Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

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Significance

The sperm−oocyte fusion step is important to transport the male genome into oocytes. So far, IZUMO1 and FIMP have been identified as fusion-related proteins in spermatozoa, but the molecular mechanisms underpinning sperm−oocyte fusion and all of the proteins required for this essential process remain unclear. In this study, using CRISPR-Cas9−mediated gene knockouts in mice, we discover that sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion and male fertility. As these genes are conserved among mammals including human, they may explain not only the sperm−oocyte fusion process but also idiopathic male infertility and be unique targets for contraception.

Abstract

Sperm−oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm−oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm−oocyte fusion required 1 ( Sof1/ Llcfc1/ 1700034O15Rik), transmembrane protein 95 ( Tmem95), and sperm acrosome associated 6 ( Spaca6), encode sperm proteins required for sperm−oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouse Sof1, Tmem95, and Spaca6 rescued the sterility of Sof1, Tmem95, and Spaca6 KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm−oocyte fusion.

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Most cited references 40

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The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids

Brian P Hermann, Keren Cheng, Anukriti Singh … (2018)

Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermato- genic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro . Hermann et al. present single-cell transcriptomes from >62,000 individual spermatogenic cells from immature and adult male mice and adult men. Their analysis facilitates resolution of SSCs and progenitor spermatogonia, elucidates the full range of gene expression changes during male meiosis and spermiogenesis, and derives unique gene expression signatures for eleven mouse and human spermatogenic cell types.

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Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Daisuke Mashiko, Yoshitaka Fujihara, Yuhkoh Satouh … (2013)

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

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The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.

Naokazu Inoue, Masahito Ikawa, Ayako Isotani … (2005)

Representing the 60 trillion cells that build a human body, a sperm and an egg meet, recognize each other, and fuse to form a new generation of life. The factors involved in this important membrane fusion event, fertilization, have been sought for a long time. Recently, CD9 on the egg membrane was found to be essential for fusion, but sperm-related fusion factors remain unknown. Here, by using a fusion-inhibiting monoclonal antibody and gene cloning, we identify a mouse sperm fusion-related antigen and show that the antigen is a novel immunoglobulin superfamily protein. We have termed the gene Izumo and produced a gene-disrupted mouse line. Izumo-/- mice were healthy but males were sterile. They produced normal-looking sperm that bound to and penetrated the zona pellucida but were incapable of fusing with eggs. Human sperm also contain Izumo and addition of the antibody against human Izumo left the sperm unable to fuse with zona-free hamster eggs.

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Author and article information

Journal

Journal ID (nlm-ta): Proc Natl Acad Sci U S A

Journal ID (iso-abbrev): Proc. Natl. Acad. Sci. U.S.A

Journal ID (hwp): pnas

Journal ID (pmc): pnas

Journal ID (publisher-id): PNAS

Title: Proceedings of the National Academy of Sciences of the United States of America

Publisher: National Academy of Sciences

ISSN (Print): 0027-8424

ISSN (Electronic): 1091-6490

Publication date (Print): 26 May 2020

Publication date (Electronic): 11 May 2020

Publication date PMC-release: 11 May 2020

Volume: 117

Issue: 21

Pages: 11493-11502

Affiliations

[1] ^aResearch Institute for Microbial Diseases, Osaka University , 565-0871 Osaka, Japan;

[2] ^bGraduate School of Pharmaceutical Sciences, Osaka University , 565-0871 Osaka, Japan;

[3] ^cDivision of Molecular Genetics, Shigei Medical Research Institute , 701-0202 Okayama, Japan;

[4] ^dCenter for Drug Discovery, Baylor College of Medicine , Houston, TX 77030;

[5] ^eDepartment of Pathology & Immunology, Baylor College of Medicine , Houston, TX 77030;

[6] ^fThe Institute of Medical Science, The University of Tokyo , 108-8639 Tokyo, Japan

Author notes

³To whom correspondence may be addressed. Email: mmatzuk@ 123456bcm.edu or ikawa@ 123456biken.osaka-u.ac.jp .

Contributed by Martin M. Matzuk, March 19, 2020 (sent for review December 27, 2019; reviewed by Matteo Avella and Andrea Pauli)

Author contributions: T.N., Y.L., M.M.M., and M.I. designed research; T.N., Y.L., Y.F., S.O., T.K., and S.K. performed research; T.N., Y.L., M.M.M., and M.I. analyzed data; and T.N., Y.L., M.M.M., and M.I. wrote the paper.

Reviewers: M.A., University of Tulsa; and A.P., Vienna Biocenter.

¹T.N. and Y.L. contributed equally to this work.

²Present address: Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, 564-8565 Osaka, Japan.

Author information

Taichi Noda https://orcid.org/0000-0003-0260-7861

Yonggang Lu https://orcid.org/0000-0003-0198-8906

Yoshitaka Fujihara https://orcid.org/0000-0001-8332-3507

Seiya Oura https://orcid.org/0000-0002-7606-732X

Takayuki Koyano https://orcid.org/0000-0003-1675-2525

Masahito Ikawa https://orcid.org/0000-0001-9859-6217

Article

Publisher ID: 201922650

DOI: 10.1073/pnas.1922650117

PMC ID: 7261011

PubMed ID: 32393636

SO-VID: 908671c2-a0a3-42e9-a3c7-02a0c040a8f9

License:

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).