The Function And Mechanism Study Of Cabs1 In Mice Spermiogenesis

Background:Spermiogenesis is the key step of spermatogenesis,mainly referring to the process by which spherical spermatids differentiate into spermatozoa.The abnormality of spermiogenesis will lead to sperm deformity or sperm dysfunction,which are the inducements of male infertility.Therefore,elucidating the precise mechanism of spermiogenesis is of great significance for the study of male infertility.At present,the researches about the early stages of spermatogenesis have been thoroughly studied.While the investigations about spermiogenesis were relatively scarce.In our previous study,we selected a sperm-specific calcium ion binding protein Cabsl(Calcium binding protein,spermatid specific 1)and its antisense lncRNA(long noncoding RNA),AntiCabsl,as candidates in our research.The knockout mouse models and overexpression cell line were built to study the mutual regulation between AntiCabs1 and Cabsl,then further explore their role and mechanism in the regulation of spermiogenesis and sperm function.Method:After indicated the mouse KO models were built successful,we verified whether the male reproductive ability was normal or not by the experiments below:the observation of mating days before female mice reproduction and the size of each litter,male reproductive organs and tissues morphological changes,motility and morphology of spermatozoa in cauda epididymis.Based on the deformation of Cabs1 null mice sperm,we investigated the testis spermatogenesis,the epididymis sperm maturation,the sperm structure and the influence of epididymis microenvironment to sperm deformation.The overexpression of AntiCabsl in H293 cell line and the AntiCabs1-deficient mice were employed to explore the changes of Cabsl expression.Protein mass spectrometry was used to analyze those proteins which were interacted with Cabsl in epididymis and spermatozoa.Meanwhile the genes with the similar sperm deformation of KO models were investigated.We selected molecules from the two ways above,to confirm whether they participated in the mechanism of Cabs1 KO tail structure abnormality and the process of sperm tail bending in the transit of epididymis.Result:1.This research has determined that AntiCabs1 and Cabs1 knockout mouse models were successfully established.2.The relative phenotypes of male mice reproduction were observed in the two lines of KO mouse.AntiCabs1 null male mouse was proved to be normal in fertility,reproductive organ morphology,spermatogenesis and sperm motility.While the Cabs1 KO male mouse showed impaired fertility,and the Cabsl-deficient sperm were not normal in motility and morphology.3.The entire process of deficient-Cabs1 spermatogenesis was complete,and even the form of elongated sperm was normal.However,when sperm went into the epididymis,the rate of abnormal sperm become higher and higher through caput,corpus to cauda.Wen sperm passing through the epididymis,the deformed sperm changed from half-folded tail(<180°)to hairpin-shaped tail(180°).The 430 mOsm HS(Hepes-buffered saline)were used to swim sperm out,which modified the Osmotic environment in the epididymis.After that,the 430 mOsm HS were adjusted into 310 mOsm,which tested the rapid osmoadaptation of sperm during the environment changing from epididymis into the vitro 310 mOsm HS.The rate of sperm with the folded tail didn't change dramatically in such an osmatic transformation.Those results indicated that the lack of Cabs1 caused the deformation of sperm in the transit through the epididymis.Meanwhile,almost half of Cabs1 null sperm displayed the thinning of annulus between the middle piece and principal piece(which was named as Jenson ring).The rate of structurally deficient sperm in caput was nearly equal with the rate of total deformed sperm in cauda.4.Mass spectrometry authenticated the proteins interacting with Cabs1,some of which were relative with spermatogenesis and cytoskeleton formation.Based on bioinformatics and literature analysis,we further confirmed the possible interacted molecules of Cabsl,and found significant differences in the expression of some interacted proteins between KO and WT groups.5.Over-expression of AntiCabs1 didn't affect the expression level of Cabsl protein in Cabsl stably transfected H293 cell line.Compared with WT testis,the deletion of AntiCabsl in testis didn't increase the expression of Cabs1 notably.Conclusion:We didn't find any evidence to show that AntiCabs 1 influenced the expression of Cabs1.It was normal for AntiCabs1 null mouse in male fertility and reproduction,while Cabs1 was found to be vital for spermiogenesis.The deficiency of Cabsl induced the low motility and deformation of spermatozoa,which further caused the impaired male fertility.The null of Cabsl and the disfunction of the spermiogenesis-related Cabsl interacted proteins might be the key points for the incomplete fertility and reproduction.