{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:07:17Z","timestamp":1760144837795,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2024,5,22]],"date-time":"2024-05-22T00:00:00Z","timestamp":1716336000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Scientific research project of Health Committee of Shaanxi province","award":["2022A012"],"award-info":[{"award-number":["2022A012"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>A low-calcium microenvironment is imperative for spermatozoa maturation within the epididymis. Our previous work has shown that \u03b3-glutamyl carboxylase (GGCX), the carboxylation enzyme of the matrix Gla protein (MGP), plays an essential role in epididymal calcium homeostasis and sperm maturation in rats and that the GGCX SNP mutation rs699664 was associated with asthenozoospermia (AZS) in humans. Here, we investigated the expression patterns of GGCX and MGP in the mouse epididymis and generated GgcxK325Q knock-in (KI) mice. We also tested the effects of this mutation on epididymal calcium homeostasis, sperm function, and male fertility in GgcxK325Q\u2212\/\u2212 mice. The results showed that both GGCX and MGP were enriched in all regions of the mouse epididymis, especially in the initial segment of the epididymis. Double immunofluorescence staining revealed that GGCX colocalized with MGP in the epithelial cells of the initial segment and caput regions as well as in the lumen of the corpus and cauda regions of the mouse epididymis. However, the GgcxK325Q\u2212\/\u2212 mice were fertile with normal epididymal morphology, sperm functions, and epididymal calcium concentration. Overall, our findings revealed that the GgcxK325Q mutation does not exert any discernible effect on male fertility in mice.<\/jats:p>","DOI":"10.3390\/cimb46060303","type":"journal-article","created":{"date-parts":[[2024,5,22]],"date-time":"2024-05-22T11:03:47Z","timestamp":1716375827000},"page":"5052-5065","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The GgcxK325Q Mutation Does Not Affect the Calcium Homeostasis of the Epididymis and Male Fertility in Mice"],"prefix":"10.3390","volume":"46","author":[{"given":"Mingxiang","family":"Xiong","sequence":"first","affiliation":[{"name":"College of Life Sciences, Northwest University, Xi\u2019an 710069, China"},{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1051-2208","authenticated-orcid":false,"given":"Pang","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8085-2302","authenticated-orcid":false,"given":"Yanqiu","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0910-0696","authenticated-orcid":false,"given":"Ting","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4944-4457","authenticated-orcid":false,"given":"Zhen","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Human Anatomy, Histology and Embryology, Air Force Medical University, Xi\u2019an 710038, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,5,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Ali, W., Deng, K., Bian, Y., Liu, Z., and Zou, H. (2023). Spectacular role of epididymis and bio-active cargo of nano-scale exosome in sperm maturation: A review. Biomed. Pharmacother., 164.","DOI":"10.1016\/j.biopha.2023.114889"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Fang, F., Ni, K., Cai, Y., Zhao, Q., Shang, J., Zhang, X., Shen, S., and Xiong, C. (2017). Busulfan administration produces toxic effects on epididymal morphology and inhibits the expression of ZO-1 and vimentin in the mouse epididymis. Biosci. Rep., 37.","DOI":"10.1042\/BSR20171059"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"7938","DOI":"10.1074\/jbc.M110.142836","article-title":"Switch of PMCA4 splice variants in bovine epididymis results in altered isoform expression during functional sperm maturation","volume":"286","author":"Brandenburger","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"ra27","DOI":"10.1126\/scisignal.2001791","article-title":"Male fertility depends on Ca2+ absorption by TRPV6 in epididymal epithelia","volume":"4","author":"Weissgerber","year":"2011","journal-title":"Sci. Signal."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1093\/molehr\/gar075","article-title":"Regucalcin, a calcium-binding protein with a role in male reproduction?","volume":"18","author":"Laurentino","year":"2012","journal-title":"Mol. Hum. Reprod."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"442","DOI":"10.1126\/science.aad0836","article-title":"Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive","volume":"350","author":"Miyata","year":"2015","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Tello-Mora, P., Hern\u00e1ndez-Cadena, L., Pedraza, J., L\u00f3pez-Bayghen, E., and Quintanilla-Vega, B. (2018). Acrosome reaction and chromatin integrity as additional parameters of semen analysis to predict fertilization and blastocyst rates. Reprod. Biol. Endocrinol. RBE, 16.","DOI":"10.1186\/s12958-018-0408-0"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Shum, W., Zhang, B.L., Cao, A.S., Zhou, X., Shi, S.M., Zhang, Z.Y., Gu, L.Y., and Shi, S. (2022). Calcium Homeostasis in the Epididymal Microenvironment: Is Extracellular Calcium a Cofactor for Matrix Gla Protein-Dependent Scavenging Regulated by Vitamins. Front. Cell Dev. Biol., 10.","DOI":"10.3389\/fcell.2022.827940"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1038\/386078a0","article-title":"Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein","volume":"386","author":"Luo","year":"1997","journal-title":"Nature"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"11400","DOI":"10.1074\/jbc.M116.769802","article-title":"Matrix Gla protein deficiency impairs nasal septum growth, causing midface hypoplasia","volume":"292","author":"Marulanda","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ghosh, S., Oldenburg, J., and Czogalla-Nitsche, K.J. (2022). The Role of GRP and MGP in the Development of Non-Hemorrhagic VKCFD1 Phenotypes. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23020798"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1016\/j.isci.2019.03.030","article-title":"Vitamin K2-Dependent GGCX and MGP Are Required for Homeostatic Calcium Regulation of Sperm Maturation","volume":"14","author":"Ma","year":"2019","journal-title":"iScience"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e00404-20","DOI":"10.1128\/MCB.00404-20","article-title":"Vitamin K-Dependent \u03b3-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice","volume":"41","author":"Shiba","year":"2021","journal-title":"Mol. Cell. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Azuma, K., and Inoue, S. (2019). Multiple Modes of Vitamin K Actions in Aging-Related Musculoskeletal Disorders. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20112844"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1111\/jth.13217","article-title":"Structural and functional insights into enzymes of the vitamin K cycle","volume":"14","author":"Tie","year":"2016","journal-title":"J. Thromb. Haemost. JTH"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5270","DOI":"10.1182\/blood-2006-12-064188","article-title":"Fatal hemorrhage in mice lacking gamma-glutamyl carboxylase","volume":"109","author":"Zhu","year":"2007","journal-title":"Blood"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2410","DOI":"10.1111\/jth.13517","article-title":"Uniparental disomy causes deficiencies of vitamin K-dependent proteins","volume":"14","author":"Dasi","year":"2016","journal-title":"J. Thromb. Haemost. JTH"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/j.cell.2010.06.003","article-title":"Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism","volume":"142","author":"Ferron","year":"2010","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1016\/j.cell.2007.05.047","article-title":"Endocrine regulation of energy metabolism by the skeleton","volume":"130","author":"Lee","year":"2007","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Lacombe, J., and Ferron, M. (2018). VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues. Nutrients, 10.","DOI":"10.3390\/nu10080970"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1002\/mnfr.201300743","article-title":"The realm of vitamin K dependent proteins: Shifting from coagulation toward calcification","volume":"58","author":"Willems","year":"2014","journal-title":"Mol. Nutr. Food Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2503","DOI":"10.1111\/j.1538-7836.2007.02758.x","article-title":"Post-translational modifications regulate matrix Gla protein function: Importance for inhibition of vascular smooth muscle cell calcification","volume":"5","author":"Schurgers","year":"2007","journal-title":"J. Thromb. Haemost. JTH"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Okubo, Y., Masuyama, R., Iwanaga, A., Koike, Y., Kuwatsuka, Y., Ogi, T., Yamamoto, Y., Endo, Y., Tamura, H., and Utani, A. (2017). Calcification in dermal fibroblasts from a patient with GGCX syndrome accompanied by upregulation of osteogenic molecules. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0177375"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.molmed.2012.12.008","article-title":"Vitamin K-dependent carboxylation of matrix Gla-protein: A crucial switch to control ectopic mineralization","volume":"19","author":"Schurgers","year":"2013","journal-title":"Trends Mol. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1111\/j.1440-1797.2006.00660.x","article-title":"Molecular mechanisms mediating vascular calcification: Role of matrix Gla protein","volume":"11","author":"Proudfoot","year":"2006","journal-title":"Nephrology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"856","DOI":"10.1016\/j.thromres.2014.07.004","article-title":"Bleeding and non-bleeding phenotypes in patients with GGCX gene mutations","volume":"134","author":"Watzka","year":"2014","journal-title":"Thromb. Res."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vlasschaert, C., Goss, C.J., Pilkey, N.G., McKeown, S., and Holden, R.M. (2020). Vitamin K Supplementation for the Prevention of Cardiovascular Disease: Where Is the Evidence? A Systematic Review of Controlled Trials. Nutrients, 12.","DOI":"10.3390\/nu12102909"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/s41419-023-05655-w","article-title":"MLKL signaling regulates macrophage polarization in acute pancreatitis through CXCL10","volume":"14","author":"Peng","year":"2023","journal-title":"Cell Death Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1111\/andr.12434","article-title":"The dynamic metabolomic changes throughout mouse epididymal lumen fluid potentially contribute to sperm maturation","volume":"6","author":"Hu","year":"2018","journal-title":"Andrology"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3187","DOI":"10.1007\/s00018-012-1231-y","article-title":"Regulatory circuits controlling vascular cell calcification","volume":"70","author":"Sallam","year":"2013","journal-title":"Cell. Mol. Life Sci. CMLS"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Wan, Z., Bai, X., Wang, X., Guo, X., Wang, X., Zhai, M., Fu, Y., Liu, Y., Zhang, P., and Zhang, X. (2024). Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen\/Nanohydroxyapatite-Mediated Bone Regeneration. Adv. Sci., e2308986.","DOI":"10.1002\/advs.202308986"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e171","DOI":"10.1111\/aos.14533","article-title":"High expression of Matrix Gla Protein in Schnyder corneal dystrophy patients points to an active role of vitamin K in corneal health","volume":"99","author":"Sarosiak","year":"2021","journal-title":"Acta Ophthalmol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2393","DOI":"10.1172\/JCI117246","article-title":"High expression of genes for calcification-regulating proteins in human atherosclerotic plaques","volume":"93","author":"Shanahan","year":"1994","journal-title":"J. Clin. Investig."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"28220","DOI":"10.1074\/jbc.M312599200","article-title":"Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility","volume":"279","author":"Schuh","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1095\/biolreprod55.1.207","article-title":"Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa","volume":"55","author":"Luconi","year":"1996","journal-title":"Biol. Reprod."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"33742","DOI":"10.1074\/jbc.M404628200","article-title":"Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4","volume":"279","author":"Okunade","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1093\/humupd\/dmaa034","article-title":"Molecular genetics of infertility: Loss-of-function mutations in humans and corresponding knockout\/mutated mice","volume":"27","author":"Jiao","year":"2021","journal-title":"Hum. Reprod. Update"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1182\/blood.2020006329","article-title":"\u03b3-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K-dependent proteins","volume":"137","author":"Hao","year":"2021","journal-title":"Blood"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.ajhg.2013.11.017","article-title":"Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm flagella","volume":"94","author":"Coutton","year":"2014","journal-title":"Am. J. Hum. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1093\/hmg\/10.11.1117","article-title":"Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency","volume":"10","author":"Neesen","year":"2001","journal-title":"Hum. Mol. Genet."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1395","DOI":"10.1002\/nbm.2965","article-title":"An embedded four-channel receive-only RF coil array for fMRI experiments of the somatosensory pathway in conscious awake marmosets","volume":"26","author":"Papoti","year":"2013","journal-title":"NMR Biomed."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/46\/6\/303\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:46:10Z","timestamp":1760107570000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/46\/6\/303"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,5,22]]},"references-count":41,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["cimb46060303"],"URL":"https:\/\/doi.org\/10.3390\/cimb46060303","relation":{},"ISSN":["1467-3045"],"issn-type":[{"type":"electronic","value":"1467-3045"}],"subject":[],"published":{"date-parts":[[2024,5,22]]}}}