{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T09:08:34Z","timestamp":1790759314828,"version":"4.1.0"},"reference-count":63,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T00:00:00Z","timestamp":1711324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Texas Department of Agriculture"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome\/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a\u2013Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20\/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.<\/jats:p>","DOI":"10.3390\/ijms25073652","type":"journal-article","created":{"date-parts":[[2024,3,25]],"date-time":"2024-03-25T12:47:11Z","timestamp":1711370831000},"page":"3652","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["SMA20\/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization"],"prefix":"10.3390","volume":"25","author":[{"given":"Nathaly","family":"Cormier","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Wisconsin-Whitewater, Whitewater, WI 53190, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asha E.","family":"Worsham","sequence":"additional","affiliation":[{"name":"Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kinsey A.","family":"Rich","sequence":"additional","affiliation":[{"name":"Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7297-3093","authenticated-orcid":false,"given":"Daniel M.","family":"Hardy","sequence":"additional","affiliation":[{"name":"Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,3,25]]},"reference":[{"key":"ref_1","unstructured":"Knobil, E., and Neill, J.D. (1994). The Physiology of Reproduction, Raven Press."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1093\/biolre\/ioac037","article-title":"Mysteries and unsolved problems of mammalian fertilization and related topics","volume":"106","author":"Yanagimachi","year":"2022","journal-title":"Biol. Reprod."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/S0092-8674(00)80558-9","article-title":"Mammalian fertilization: Molecular aspects of gamete adhesion, exocytosis, and fusion","volume":"96","author":"Wassarman","year":"1999","journal-title":"Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4471","DOI":"10.1242\/dev.090613","article-title":"The cell biology of mammalian fertilization","volume":"140","author":"Okabe","year":"2013","journal-title":"Development"},{"key":"ref_5","unstructured":"Hardy, D.M. (2002). Fertilization, Academic Press."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"984","DOI":"10.1172\/JCI41585","article-title":"Fertilization: A sperm\u2019s journey to and interaction with the oocyte","volume":"120","author":"Ikawa","year":"2010","journal-title":"J. Clin. Investig."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1095\/biolreprod38.2.423","article-title":"Purification and characterization of the primary acrosomal autoantigen of guinea pig epididymal spermatozoa","volume":"38","author":"Hardy","year":"1988","journal-title":"Biol. Reprod."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1002\/(SICI)1098-2795(199606)44:2<221::AID-MRD11>3.0.CO;2-5","article-title":"Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family","volume":"44","author":"Foster","year":"1996","journal-title":"Mol. Reprod. Dev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1002\/mrd.23180","article-title":"An update of the regulatory factors of sperm migration from the uterus into the oviduct by genetically manipulated mice","volume":"86","author":"Xiong","year":"2019","journal-title":"Mol. Reprod. Dev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1093\/humupd\/dmaa016","article-title":"The functions of CAP superfamily proteins in mammalian fertility and disease","volume":"26","author":"Gaikwad","year":"2020","journal-title":"Hum. Reprod. Update"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"33509","DOI":"10.1074\/jbc.271.52.33509","article-title":"Sperad is a novel sperm-specific plasma membrane protein homologous to a family of cell adhesion proteins","volume":"271","author":"Quill","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Whitaker, J.R., and Sonnet, P. (1989). Biocatalysis in Agricultural Biotechnology, CRC Press.","DOI":"10.1021\/bk-1989-0389"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1006\/dbio.1995.1152","article-title":"ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain","volume":"169","author":"Wolfsberg","year":"1995","journal-title":"Dev. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3415","DOI":"10.1074\/jbc.273.6.3415","article-title":"Species diversity in the structure of zonadhesin, a sperm-specific membrane protein containing multiple cell adhesion molecule-like domains","volume":"273","author":"Gao","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"24863","DOI":"10.1074\/jbc.M110.123125","article-title":"Zonadhesin is essential for species specificity of sperm adhesion to the egg zona pellucida","volume":"285","author":"Tardif","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1038\/nature03362","article-title":"The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs","volume":"434","author":"Inoue","year":"2005","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"150296","DOI":"10.1098\/rsos.150296","article-title":"Izumo1 and Juno: The evolutionary origins and coevolution of essential sperm-egg binding partners","volume":"2","author":"Grayson","year":"2015","journal-title":"R. Soc. Open Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12527","DOI":"10.1073\/pnas.221454998","article-title":"A voltage-gated ion channel expressed specifically in spermatozoa","volume":"98","author":"Quill","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1038\/35098027","article-title":"A sperm ion channel required for sperm motility and male fertility","volume":"413","author":"Ren","year":"2001","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1016\/j.jri.2009.07.014","article-title":"Diagnosis and treatment of immunologically infertile women with sperm-immobilizing antibodies in their sera","volume":"83","author":"Shibahara","year":"2009","journal-title":"J. Reprod. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1839","DOI":"10.1095\/biolreprod63.6.1839","article-title":"Systematic characterization of sperm-specific membrane proteins in swine","volume":"63","author":"Haden","year":"2000","journal-title":"Biol. Reprod."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Cormier, N., McGlone, J.J., Leszyk, J., and Hardy, D.M. (2018). Immunocontraceptive target repertoire defined by systematic identification of sperm membrane alloantigens in a single species. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0190891"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1083\/jcb.108.2.229","article-title":"The scanning model for translation: An update","volume":"108","author":"Kozak","year":"1989","journal-title":"J. Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1111\/j.1432-1033.1983.tb07424.x","article-title":"Patterns of amino acids near signal-sequence cleavage sites","volume":"133","year":"1983","journal-title":"Eur. J. Biochem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2216","DOI":"10.1073\/pnas.91.6.2216","article-title":"PCR amplification of up to 35-kb DNA with high fidelity and high yield from L bacteriophage templates","volume":"91","author":"Barnes","year":"1994","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1042\/bj20030753","article-title":"Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome","volume":"375","author":"Bi","year":"2003","journal-title":"Biochem. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1002\/j.1939-4640.2000.tb02111.x","article-title":"Human glyceraldehyde 3-phosphate dehydrogenase-2 gene is expressed specifically in spermatogenic cells","volume":"21","author":"Welch","year":"2000","journal-title":"J. Androl."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1038\/nature05634","article-title":"The delayed rise of present-day mammals","volume":"446","author":"Cardillo","year":"2007","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Roberts, E.K., Tardif, S., Wright, E.A., Platt II, R.N., Bradley, R.D., and Hardy, D.M. (2022). Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria. Genome Biol., 23.","DOI":"10.1186\/s13059-022-02721-y"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Foley, N.M., Springer, M.S., and Teeling, E.C. (2016). Mammal madness: Is the mammal tree of life not yet resolved?. Philos. Trans. R. Soc. Lond. B Biol. Sci., 371.","DOI":"10.1098\/rstb.2015.0140"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2671","DOI":"10.1091\/mbc.e11-12-1025","article-title":"Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa","volume":"23","author":"Yamaguchi","year":"2012","journal-title":"Mol. Biol. Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1038\/42484","article-title":"The putative chaperone calmegin is required for sperm fertility","volume":"387","author":"Ikawa","year":"1997","journal-title":"Nature"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4703","DOI":"10.1073\/pnas.1102296108","article-title":"Site of the mammalian sperm physiological acrosome reaction","volume":"108","author":"Bedford","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4892","DOI":"10.1073\/pnas.1018202108","article-title":"Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization","volume":"108","author":"Jin","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Kellogg, M.K., Miller, S.C., Tikhonova, E.B., and Karamyshev, A.L. (2021). SRPassing Co-translational Targeting: The Role of the Signal Recognition Particle in Protein Targeting and mRNA Protection. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22126284"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Tirincsi, A., Sicking, M., Hadzibeganovic, D., Ha\u00dfdenteufel, S., and Lang, S. (2021). The molecular biodiversity of protein targeting and protein transport related to the endoplasmic reticulum. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23010143"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Farkas, \u00c1., and Bohnsack, K.E. (2021). Capture and delivery of tail-anchored proteins to the endoplasmic reticulum. J. Cell Biol., 220.","DOI":"10.1083\/jcb.202105004"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/0092-8674(88)90009-8","article-title":"Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence","volume":"55","author":"Wessels","year":"1988","journal-title":"Cell"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1074\/jbc.273.1.568","article-title":"Co- and posttranslational translocation mechanisms direct cystic fibrosis transmembrane conductance regulator N terminus transmembrane assembly","volume":"273","author":"Lu","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1021\/bi991740r","article-title":"Transmembrane biogenesis of Kv1.3","volume":"39","author":"Tu","year":"2000","journal-title":"Biochemistry"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/0014-5793(95)00551-J","article-title":"Heads or tails-what determines the orientation of proteins in the membrane","volume":"369","author":"Spiess","year":"1995","journal-title":"FEBS Lett."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/S0014-5793(01)02712-0","article-title":"Topogenesis of membrane proteins: Determinants and dynamics","volume":"504","author":"Goder","year":"2001","journal-title":"FEBS Lett."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"909","DOI":"10.1038\/nrm2063","article-title":"Membrane-protein topology","volume":"7","year":"2006","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"5786","DOI":"10.1073\/pnas.86.15.5786","article-title":"Predicting the orientation of eukaryotic membrane-spanning proteins","volume":"86","author":"Hartmann","year":"1989","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/367654a0","article-title":"Evolutionary conservation of components of the protein translocation complex","volume":"367","author":"Hartmann","year":"1994","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1038\/367243a0","article-title":"The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1","volume":"367","author":"Picot","year":"1994","journal-title":"Nature"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1038\/nsb1294-835","article-title":"Backwards and forwards binding","volume":"1","author":"Saraste","year":"1994","journal-title":"Nat. Struct. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1096\/fasebj.14.2.231","article-title":"The importance of being proline: The interaction of proline-rich motifs in signaling proteins with their cognate domains","volume":"14","author":"Kay","year":"2000","journal-title":"FASEB J."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1042\/bj2970249","article-title":"The structure and function of proline-rich regions in proteins","volume":"297","author":"Williamson","year":"1994","journal-title":"Biochem. J."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1126\/science.1083653","article-title":"Assembly of cell regulatory systems through protein interaction domains","volume":"300","author":"Pawson","year":"2003","journal-title":"Science"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/S0898-6568(98)00059-X","article-title":"Regulatory role of SH3 domain-mediated protein-protein interactions in synaptic vesicle endocytosis","volume":"11","author":"McPherson","year":"1999","journal-title":"Cell Signal"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1093\/humupd\/dmi047","article-title":"Sperm transport in the female reproductive tract","volume":"12","author":"Suarez","year":"2006","journal-title":"Hum. Reprod. Update"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1038\/nrg733","article-title":"The rapid evolution of reproductive proteins","volume":"3","author":"Swanson","year":"2002","journal-title":"Nat. Rev. Genet."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Clark, N.L., Gasper, J., Sekino, M., Springer, S.A., Aquadro, C.F., and Swanson, W.J. (2009). Coevolution of interacting fertilization proteins. PLoS Genet., 5.","DOI":"10.1371\/journal.pgen.1000570"},{"key":"ref_55","unstructured":"Hardy, D.M., Wright, E.A., Worsham, A.E., Bradley, R.D., and Roberts, E.K. (2024). Zan Til+E Domain Duplication and Divergence Reflect Unique Evolution of a Speciation Gene in Muroid Rodents, Texas Tech University Health Sciences Center. In Preparation."},{"key":"ref_56","unstructured":"Ashton, M.N., Worsham, A.E., Cheung, T.L., Grozdanov, P.N., and Hardy, D.M. (2024). Heterogeneous RNA Splicing and Permissive Surveillance in Human Male Germ Cells Drive Individual Variation in Expressed Transcripts of the Speciation Gene ZAN, Texas Tech University Health Sciences Center. In Preparation."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1146\/annurev-genet-111212-133301","article-title":"New gene evolution: Little did we know","volume":"47","author":"Long","year":"2013","journal-title":"Annu. Rev. Genet."},{"key":"ref_58","doi-asserted-by":"crossref","unstructured":"Andersson, D.I., Jerlstr\u00f6m-Hultqvist, J., and N\u00e4svall, J. (2015). Evolution of new functions de novo and from preexisting genes. Cold Spring Harb. Perspect. Biol., 7.","DOI":"10.1101\/cshperspect.a017996"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction","volume":"162","author":"Chomczynski","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"1991","DOI":"10.1073\/pnas.81.7.1991","article-title":"Genomic sequencing","volume":"81","author":"Church","year":"1984","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"41502","DOI":"10.1074\/jbc.M106795200","article-title":"Heterogeneous processing and zona pellucida binding activity of pig zonadhesin","volume":"276","author":"Hickox","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1095\/biolreprod58.2.361","article-title":"Acrosome biogenesis in the hamster: Ultrastructurally distinct matrix regions are assembled from a common precursor polypeptide","volume":"58","author":"Olson","year":"1998","journal-title":"Biol. Reprod."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1002\/jemt.10316","article-title":"Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis","volume":"61","author":"Olson","year":"2003","journal-title":"Microsc. Res. Tech."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/7\/3652\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:18:16Z","timestamp":1760105896000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/25\/7\/3652"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,3,25]]},"references-count":63,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2024,4]]}},"alternative-id":["ijms25073652"],"URL":"https:\/\/doi.org\/10.3390\/ijms25073652","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,3,25]]}}}