Studies On The Function And Mechanism Of DNA Damage Repair Protein BRCA1 In Spermatogenesis

Mammalian spermatogenesis is a complex and continuous process,which is composed of the following three stages:1.Self-renewal and differentiation of spermatogonial stem cells;2.Meiosis;3.Transformation and maturation of spermatid.Precise regulation by a great many genes is required for normal and orderly spermatogenesis,which includes a series of DNA damage repair-related genes.BRCA1?Breast cancer 1,early onset?is a key protein involved in DNA double-strand break?DSB?repair in somatic cells.Just for this reason,BRCA1 is widely recognized as a tumor suppressor,which prevents tumorigenesis by participating in homologous recombination repair,an error-free method of DSB repair.Previous studies have reported that BRCA1 localizes to unsynapsed axes of sex chromosome in pachytene spermatocyte of meiosis I and is responsible for establishment of meiotic sex chromosome inactivation?MSCI?.However,the BRCA1 mutant mice they used are hypomorphic,the function of BRCA1 is partially retained in these mice.Therefore,the full spectrum of BRCA1's function in spermatogenesis requires further investigation.Because Brca1 knockout mice are embryonic lethal,we utilized cre-loxp technique and conditionally deleted exon 5-13 of Brca1 specifically in germ cells using well-recognized Vasa-cre mice.BRCA1?5-13 destroys the N-terminal RING domain and results in frame-shift mutation and then a premature stop codon,eventually producing a short peptide comprising only 52 amino acids.Thus,BRCA1?5-13destroys all functional domains of BRCA1,which provides opportunity for us to investigate the exact role of BRCA1 in spermatogenesis.We observed that Brca1^flox5-13/-;Vasa-Cre mice grew normally and females were fertile.However,Brca1^flox5-13/-;Vasa-Cre males were infertile,without any mature spermatozoa in their epididymides.Further examination indicates that the testes of Brca1^flox5-13/-;Vasa-Cre males were remarkably smaller than controls and germ cells were seriously depleted,all of which were located in the basal membrane of seminiferous tubule.Immunofluorescence staining shows that meiosis and differentiating spermatogonia were completely absent in testes of Brca1^flox5-13/-;Vasa-Cre males,suggesting that BRCA1 plays an important role in spermatogonial differentiation and meiosis initiation.Consistently,all the remaining germ cells were PLZF-positive undifferentiated spermatogonia(A_undiff).A_undiffincludes GFR?1-positive spermatogonial stem cell?SSC?population and SOX3/RAR?-positive differentiation-primed A_undiff population,and the latter derives from the former.Interestingly,at postnatal day 21?P21?,the number of SOX3/RAR?-positive differentiation-primed A_undiff per seminiferous tubule in Brca1^flox5-13/-;Vasa-Cre males was sharply decreased,while the number of SSCs was significantly increased.Analysis of proliferation and apoptosis status of these two population shows that elevated apoptosis and declined proliferation were observed with Brca1 knockout SOX3-positive differentiation-primed A_undiff in a p53-dependent manner,but the SSCs were hyper-proliferative.The above results indicate that complete knockout of Brca1 leads to loss of homeostasis in undifferentiated spermatogonia.Mechanistically,BRCA1 complete loss results in accumulation of DSBs in both SSCs and differentiation-primed A_undiff.However,due to vulnerability to DNA damage,SOX3-positive differentiation-primed A_undiff first underwent programed cell death and were dramatically depleted.Then,SSCs increased proliferation to compensate for massive loss of SPCs,which was accompanied by a slight but significant increase in their apoptosis.Accordingly,the number of SSCs reduced remarkably in Brca1^flox5-13/-;Vasa-Cre testes at P90.Preventing cell death by concomitant deletion of p53 or restoring DSB repair by concomitant deletion of 53bp1 largely overcame the spermatogonial differentiation defects and restored their meiosis and homeostasis of undifferentiated spermatogonia.Collectively,our study not only reveals a novel role of DNA damage repair protein BRCA1 in spermatogenesis,but also demonstrates that DSB repair is important for maintaining homeostasis of undifferentiated spermatogonia.