Abstract
Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3 −/−) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3 −/− mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3 −/− mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3 −/− mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3 −/− mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm–egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm–egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.
Similar content being viewed by others
References
Alexander NY, Derek SO, Angshumoy, Paola AAM, Ruihong C, Lata JM, Dolores JL, Martin MM (2012) Association of mutations in the zona pellucida binding protein 1 (ZPBP1) gene with abnormal sperm head morphology in infertile men. Mol Hum Reprod 18(1):14–21. doi:10.1093/molehr/gar057
Araya K, Fukumoto S, Backenroth R, Takeuchi Y, Nakayama K, Ito N, Yoshii N, Yamazaki Y, Yamashita T, Silver J, Igarashi T, Fujita T (2005) A novel mutation in fibroblast growth factor 23 gene as a cause of tumoral calcinosis. J Clin Endocrinol Metab 90(10):5523–5527. doi:10.1210/jc.2005-0301
Bedford JM, Moore HD, Franklin LE (1979) Significance of the equatorial segment of the acrosome of the spermatozoon in eutherian mammals. Exp Cell Res 119(1):119–126
Benet-Pages A, Orlik P, Strom TM, Lorenz-Depiereux B (2005) An FGF23 missense mutation causes familial tumoral calcinosis with hyperphosphatemia. Hum Mol Genet 14(3):385–390. doi:10.1093/hmg/ddi034
Bennett EP, Hassan H, Clausen H (1996) cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-d-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, GalNAc-t3. J Biol Chem 271(29):17006–17012
Campagnoli MF, Pucci A, Garelli E, Carando A, Defilippi C, Lala R, Ingrosso G, Dianzani I, Forni M, Ramenghi U (2006) Familial tumoral calcinosis and testicular microlithiasis associated with a new mutation of GALNT3 in a white family. J Clin Pathol 59:440–442. doi:10.1136/jcp.2005.026369
Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I (1989) An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 86(2):679–688
Chefetz I, Heller R, Galli-Tsinopoulou A, Richard G, Wollnik B, Indelman M, Koerber F, Topaz O, Bergman R, Sprecher E, Schoenau E (2005) A novel homozygous missense mutation in FGF23 causes familial tumoral calcinosis associated with disseminated visceral calcification. Hum Genet 118(2):261–266. doi:10.1007/s00439-005-0026-8
Dam AH, Feenstra I, Westphal JR, Ramos L, van Golde RJ, Kremer JA (2007a) Globozoospermia revisited. Hum Reprod Update 13(1):63–75. doi:10.1093/humupd/dml047
Dam AH, Koscinski I, Kremer JA, Moutou C, Jaeger AS, Oudakker AR, Tournaye H, Charlet N, Lagier-Tourenne C, van Bokhoven H, Viville S (2007b) Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia. Am J Hum Genet 81(4):813–820. doi:10.1086/521314
Escalier D, Silvius D, Xu X (2003) Spermatogenesis of mice lacking CK2α′: Failure of germ cell survival and characteristic modifications of the spermatid nucleus. Mol Reprod Dev 66(2):190–201. doi:10.1002/mrd.10346
Frishberg Y, Topaz O, Bergman R, Behar D, Fisher D, Gordon D, Richard G, Sprecher E (2005) Identification of a recurrent mutation in GALNT3 demonstrates that hyperostosis-hyperphosphatemia syndrome and familial tumoral calcinosis are allelic disorders. J Mol Med (Berl) 83(1):33–38. doi:10.1007/s00109-004-0610-8
Frishberg Y, Ito N, Rinat C, Yamazaki Y, Feinstein S, Urakawa I, Navon-Elkan P, Becker-Cohen R, Yamashita T, Araya K, Igarashi T, Fujita T, Fukumoto S (2007) Hyperostosis-hyperphosphatemia syndrome: a congenital disorder of O-glycosylation associated with augmented processing of fibroblast growth factor 23. J Bone Miner Res 22(2):235–242. doi:10.1359/jbmr.061105
Gu C, Oyama T, Osaki T, Li J, Takenoyama M, Izumi H, Sugio K, Kohno K, Yasumoto K (2004) Low expression of polypeptide GalNAc N-acetylgalactosaminyl transferase-3 in lung adenocarcinoma: impact on poor prognosis and early recurrence. Br J Cancer 90(2):436–442. doi:10.1038/sj.bjc.6601531
Harbuz R, Zouari R, Pierre V, Ben Khelifa M, Kharouf M, Coutton C, Merdassi G, Abada F, Escoffier J, Nikas Y, Vialard F, Koscinski I, Triki C, Sermondade N, Schweitzer T, Zhioua A, Zhioua F, Latrous H, Halouani L, Ouafi M, Makni M, Jouk PS, Sele B, Hennebicq S, Satre V, Viville S, Arnoult C, Lunardi J, Ray PF (2011) A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation. Am J Hum Genet 88(3):351–361. doi:10.1016/j.ajhg.2011.02.007
Ichikawa S, Sorenson AH, Austin AM, Mackenzie DS, Fritz TA, Moh A, Hui SL, Econs MJ (2009) Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression. Endocrinology 150(6):2543–2550. doi:10.1210/en.2008-0877
Ichikawa S, Baujat G, Seyahi A, Garoufali AG, Imel EA, Padgett LR, Austin AM, Sorenson AH, Pejin Z, Topouchian V, Quartier P, Cormier-Daire V, Dechaux M, Malandrinou F, Singhellakis PN, Le Merrer M, Econs MJ (2010) Clinical variability of familial tumoral calcinosis caused by novel GALNT3 mutations. Am J Med Genet A 152A(4):896–903. doi:10.1002/ajmg.a.33337
Ichikawa S, Austin AM, Gray AK, Allen MR, Econs MJ (2011) Dietary phosphate restriction normalizes biochemical and skeletal abnormalities in a murine model of tumoral calcinosis. Endocrinology 152(12):4504–4513. doi:10.1210/en.2011-1137
Ito C, Suzuki-Toyota F, Maekawa M, Toyama Y, Yao R, Noda T, Toshimori K (2004) Failure to assemble the peri-nuclear structures in GOPC deficient spermatids as found in round-headed spermatozoa. Arch Histol Cytol 67(4):349–360
Juneja SC, van Deursen JM (2005) A mouse model of familial oligoasthenoteratozoospermia. Hum Reprod 20(4):881–893. doi:10.1093/humrep/deh719
Kang-Decker N, Mantchev GT, Juneja SC, McNiven MA, van Deursen JM (2001) Lack of acrosome formation in Hrb-deficient mice. Science 294(5546):1531–1533. doi:10.1126/science.1063665
Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H (2006) Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 281(27):18370–18377. doi:10.1074/jbc.M602469200
Kato K, Takeuchi H, Ohki T, Waki M, Usami K, Hassan H, Clausen H, Irimura T (2008) A lectin recognizes differential arrangements of O-glycans on mucin repeats. Biochem Biophys Res Commun 371(4):698–701. doi:10.1016/j.bbrc.2008.04.120
Koscinski I, Elinati E, Fossard C, Redin C, Muller J, Velez de la Calle J, Schmitt F, Ben Khelifa M, Ray PF, Kilani Z, Barratt CL, Viville S (2011) DPY19L2 deletion as a major cause of globozoospermia. Am J Hum Genet 88(3):344–350. doi:10.1016/j.ajhg.2011.01.018
Larsson T, Yu X, Davis SI, Draman MS, Mooney SD, Cullen MJ, White KE (2005) A novel recessive mutation in fibroblast growth factor-23 causes familial tumoral calcinosis. J Clin Endocrinol Metab 90(4):2424–2427. doi:10.1210/jc.2004-2238
Liu G, Shi QW, Lu GX (2010) A newly discovered mutation in PICK1 in a human with globozoospermia. Asian J Androl 12(4):556–560. doi:10.1038/aja.2010.47
Meyts ER-D, Poll SN, Goukasian I, Jeanneau C, Herlihy AS, Bennett EP, Skakkebæk NE, Clausen H, Giwercman A, Mandel U (2007) Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation. Virchows Arch 451:805–814. doi:10.1007/s00428-007-0478-4
Mori E, Baba T, Iwamatsu A, Mori T (1993) Purification and characterization of a 38-kDa protein, sp38, with zona pellucida-binding property from porcine epididymal sperm. Biochem Biophys Res Commun 196(1):196–202
Mori E, Kashiwabara S, Baba T, Inagaki Y, Mori T (1995) Amino acid sequences of porcine sp38 and proacrosin required for binding to the zona pellucida. Dev Biol 168(2):575–583
Puri KD, Gopalakrishnan B, Surolia A (1992) Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4). FEBS Lett 312(2–3):208–212
Stevens A, Lowe J (1992) Histology. Gower Medical Publishing, London
Topaz O, Shurman DL, Bergman R, Indelman M, Ratajczak P, Mizrachi M, Khamaysi Z, Behar D, Petronius D, Friedman V, Zelikovic I, Raimer S, Metzker A, Richard G, Sprecher E (2004) Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat Genet 36(6):579–581. doi:10.1038/ng1358
Toshimori K (2009) Dynamics of the mammalian sperm head: modifications and maturation events from spermatogenesis to egg activation. Adv Anat Embryol Cell Biol 204:5–94
Toshimori K, Tanii I, Araki S, Oura C (1992) Characterization of the antigen recognized by a monoclonal antibody MN9: unique transport pathway to the equatorial segment of sperm head during spermiogenesis. Cell Tissue Res 270(3):459–468
Toshimori K, Saxena DK, Tanii I, Yoshinaga K (1998) An MN9 antigenic molecule, equatorin, is required for successful sperm–oocyte fusion in mice. Biol Reprod 59(1):22–29
Wu AM, Sugii S, Herp A (1988) A guide for carbohydrate specificities of lectins. Adv Exp Med Biol 228:819–847
Xiao N, Kam C, Shen C, Jin W, Wang J, Lee KM, Jiang L, Xia J (2009) PICK1 deficiency causes male infertility in mice by disrupting acrosome formation. J Clin Invest 119(4):802–812. doi:10.1172/JCI36230
Xu X, Toselli PA, Russell LD, Seldin DC (1999) Globozoospermia in mice lacking the casein kinase II alpha’ catalytic subunit. Nat Genet 23(1):118–121. doi:10.1038/12729
Yamamoto S, Nakamori S, Tsujie M, Takahashi Y, Nagano H, Dono K, Umeshita K, Sakon M, Tomita Y, Hoshida Y, Aozasa K, Kohno K, Monden M (2004) Expression of uridine diphosphate N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl transferase 3 in adenocarcinoma of the pancreas. Pathobiology 71(1):12–18. doi:10.1159/000072957
Yamatoya K, Yoshida K, Ito C, Maekawa M, Yanagida M, Takamori K, Ogawa H, Araki Y, Miyado K, Toyama Y, Toshimori K (2009) Equatorin: identification and characterization of the epitope of the MN9 antibody in the mouse. Biol Reprod 81(5):889–897. doi:10.1095/biolreprod.109.077438
Yanagimachi R, Noda YD (1970) Physiological changes in the postnuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells. J Ultrastruct Res 31:486–493
Yao R, Ito C, Natsume Y, Sugitani Y, Yamanaka H, Kuretake S, Yanagida K, Sato A, Toshimori K, Noda T (2002) Lack of acrosome formation in mice lacking a Golgi protein, GOPC. Proc Natl Acad Sci USA 99(17):11211–11216. doi:10.1073/pnas.162027899
Yi-Nan L, Angshumoy R, Wei Y, Kathleen HB, Martin MM (2007) Loss of zona pellucida binding proteins in the acrosomal matrix disrupts acrosome biogenesis and sperm morphogenesis. Mol Cell Biol 27(19):6794–6805. doi:10.1128/MCB.01029-07
Yoshida K, Ito C, Yamatoya K, Maekawa M, Toyama Y, Suzuki-Toyota F, Toshimori K (2010) A model of the acrosome reaction progression via the acrosomal membrane-anchored protein equatorin. Reproduction 139(3):533–544. doi:10.1530/REP-09-0434
Yoshinaga K, Saxena DK, Oh-oka T, Tanii I, Toshimori K (2001) Inhibition of mouse fertilization in vivo by intra-oviductal injection of an anti-equatorin monoclonal antibody. Reproduction 122(4):649–655
Acknowledgments
We thank Y. Matsuo, Y. Date, and M. Hirakawa for technical assistance and C. Fukuda for secretarial assistance. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Author information
Authors and Affiliations
Corresponding author
Additional information
T. Miyazaki, M. Mori, and C. A. Yoshida contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Miyazaki, T., Mori, M., Yoshida, C.A. et al. Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia. Histochem Cell Biol 139, 339–354 (2013). https://doi.org/10.1007/s00418-012-1031-3
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-012-1031-3