM showed that mitochondrias were swelling,and endometrial formed vesicles-like crest and gathered clusters around the nucleus,suggesting some defects in mitochondrial bioenergetics aspects.In Mfnl CKO Stra8-cre,the generative capacity decreased,secondary spermatocytes differentiation to sperm cell development is blocked.And Mfn2 Cre mice also showed germ cell developmental disorders;this explains functional relevance between Mfnl and Mfn2.In summary,we identified the mitochondrial localization of Aszl is extremely important in germ cell development,and showed its close relationship with Mfnl/2 in germ cells.And we will continue to explore the molecular mechanism of Aszl/Mfn 1/2 mediated mitochondrial fusion in germ cell function.