Mechanism Of Male Sterility In Type 3 Adenyly Lcyclase Knockout Mice

Human infertility has become a worldwide health and social health problems,and its incidence is rising year by year.Therefore,the study of male reproductive health is of great physiological and social importance.Adenylyl cyclase 3(AC3)can catalyze the production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).c AMP plays an important role in regulating basic functions such as sperm maturation,motility,acrosome reaction and the fertilization process by changing the intracellular p H and calcium ion concentration.Previous studies have suggested that male infertility in AC3^-/-mice is due to a reduced ability of sperm to cross the zona pellucida.However,their such findings are only superficial explanations,and the effects of AC3deficiency on the functional structure of the testis and epididymis and the molecular mechanisms by which AC3 regulates male infertility are unclear.This study aimed to investigate the biological functions and molecular mechanisms of AC3 in the blood-testis barrier and epididymis of mice,and to provide molecular-level information for solving male infertility caused by abnormal sperm maturation due to blood-testis barrier.In this study,we analyzed the biological functions and molecular mechanisms of AC3 in BTB and epididymis of mice.The research is mainly performed in the following three parts.1.Effects on testicular morphology and spermatogenesis processes after AC3 deletion in miceTo investigate,whether the deletion of AC3 affects the process of spermatogenesis and the acrosome formation.The testis or epididymis/body weight of wild type(WT)and AC3knockout(AC3^-/-)mice were first compared.Afterward,hematoxylin and eosin staining assay was used to observe the differences in testicular histomorphology between WT and AC3^-/-mice after AC3 deletion.The effect of AC3 deletion on acrosome biogenesis in AC3^-/-mice were examined using acrosome specific marker FITC-PNA.The results showed that AC3 deletion resulted in reduced gonadal index in AC3^-/-mice.The process of acrosome formation was normal in AC3^-/-mice by immunofluorescence(IF)labeled with FITC-PNA.Furthermore,histopathological evaluation revealed that AC3^-/-mice exhibited mild testicular impairment.A photomicrograph of the testis stained with H&E showing degeneration of germinal cell with double cell layers.Disorganised germ cells with deeply stained pyknotic nuclei and SCs cytoplasmic vacuolation were visible in the seminiferous tubules.Beside multinucleated giant cells were occasionally observed in the seminiferous tubules.Some germinal cells were prematurely sloughed off into the lumen of seminiferous tubules,leading to the degeneration and/or apoptosis of germ cells in lumen.These results showed that the deletion of AC3 in mice will result in abnormal characteristics during spermatogenesis.2.Effects on epididymal morphology and spermatozoa after AC3 deletion in miceDifferences in epididymal histomorphology were observed using H&E staining assay between WT and AC3^-/-mice.The morphology and the number of mature spermatozoa in the epididymis of WT and AC3^-/-mice were compared.Subsequently,mass spectrometry was used to screen for different expression proteins in caput,corpus,and cauda between WT and AC3^-/-mice.Finally,molecular biology techniques such as RT-PCR,q RT-PCR,western blot,and IF were used to investigate the expression and localization of AC3 in the epididymis at the molecular and cellular levels.The results of hematoxylin and eosin staining assay showed that luminal epithelium of the initial segment,caput,corpus,and cauda of the epididymis were normal after deletion of AC3.The number of mature spermatozoa in the epididymis of AC3^-/-mice were significantly decreased,and the head of spermatozoa was malformed.The results of label-free quantification proteomics showed that multiple epididymosomes were significantly changed in AC3^-/-mice.The results of IF showed that the expression of AC3 in the mouse epididymis showed a zonal pattern,with a gradual decrease in the expression of the initial segment,caput,corpus,and cauda.AC3 was not only localized in the narrow cells,clear cells,basal cells,smooth cells,and interstitial tissue of the epididymis,but is also expressed in the stereocilium of the initial segment and on the primary cilia of the epithelial cells of the epididymis.3.Effects of AC3 deletion on the structure and function of blood-testis barrier(BTB)To investigate the key signaling pathways of AC3 that regulate testicular function and spermatogenesis.Mass spectrometry was used to screen for different expression proteins in WT and AC3^-/-mice testes.Afterward,the integrity of BTB in AC3^-/-mice testis was assessed by using a cell biology technique,FITC fluorescence tracer.Molecular and cell biology techniques such as q RT-PCR,western blot,and IF were used to validate the expression of different expression proteins related to BTB function.To further investigate how AC3 regulates BTB function in the testis,proteins interacting with AC3 were screened and validated using CO-IP-MS technique.Subsequently,we used western blot as well as IF techniques to verify the effects of AC3 deletion on germ cell proliferation and apoptosis.BTB can regulate the transport of various substances,including lipids,in the testis.We next examined the changes in lipid metabolism in the testes of AC3^-/-mice by oil red O staining.Finally,we used western blot as well as IF techniques to validate multiple different expression proteins screened by label-free quantification proteomics.The results of label-free quantification proteomics showed that a significant change in signaling pathways associated with adhesion was found in AC3^-/-mice.The results of FITC showed that the blood-testis barrier structure was disrupted after AC3 deletion.Moreover,the expression of tight junction proteins Occludin and Claudin-1 and basal cell ectoplasmic specialization(?-catenin)-related proteins were reduced in AC3^-/-mice,which lead to mild disruption of the BTB components.We confirmed that Basigin and AC3 have certain interactions in the testis using CO-IP-MS and CO-IP.Results from western blot demonstrated that the expression of Basigin was significantly reduced in AC3^-/-mice.In addition,the deletion of AC3 suppressed the germ cell proliferation by measuring positive germ cells of Brdu⁺,Edu⁺,and Ki67⁺.Caspase 3 and Tunel staining showed that the apoptosis of germ cells were increased in AC3^-/-mice.The oil red O staining of testicular tissue showed that testicular lipid metabolism was disturbed after AC3 deletion.Western blot and IF were performed to validate multiple proteins(H3.3,H2AX,and PRM2)screened in the LFQ proteomics.Our results indicated that the process of histone replacement was abnormal during spermatogenesis in AC3^-/-mice.These results suggested that the deletion of AC3 disrupts the structure and function of the blood-testis barrier by reducing the expression of TJ and ES-related proteins,which in turn triggers dysfunction in spermatogenesis,such as diminished proliferation of germ cells,increased apoptosis of germ cells,lipid metabolism disorders,and abnormal histone replacement during spermatogenesis.In conclusion,our findings demonstrated that AC3 is critical for the maintenance of BTB structure and function.These findings advance the previous understanding of male infertility in AC3^-/-mice,from sperm function to BTB function leading to male infertility.This study has broad reference value and important social implications in addressing male infertility caused by abnormal testicular blood-testis barrier function.