Study Of The Role And Mechanism Of Rad51 In Mouse Male Germ Cell Development

[Background]Male infertility increases rapidly in recent years,30%of the patients the disease remains idiopathic(eg non-obstructive oligo-/azoospermia and severe oligozoospermia)and and clinical treatment is limited.Therefore,study of the molecular mechanism of spermatogenesis has great significance for elucidating the cause of infertility.Spermatogenesis includes spermatogonial mitosis,spermatocyte meiosis and sperm metamorphosis and maturation.It is regulated by many hormones and many genes.Meiosis is indispensable for the formation of haploid spermatozoa,and Rad51 maintain the homologous chromosome pair.The single-strand intrusion,homologous pairing and chain exchange catalyzed by the recombinases Rad51 and Dmc1 are the core events in meiotic recombination.In the early stage of Meiosis I,the endonuclease SPO11 catalyzed a large number of programmed-DNA double-strand DNA breaks(DSBs),initiating meiotic recombination.DSBs are excised to form 3'-end single-stranded DNA(ssDNA)tail;Rad51 and Dmc1 bind to ssDNA to form nuclear protein fibers and mediate invasion of ssDNA into homoduplex regions to form D-loop intermediates.After DSBs are produced,homologous chromosomes begin to pair and form synaptonemal complexes(SCs).SC is a complex composed of many proteins such as SCP-1?3,which can not only maintain the homologous chromosome pair but also act as a scaffold to bind Rad51 and DMC1,promote the formation of recombinant complex and participate in meiotic recombination process.Rad51 null mouse embryos are lethal,which preclude their use for study of the mechanism of action of the Rad51 gene during spermatogenesis.Conditional knockout mouse models can avoid embryonic lethality in Rad51 null mouse,but no reported about this.In order to explore the role and mechanism of Rad51 in spermatogenesis,we characterized spermatogenesis in Rad51 germ cell conditional knockout mice:1.Expression of Rad51 in mouse testisWestern blotting demonstrated that Rad51 expression level increased from the 8th day of age,and the expression level increased significantly at the 42nd day of age,there was no significant difference in the expression level of Rad51 protein between 42nd and 96th day.Immunohistochemistry and Real-time PCR demonstrated that Rad51 is highly expressed in mouse testicular germ cells.2.Rad51 conditional knockout mice are sterileWe constructed a Rad51 conditional knockout mouse and mated it with a DDX4-Cre transgenic mouse to obtain a male mouse having the Rad51 gene deleted from the embryonic day 15 germ cell.We used the DDX4-Cre to specifically-delete Rad51 in germ cells.The germ cell specific deletion was confirmed by Western blotting and immunohistochemistry;we found that the testicle volume was reduced by 2/3 compared to the control and this significant difference occurred in the early stage of the individual's growth.Rad51 males are sterile.3.Absence of meiosis and spermatogonia cells in Rad51 conditional knockout miceExamination of chromosome spreads and testicular tissue by immunofluorescence,immunohistochemistry and Western blotting showed no staining of SCP-3 in Rad51fl/-;DDX4-Cre mice,indicating absence of meiosis.Ki-67 positive germ cells are also reduced in the testis of Rad51fl/-;DDX4-Cre mice,indicating that proliferation was impaired in the absence of Rad51.Furthermore,we failed to detect PLZF,a marker for spermatogonia in Rad51fl/-;DDX4-Cre mice,indicating that the generation of the most primitive germ cells is blocked.4.Examination of Sertoli cells in Rad51 conditional knockout miceBecause testicular seminiferous tubules contain only one layer of cells,and spermatogonia mitosis process is blocked in Rad51fl/-;DDX4-Cre mice.By immunofluorescence and immunohistochemical staining of the Sertoli cells marker SOX9 confirmed only Sertoli cells may exist in testicular in Rad51fl/-;DDX4-Cre mice.Statistical analysis of the number of Sertoli cells revealed a significant increased only Sertoli cells may exist in testicular in Rad51fl/;DDX4-Cre mice;transmission electron microscopy showed abnormal morphology of the Sertoli cells,the cells extended to the middle of the seminiferous tubule lumen,but no abnormalities were found in the organelles.5.Germ cells in Rad51fl/+,DDX4-Cre mice have have higher basal DNA damage level and are more sensitive to X-rayWe also examined the level of ?-H2AX in Rad51fl/-;DDX4-Cre mice and found that the basal y-H2AX level was higher in the germline heterozygous mice than in wild-type control.When the mice were exposed to 4Gy X-rays and were examined for ?-H2AX 24 hours later,it was found that Rad51fl/-;DDX4-Cre mice were more sensitive to X-rays compared to controls.