Establishment Of Animal Model With Ablation Of Endogenous Spermatogenesis And SSCS Transplantation

Pork is the main source of meat consumption in China.In modern pig industry,the top boars can reproduce a large number of offspring through artificial insemination technology,which plays an important role in improving the breeding and economic benefits.How to reproduce the top breed boars or their genetic materials in large quantities?Somatic cell cloning is the main technical means at present,but because of the low birthrates of cloning animals,it can not be applied on a large scale.A new approach similar to somatic cell cloning is spermatogonial stem cells(SSCs)transplantation,that transplanting exogenous SSCs into infertility recipient testis to reproduce sperm,which enables the complete replication of exogenous genetic materials.The aim of this paper is to establish the model animals that have their own SSCs ablation but can reproduce exogenous spermatogenesis until to spermatozoon production after transplantation,and then lay a solid foundation for the next step to establish a new system,which can replace cloning technology to replicate donor animals genetic materials.The main research contents include:With the clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9)artificial nuclease system,we firstly established mouse model by knocking out its ETV5 gene,then transplanted normal SSCs cultured in vitro into model's testis,and further evaluated the feasibility of this technique applying in mouse by observing donor-derived SSCs spermatogenesis.We lastly performed the preparation of ETV5 gene knock out pig using mouse as a reference.The main results are as follows:1.Preparation of ETV5 gene knock out mice:(1)Comparing to WT groups from 3weeks of age,the testes in ETV5 gene homozygous knockout mice(ETV5^-/-)using the CRISPR/Cas9 system,were much smaller and testes weights were likewise significantly lower(P<0.01).(2)ETV5^-/-mice had a degenerative loss of germ cells in the seminiferous tubules,and Sertoli cell-only syndrome at 12 weeks of age.(3)After sexual maturity,ETV5^-/-mice had a significant reduction of testosterone levels in the blood compared to that of WT groups(P<0.01),and there were no sperm in the epididymides at 12 weeks of age,causing them to be infertile.(4)However,both testicular morphology and development,as well as fertility in the mice with ETV5 gene heterozygous knockout were consistent with that in WT controls.2.Establishment of mouse SSCs culture system in vitro:(1)Testicular single-cell suspensions with viability greater than 99%were obtained by two-step enzyme digestion,and SSCs with purity higher than 79%could be achieved by differential adherent method.(2)Using mouse fetal fibroblasts treated with mitomycin C as feeder layer,stempro-34(containing SFM)as base medium,and adding GDNF,LIF,BFGF,IGF1 growth factors,the proliferation and long-term culture of SSCs in vitro were achieved.(3)The mouse SSCs could be frozen and resuscitated normally.SSC colonies from in vitro culture and enrichment showed positive signals by AKP immunostaining and PLZF immunofluorescence,and in which marker gene expression indicated stable proliferation and stemmed maintenance of SSCs by RT-PCR and Q-PCR testing.3.Transplantation of mouse SSCs:(1)Immature ETV5^-/-mice(3 weeks of age)were more likely to regenerate exogenous sperm as transplant recipients than mature ones(7weeks of age).Two months post-transplantation,complete spermatogenesis was observed in all immature testes with spermatozoa observed in the epididymides(approximately9.58±5.05×10⁴),however,no spermatozoa were detected in adult testes.(2)Under fluorescence stereomicroscope,the head of the sperm produced in the ETV5^-/-epididymides emitted green light,indicating the sperm originated from the donor-SSCs(with the EGFP reporter gene using the lentivirus infection).Moreover,allogeneic ETV5 and EGFP genes were detected in the epididymal sperm,illustrating that the recovered sperm were derived from exogenous donor-SSCs,rather than endogenous production.4.Discussion about animal model of ETV5 knockout pig:(1)The constructed plasmid of p X330-g RNA had a knockout efficiency approximately 41.92%,when it was transfected into Laiwu pig fetal fibroblasts.(2)Eight cloned pigs were born after somatic cell nuclear transplantation,the results of genotypic detection showed that ETV5 gene was knocked out at the target site,and Western blot verified the loss of ETV5 protein.(3)Using H.E.staining and recognizing the expression of UCHL-1 protein,the results showed that both morphology of seminiferous tubules and germ cells in ETV5^-/-pig testes were almost similar with those in WT controls at one month of age,but the former germ cells had a tendency to decline at2.5 month of age.Furthermore,UCHL-1 positive SSCs in ETV5^-/-pig testes were fewer compared to WT groups.Which indicated that ETV5 knock out had an effect on the development of porcine germ cells.In conclusion,ETV5 gene homozygous knockout mouse model with SSCs-deficient and no fertility was generated.Subsequently,the fertility in recipient testis in which being transplanted allogeneic SSCs was restored.Finally,this method was used to pig and hoped to provide scientific basis for the high reproduction of quality individual genetic material,species conservation and spermatogonial stem cell research.