Abstract
Soon after fertilization, the block mechanisms are developed in the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, penetration, and fusion. However, the molecular basis and underlying mechanism for the post-fertilization block to sperm penetration through ZP has not yet been determined. Here, we find that transglutaminase 2 (Tgm2), an enzyme that catalyzes proteins by the formation of an isopeptide bond within or between polypeptide chains, crosslinks zona pellucida glycoprotein 3 (ZP3) to result in the ZP hardening after fertilization and thus prevents polyspermy. Tgm2 abundantly accumulates in the subcortical region of the oocytes and vanishes upon fertilization. Both inhibition of Tgm2 activity in oocytes by the specific inhibitor in vitro and genetic ablation of Tgm2 in vivo cause the presence of additional sperm in the perivitelline space of fertilized eggs, consequently leading to the polyploid embryos. Biochemically, recombinant Tgm2 binds to and crosslinks ZP3 proteins in vitro, and incubation of oocytes with recombinant Tgm2 protein inhibits the polyspermy. Altogether, our data identify Tgm2 as a participant of zona block to the post-fertilization sperm penetration via hardening ZP surrounding fertilized eggs, extending our current understanding about the molecular basis of block to polyspermy.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Data availability
All relevant data are within the paper and its Supplementary Information files.
References
Bianchi E, Wright GJ. Sperm meets egg: the genetics of mammalian fertilization. Annu Rev Genet. 2016;50:93–111.
Ikawa M, Inoue N, Benham AM, Okabe M. Fertilization: a sperm’s journey to and interaction with the oocyte. J Clin Invest. 2010;120:984–94.
Bhakta HH, Refai FH, Avella MA. The molecular mechanisms mediating mammalian fertilization. Development. 2019;146:dev176966.
Wong JL, Wessel GM. Defending the zygote: search for the ancestral animal block to polyspermy. Curr Top Dev Biol. 2006;72:1–151.
Clift D, Schuh M. Restarting life: fertilization and the transition from meiosis to mitosis. Nat Rev Mol Cell Biol. 2013;14:549–62.
Okabe M. The cell biology of mammalian fertilization. Development. 2013;140:4471–9.
Gardner AJ, Evans JP. Mammalian membrane block to polyspermy: new insights into how mammalian eggs prevent fertilisation by multiple sperm. Reprod Fertil Dev. 2006;18:53–61.
Avella MA, Xiong B, Dean J. The molecular basis of gamete recognition in mice and humans. Mol Hum Reprod. 2013;19:279–89.
Li L, Lu X, Dean J. The maternal to zygotic transition in mammals. Mol ASP Med. 2013;34:919–38.
Fahrenkamp E, Algarra B, Jovine L. Mammalian egg coat modifications and the block to polyspermy. Mol Reprod Dev. 2020;87:326–40.
Bianchi E, Doe B, Goulding D, Wright GJ. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature. 2014;508:483–7.
Xiong B, Zhao Y, Beall S, Sadusky AB, Dean J. A unique egg cortical granule localization motif is required for ovastacin sequestration to prevent premature ZP2 cleavage and ensure female fertility in mice. PLoS Genet. 2017;13:e1006580.
Avella MA, Baibakov B, Dean J. A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans. J Cell Biol. 2014;205:801–9.
Burkart AD, Xiong B, Baibakov B, Jimenez-Movilla M, Dean J. Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. J Cell Biol. 2012;197:37–44.
Gundemir S, Colak G, Tucholski J, Johnson GV. Transglutaminase 2: a molecular Swiss army knife. Biochim Biophys Acta. 2012;1823:406–19.
Wilhelmus MM, de Jager M, Bakker EN, Drukarch B. Tissue transglutaminase in Alzheimer’s disease: involvement in pathogenesis and its potential as a therapeutic target. J Alzheimers Dis. 2014;42:S289–303.
Zemskov EA, Mikhailenko I, Hsia RC, Zaritskaya L, Belkin AM. Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes. PLoS One. 2011;6:e19414.
Belkin AM. Extracellular TG2: emerging functions and regulation. FEBS J. 2011;278:4704–16.
Harper JC, Schatten G. Are we ready for genome editing in human embryos for clinical purposes? Eur J Med Genet. 2019;62:103682.
Okabe M. Sperm-egg interaction and fertilization: past, present, and future. Biol Reprod. 2018;99:134–46.
Grau N, Escrich L, Martin J, Rubio C, Pellicer A, Escriba MJ. Self-correction in tripronucleated human embryos. Fertil Steril. 2011;96:951–6.
Chen Z, Yan J, Feng HL. Aneuploid analysis of tripronuclear zygotes derived from in vitro fertilization and intracytoplasmic sperm injection in humans. Fertil Steril. 2005;83:1845–8.
Evans JP. Preventing polyspermy in mammalian eggs-contributions of the membrane block and other mechanisms. Mol Reprod Dev. 2020;87:341–9.
Liu M. The biology and dynamics of mammalian cortical granules. Reprod Biol Endocrinol. 2011;9:149.
Wang Y, Chen F, He J, Xue G, Chen J, Xie P. Cellular and molecular modification of egg envelope hardening in fertilization. Biochimie 2021;181:134–44.
Tokuhiro K, Dean J. Glycan-independent gamete recognition triggers egg zinc sparks and ZP2 cleavage to prevent polyspermy. Dev Cell. 2018;46:627–40 e5.
Que EL, Bleher R, Duncan FE, Kong BY, Gleber SC, Vogt S, et al. Quantitative mapping of zinc fluxes in the mammalian egg reveals the origin of fertilization-induced zinc sparks. Nat Chem. 2015;7:130–9.
Que EL, Duncan FE, Bayer AR, Philips SJ, Roth EW, Bleher R, et al. Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy. Integr Biol. 2017;9:135–44.
Roth EB, Sjoberg K, Stenberg P. Biochemical and immuno-pathological aspects of tissue transglutaminase in coeliac disease. Autoimmunity. 2003;36:221–6.
Stenberg P, Roth B. Zinc is the modulator of the calcium-dependent activation of post-translationally acting thiol-enzymes in autoimmune diseases. Med Hypotheses. 2015;84:331–5.
Acknowledgements
We thank Dr. Jurrien Dean very much for providing antibodies.
Author information
Authors and Affiliations
Contributions
BX designed the study; ZC, YL, YM, XD and YZ performed the experiments; ZC, YL, YM, and BX analyzed the data; ZC, YL, and BX wrote the manuscript. This work was supported by the National Key Research and Development Program of China (2021YFC2700100, 2018YFC1004002).
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Ethics statement
No experiments involving human subjects were performed in this study. All animal experiments in this article were approved by the Animal Research Institute Committee of Nanjing Agricultural University, China.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by M Piacentini
Supplementary information
Rights and permissions
About this article
Cite this article
Cui, Z., Lu, Y., Miao, Y. et al. Transglutaminase 2 crosslinks zona pellucida glycoprotein 3 to prevent polyspermy. Cell Death Differ 29, 1466–1473 (2022). https://doi.org/10.1038/s41418-022-00933-0
Received:
Revised:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41418-022-00933-0
