Studies On The Function And Mechanism Of Ggnbp2 At The Spermiogenesis Stage In Mice

Male infertility is not only a medical problem,but also a social problem.But the pathogenesis of some male infertility diseases is still not clear so far.Azoospermia in male infertility patients is in the proportion of about 10%-20%.according to whether the obstruction of the vas deferens azoospermia can be divided into obstructive and non-obstructive.Non-obstructive azoospermia refers to spermatozoa that are not detected in semen for more than 3 times,and testicular biopsy presents germline dysfunction due to germ cell dysplasia,and genetic abnormalities are generally considered to be a major risk factor.Gametogenetin binding protein 2(GGNBP2)is an evolutionarily conserved zinc finger protein.To elucidate the function of GGNBP2 in vivo,we generated Ggnbp2 knockout mice 6.8% of Ggnbp2 null mice in the B6/129 mixed background were viable and continued to adulthood.Adult Ggnbp2 null males were sterile with smaller testes and an azoospermic phenotype,while mutant females were fertile.Methods:1.A Ggnbp2 knockout mouse model was established using B6 / 129 hybrid mice,and the offspring were obtained by crossbreeding.The mouse DNA was extracted and the genotype was identified by PCR.The testis of Ggnbp2 knockout mice were obtained and the ultrastructures of spermatogenic cells were analyzed under electron microscopy2.PAS staining was performed on testicular sections of wild-type and Ggnbp2 knockout mice.The spermatogenesis stage of spermatogenic epithelium was determined by the acrosome shape of sperm cells and the composition of whole spermatogenic epithelium and counted respectively3.LH(luteinizing hormone),FSH(follicle stimulating hormone)and testosterone levels in serum of wild type and Ggnbp2 knockout mice were measured4.The specific antibodies to spermatogonia,spermatocytes,testicular stromal cells,testicular support cells and blood testis were selected for the testis sections of wild type and Ggnbp2 knockout mice.Immunofluorescence staining was performed on the cells.5.The morphological differences of spermatogenic cells were observed by immunostaining and fluorescence microscopy after separated by gravity sedimentation method.Real-time PCR was used to analyze the specific marker m RNA of spermatocytes and round sperm cells.6.we injected solution containg sfln5 os plasmid to one side testis of WT mice and injected saline as control.Then examine the histological score and DNA expression of the testis between these two groups after72 h.Results:1.Histopathological analysis of 2-month old Ggnbp2 null testes revealed absence of mature spermatozoa in the seminiferous tubules and epididymides and reduction of the number of spermatids.2.Ultrastructural analysis indicated dramatic morphological defects of developing spermatids in the Ggnbp2 null testes,including irregularly shaped acrosomes,acrosome detachment,cytoplasmic remnant,ectopic manchette and illformed head shape in both elongating and elongated spermatids.3.However,the numbers of spermatogonia,spermatocytes,Leydig cells and Sertoli cells in Ggnbp2 null testes did not significantly differ from the wild-type(WT)siblings.4.Gonadotropins,testosterone and the blood-testis barrier were essentially unaffected.5.Western blot analyses showed increases in α-E-catenin,β-catenin and N-cadherin,decreases in Ecadherin,afadin and nectin-3,and were unchanged in vinculin,nectin-2,FAK and integrin-β1 protein levels in Ggnbp2 null testes comparedto WT siblings.6.The m RNA of Timp1,Cnn3,Doc2 b,Igta6,Serping1,Serpina3 n,Serpinb1a and Slfn5 os in Ggnbp2 knockout mice were highly expressed in testis,spermatocytes and sperm cells.The expression Slfn5 os is up to 40 times than WT.7.The expression of Sfln5 os was higher than that of the control group after microinjection of slfn5 os overexpression plasmid.H&E staining showed that spermatogenic tubules were similar to Ggnbp2 KO.Conclusions:1.We successfully established animal model of Ggnbp2 knockout mice,and confirmed the deletion of Ggnbp2 gene can lead to azoospermia.2.Ggnbp2 plays an indispensable role in maintaining the integrity of the seminiferous epithelium and is an important factor in maintaining normal spermatogenesis.3.Ggnbp2 plays an important role in the process of sperm cells deforming into mature spermatozoa,which could affect the sperm differentiation.4.Ggnbp2 gene plays an important role in the reproductive function of male mice and is an important factor in maintaining the normal morphology and differentiation of sperm.The research on the mechanism of Ggnbp2 gene is of great significance to explore the mechanism of male infertility.