Vitamin K2-dependent GGCK And MGP Are Required For Homeostatic Calcium Regulation In Epididymis For Male Reproduction Health

?Background?The epididymis is an important organ for sperm maturation,fertilization,storage and protection in mammalian.A low calcium environment in the epididymis lumen maintains the resting state of the spermatozoa and prevents sperm cells from being activated.Abnormal elevation of calcium concentration in the epididymal lumen will lead to premature sperm activation,decreased sperm motility,and even male fertility defects or infertility.However,the mechanism of the epididymal homeostastic calcium regulation has not been fully elucidated.Vitamin K2?vitamin K2,VK2?cycle-dependent?-glutamyl carboxylase?GGCX?and its substrate,matrix Gla protein?MGP?play an important role in the maintaince of calcium homeostasis in blood vessel and kidney.Being carboxylated by GGCX under the circumstance of suffcient VK2,MGP can directly bind calcium ions or calcic crystals to preclude the accumulation of excessive calcium ions or the despositon of calcic crystals.To explore whether this pivotal mechanism involves in the maintenance of the epididymal calcium homeostasis,we examined the expression and localization of VK cycle associated proteins in the epididymis of WT rats.Then we established a warfarin-induced vitamin K2 deficiency rat model to test the changes of fertility phenotypes as well as calcium distribution or concentration in epididymal epithelial cells and epididymal fluid.Finally,we screened the SNPs of GGCX and MGP in infertile men with asthenozoospermia to find the clinical significance of GGCX and MGP for male reproduction health.?Methods?1.Real-time quantitative PCR was used to detect the expression of GGCX,MGP,VKORC1 and VKORC1L1 in the epididymis testis and kidney in rats.The expression and localization of GGCX and MGP in rat epididymis were observed by Western blot and immunofluorescence?IF?.2.Western blot was used to detect the binding ability of MGP to its potential binding protein in epididymis and kidney with different calcium concentration in the homogenates.The in-gel digestion followed by LC-MS/MS was utilized to analyze the³2 kDa protein complex and the functional protein association network analysis was conducted to screen out the signaling pathways that the potential binding protein might be involved in.3.VK2 deficient rat model was induced by subcutaneous injection of warfarin and VK1.Body weight and organ-to-body ratios of vital organs in each group were analyzed.The mating experiment and computer assisted semen analysis system?CASA?were concluded to exmain fertility and sperm motility.Spermatozoon were collected and stained by IF.The sperm morphology was observed using confocal microscopy.4.Hematoxylin and eosin staining?H&E?was used to observe the gross anatomical morphology of the epididymis,and the markers of principle cell and clear cell,E-caherin and B1-VATPase,were labeled by IF.Moreover,the cytoskeletal protein,Phalloidin,as well as the colocalization of GGCX and MGP were observed.In addition,TUNEL staining was utilized to detect apoptosis in epididymal epithelial and lumen of each group.5.X-ray fluorescence imaging was used to detect the distribution of calcium ions in cyro-sections of the epididymis,and inductively coupled plasma mass spectrometry?ICP-MS?was conducted to analyze the relative concentration of calcium ions in the epididymal fluid from each group.In addition,the carboxylation level of MGP were detected by IF.6.High-throughput sequencing was performed to exmain all exons in GGCX and MGP.The samples were obtained from 199 patients with idiopathic asthenozoospermia and 110fertile male controls who had fathered at least one child.SNP analysis used association function of SNPassoc package in R language.?Results?1.The mRNAs of GGCX,MGP and VKORC1 were all expressed in the rat epididymis.The mRNA levels of the GGCX substrate MGP were significantly higher than that in testis,kidney and other organs.Both GGCX and MGP were initially expressed in the epididymal caput,and abundant in corpus and cauda.The colocalization of the two proteins accumulated within the vesicle-like structures in the epithelial cells and lumen environment of the caput and cauda,but negligible colocalization signial was detected in the initial segment and caput region.2.Formation of the³2 kDa MGP associated protein complex is dependent on the presence of low calcium concentration,and is inhibited by excessive calcium in both kidney and epididymis.LC-MS/MS analysis showed that the potential MGP binding protein might involve in ESCRT and Rab interaction networks.3.The body weights and organ-to-body ratios of VK2 deficiency rats were normal,but the fertility and sperm motility were significantly reduced in a warfarin dose-dependent manner.In addition,although spermatogenesis in the testis was unaffected,missing heads or a bent head wrapped around the neck displayed significantly higher percentages in VK2deficiency rats when compared to controls.4.There were some detached epithelial cells,along with reduced cytoskeleton immunofluorescent signals in the smooth muscle cells in VK2 deficiency rats.Also,signals from clear cells and principal cells in the epithelium were reduced dramatically,and the signals of clear cell as well as some apoptotic cells were visible in the detatched cells and cell debris in epididymal lumen.5.The relative Ca²⁺intensity in the epithelium and luminal area tended to be higher in VK2 deficiency rats.The Ca²⁺concentration within epididymal fluid was significantly elevated in warfarin-treated rats.Also,the signals from carboxylated MGP reduced dramatically in the epididymis of VK2 deficiency rats.6.Results from the screening for potential mutations in the exons of MGP and GGCX displayed a significant association of an SNP mutation rs699664 located in the eighth exon of GGCX with male infertility under the recessive model,but revealed no differences in MGP.?Conclusions?1.The VKD enzyme GGCX and its substrate MGP play a crucial role in the regulation of luminal microenvironment in the epididymis,especially in corpus and cauda region.2.MGP-mediated protein-complex aggregation is dependent on sub-millimolar calcium levels whereas excessive calcium levels inhibits aggregation.We propose that under the physiological low calcium microenvironment of the epididymal lumen,MGP-meditated calcium-dependent vesicle-mediated transport and membrane trafficking.3.VK cycle disruption resulted in male fertility problems and epididymal sperm defects.We attribute these phenotypes to the turbulent microenvironment in the epididymis.4.The ditached epididymal epithelial cell,impaired cytoskeleton in the smooth muscle cells,apoptotic cells in the epididymal lumen and reduced signals from clear cells and principal cells in the epithelium reveal the serious epididymal epithelial cellular dysfunction in VK2 deficiency rats.5.The elevated Ca²⁺concentration in both epididymal epithlium and epididymal fluid,along with the blocked caoboxylation of MGP suggest the disorder of low calcium microenvironment in VK2 deficiency rats and MGP might maintain the calcium homeostasis under the regulation of VK2 cycle.6.The significant difference in the GGCX SNP mutation rs699664 in the asthenozoospermic samples compared with controls remind it might be one of the causes of asthenozoospermia.Our study also discloses that VK cycle and the VKD signaling axis play a critical role in human reproduntion health.