Establishment Of MicroRNA-7 Knock-out Mouse Models And Pstudy Of Their Phenotypes On Male Fertility

MicroRNA-7 is a highly conserved microRNA sequence among various species.Until now,many studies have indicated that microRNA-7 plays an important role in the organs'development,the occurrence of diseases,the generation of cancer and other biological activities.It is also reported that microRNA-7 is abundantly expressed in endocrine tissues.However,the special role and mechanism of microRNA-7 in animal reproductive system are still unclear.In this study,we generated three types of microRNA-7 knock-out mouse models by CRISPR/Cas9,and study their phenotypes on male fertility.Firstly,we detected the expression of microRNA-7 in different tissues of adult mouse using Real-time PCR(qRT-PCR).The results showed that microRNA-7 is highly expressed in endocrine tissues such as pituitary,hypothalamus and adrenal,as well as in reproductive system tissues such as testis,uterus and epididymis.We also detected microRNA-7 expression in testis of adult mouse by using in situ hybridization(ISH).These results indicated that microRNA-7 is expressed in the sertoli cell and various differentiated staged germ cells in testis of mouse.Secondly,we established the microRNA-7 knock-out mouse model by using CRISPR/Cas9.MicroRNA-7 knock-out mouse model include three strains,namely microRNA-7al,microRNA-7a2 and microRNA-7b.We detected the gene knock-out mouse model by using PCR,sequencing and Real-time PCR.The results indicated that the microRNA-7 knock-out mouse models which generated by CRISPR/Cas9 have large fragment sequence missing,which was located in mature sequence of microRNA-7.Besides,expression of microRNA-7 in these three subtypes of microRNA-7 knockout mouse model was significantly decreased,and the expression level was close to the background value.We also detected the off-target gene in microRNA-7 knock-out mouse models by using T7EI assay,the result suggested that there were no off-target genes exist in these models.Thirdly,we found that there was no significant difference in the birth rate and survival rate in the three subtypes of microRNA-7 knock-out mouse compared with that of wild type mouse.We also found that the volume and weight of testis,epididymis and uterine of adult mouse in microRNA-7a2 knock-out mouse were significantly decreased compared with those of wild type mouse.However,the weight and volume of tissues in adult mouse with microRNA-7al and microRNA-7b knock-out mouse had no significant changes.The results of these breeding experiment showed that absence of microRNA-7a2 gene will cause both male and female mouse sterile.The microRNA-7al and microRNA-7b gene knockout mouse showed no difference in fecundity compared with wild type mouse.Histological staining of testis and epididymis of adult mouse with microRNA-7a2 knock-out mouse were also observed,the results showed that in mouse testis,the number of seminiferous tubules had decreased and cavities had appeared in some spermatogenic epithelium.This showed that the serum levels of follicle-stimulating hormone(FSH)and luteinizing hormone(LH)in microRNA-7a2 knock-out male mouse were significantly decreased.The sperm number and sperm motility of microRNA-7a2 knock-out mouse were significantly reduced.Finally,in order to study the role and mechanism of microRNA-7a2 in the reproductive system of male mice,we generated the transgenic mice with LoxP sequence flanked microRNA-7a2 lucs(microRNA-7a2flox/flox)using CRISPR/Cas9.It lays a foundation for establishing conditional knock-out(cKO)mouse model of microRNA-7a2.In sum,it is testified that the three subtypes of knock-out microRNA-7 mouse models established by CRISPR/Cas9 can be used for the study of function and mechanism of microRNA-7.In addition,the results of this study also proved that the absence of microRNA-7a2 can cause infertility in male mouse,as well as changes in reproductive hormone and reproductive organ function.These phenotypes indicated that microRNA-7a2 plays an important role in male reproduction.