Publications by authors named "Masahito Ikawa"

240 Publications

Lens-specific conditional knockout of tropomyosin 1 gene in mice causes abnormal fiber differentiation and lens opacity.

Mech Ageing Dev 2021 Apr 15;196:111492. Epub 2021 Apr 15.

Department of Ophthalmology, Kanazawa Medical University, Ishikawa, Japan. Electronic address:

Tropomyosin (Tpm) 1 and 2 are important in the epithelial mesenchymal transition of lens epithelial cells; however, the effect of Tpm1 depletion during aging remains obscure. We analyzed the age-related changes in the crystalline lens of Tpm1- conditional knockout mice (Tpm1-CKO). Floxed alleles of Tpm1 were conditionally deleted in the lens, using Pax6-cre transgenic mice. Lenses of embryonic day (ED) 14, postnatal 1-, 11-, and 48-week-old Tpm1-CKO and wild type mice were dissected to prepare paraffin sections, which subsequently underwent histological and immunohistochemical analysis. Tpm1 and α smooth muscle actin (αSMA) mRNA expression were assessed using RT-PCR. The homozygous Tpm1-CKO (Tpm1) lenses displayed a dramatic reduction in Tpm1 transcript, with no change to αSMA mRNA expression. Tpm1 mice had small lenses with disorganized, vesiculated fiber cells, and loss of epithelial cells. The lenses of Tpm1 mice had abnormal and disordered lens fiber cells with cortical and peri-nuclear liquefaction. Expression of filamentous-actin was reduced in the equator region of lenses derived from ED14, 1-, 11-, and 48-week-old Tpm1 mice. Therefore, Tpm1 plays an integral role in mediating the integrity and fate of lens fiber differentiation and lens homeostasis during aging. Age-related Tpm1 dysregulation or deficiency may induce cataract formation.
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http://dx.doi.org/10.1016/j.mad.2021.111492DOI Listing
April 2021

A novel tissue specific alternative splicing variant mitigates phenotypes in Ets2 frame-shift mutant models.

Sci Rep 2021 Apr 15;11(1):8297. Epub 2021 Apr 15.

Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-0192, Japan.

E26 avian leukemia oncogene 2, 3' domain (Ets2) has been implicated in various biological processes. An Ets2 mutant model (Ets2), which lacks the DNA-binding domain, was previously reported to exhibit embryonic lethality caused by a trophoblast abnormality. This phenotype could be rescued by tetraploid complementation, resulting in pups with wavy hair and curly whiskers. Here, we generated new Ets2 mutant models with a frame-shift mutation in exon 8 using the CRISPR/Cas9 method. Homozygous mutants could not be obtained by natural mating as embryonic development stopped before E8.5, as previously reported. When we rescued them by tetraploid complementation, these mice did not exhibit wavy hair or curly whisker phenotypes. Our newly generated mice exhibited exon 8 skipping, which led to in-frame mutant mRNA expression in the skin and thymus but not in E7.5 Ets2 embryos. This exon 8-skipped Ets2 mRNA was translated into protein, suggesting that this Ets2 mutant protein complemented the Ets2 function in the skin. Our data implies that novel splicing variants incidentally generated after genome editing may complicate the phenotypic analysis but may also give insight into the new mechanisms related to biological gene functions.
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http://dx.doi.org/10.1038/s41598-021-87751-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8050053PMC
April 2021

Thiazoline-related innate fear stimuli orchestrate hypothermia and anti-hypoxia via sensory TRPA1 activation.

Nat Commun 2021 04 6;12(1):2074. Epub 2021 Apr 6.

Department of Functional Neuroscience, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan.

Thiazoline-related innate fear-eliciting compounds (tFOs) orchestrate hypothermia, hypometabolism, and anti-hypoxia, which enable survival in lethal hypoxic conditions. Here, we show that most of these effects are severely attenuated in transient receptor potential ankyrin 1 (Trpa1) knockout mice. TFO-induced hypothermia involves the Trpa1-mediated trigeminal/vagal pathways and non-Trpa1 olfactory pathway. TFOs activate Trpa1-positive sensory pathways projecting from trigeminal and vagal ganglia to the spinal trigeminal nucleus (Sp5) and nucleus of the solitary tract (NTS), and their artificial activation induces hypothermia. TFO presentation activates the NTS-Parabrachial nucleus pathway to induce hypothermia and hypometabolism; this activation was suppressed in Trpa1 knockout mice. TRPA1 activation is insufficient to trigger tFO-mediated anti-hypoxic effects; Sp5/NTS activation is also necessary. Accordingly, we find a novel molecule that enables mice to survive in a lethal hypoxic condition ten times longer than known tFOs. Combinations of appropriate tFOs and TRPA1 command intrinsic physiological responses relevant to survival fate.
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http://dx.doi.org/10.1038/s41467-021-22205-0DOI Listing
April 2021

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization.

Protein Cell 2021 Mar 1. Epub 2021 Mar 1.

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.

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http://dx.doi.org/10.1007/s13238-021-00825-yDOI Listing
March 2021

ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility.

Proc Natl Acad Sci U S A 2021 Feb;118(6)

Research Institute for Microbial Diseases, Osaka University, 5650871 Osaka, Japan;

The mammalian sperm midpiece has a unique double-helical structure called the mitochondrial sheath that wraps tightly around the axoneme. Despite the remarkable organization of the mitochondrial sheath, the molecular mechanisms involved in mitochondrial sheath formation are unclear. In the process of screening testis-enriched genes for functions in mice, we identified armadillo repeat-containing 12 (ARMC12) as an essential protein for mitochondrial sheath formation. Here, we engineered -null mice, FLAG-tagged knock-in mice, and TBC1 domain family member 21 ()-null mice to define the functions of ARMC12 in mitochondrial sheath formation in vivo. We discovered that absence of ARMC12 causes abnormal mitochondrial coiling along the flagellum, resulting in reduced sperm motility and male sterility. During spermiogenesis, sperm mitochondria in -null mice cannot elongate properly at the mitochondrial interlocking step which disrupts abnormal mitochondrial coiling. ARMC12 is a mitochondrial peripheral membrane protein and functions as an adherence factor between mitochondria in cultured cells. ARMC12 in testicular germ cells interacts with mitochondrial proteins MIC60, VDAC2, and VDAC3 as well as TBC1D21 and GK2, which are required for mitochondrial sheath formation. We also observed that TBC1D21 is essential for the interaction between ARMC12 and VDAC proteins in vivo. These results indicate that ARMC12 uses integral mitochondrial membrane proteins VDAC2 and VDAC3 as scaffolds to link mitochondria and works cooperatively with TBC1D21. Thus, our studies have revealed that ARMC12 regulates spatiotemporal mitochondrial dynamics to form the mitochondrial sheath through cooperative interactions with several proteins on the sperm mitochondrial surface.
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http://dx.doi.org/10.1073/pnas.2018355118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8017931PMC
February 2021

Identification and characterization of the antigen recognized by the germ cell mAb TRA98 using a human comprehensive wet protein array.

Genes Cells 2021 Mar;26(3):180-189

The National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.

TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.
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http://dx.doi.org/10.1111/gtc.12832DOI Listing
March 2021

Protocol for isolation of spermatids from mouse testes.

STAR Protoc 2021 Mar 8;2(1):100254. Epub 2021 Jan 8.

Research Center for Epigenetic Code and Diseases, Department of Biological Sciences, Seoul National University, Seoul 08826, South Korea.

Post-meiotic spermatids become spermatozoa through developmental stages during spermiogenesis. Isolation of spermatid fractions is required to examine the change of protein expression during spermiogenesis. Here, we present a simple method to isolate spermatid fractions from mouse testes using unit gravity sedimentation in a BSA density gradient. Isolation of spermatid fractions can be used to analyze changes of transcript or protein during spermiogenesis. For complete details on the use and execution of this protocol, please refer to Kim et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809437PMC
March 2021

Astrocytic cAMP modulates memory via synaptic plasticity.

Proc Natl Acad Sci U S A 2021 Jan;118(3)

Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, 113-8654 Tokyo, Japan;

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo.
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http://dx.doi.org/10.1073/pnas.2016584118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826339PMC
January 2021

: an evolutionarily conserved lncRNA essential for licensing coordinated activation of p38 and NFκB in colitis.

Gut 2020 Nov 25. Epub 2020 Nov 25.

Laboratory of NFκB Signalling, Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Institute of Molecular and Cell Biology, Singapore

Objective: NFκB is the key modulator in inflammatory disorders. However, the key regulators that activate, fine-tune or shut off NFκB activity in inflammatory conditions are poorly understood. In this study, we aim to investigate the roles that NFκB-specific long non-coding RNAs (lncRNAs) play in regulating inflammatory networks.

Design: Using the first genetic-screen to identify NFκB-specific lncRNAs, we performed RNA-seq from the and mouse embryonic fibroblasts and report the identification of an evolutionary conserved lncRNA designated (mice) or (human). is upregulated in human inflammatory disorders, including UC. We generated mice, wherein deletion of two NFκB sites in the proximal promoter of abolishes its induction, to study its function in colitis.

Results: regulates inflammation via sequestering and inactivating Wip1, a known negative regulator of proinflammatory p38 kinase and NFκB subunit p65. Wip1 inactivation leads to coordinated activation of p38 and covalent modifications of NFκB, essential for its genome-wide occupancy on specific targets. enables an orchestrated response for p38 and NFκB coactivation that leads to differentiation of precursor cells into immature myeloid cells in bone marrow, recruitment of macrophages to inflamed area and expression of inflammatory genes in colitis.

Conclusion: directly regulates initiation and progression of colitis and its expression is highly correlated with NFκB activity which makes it a perfect candidate to serve as a biomarker and a therapeutic target for IBD and other inflammation-associated diseases.
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http://dx.doi.org/10.1136/gutjnl-2020-322980DOI Listing
November 2020

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

FASEB J 2020 12 15;34(12):16224-16242. Epub 2020 Oct 15.

Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4 ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3 ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4 mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4 mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4 mice. A five-fold increase in the frequency of abnormalities in Kpna4 mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4 mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4 testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.
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http://dx.doi.org/10.1096/fj.202000768RRDOI Listing
December 2020

CRISPR/CAS9-mediated amino acid substitution reveals phosphorylation residues of RSPH6A are not essential for male fertility in mice†.

Biol Reprod 2020 10;103(5):912-914

Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

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http://dx.doi.org/10.1093/biolre/ioaa161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609874PMC
October 2020

PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins.

J Biol Chem 2020 10 18;295(42):14501-14509. Epub 2020 Aug 18.

Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the Cripto-1/FRL1/Cryptic domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4, and contactin-1, in addition to CRIPTO, are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.
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http://dx.doi.org/10.1074/jbc.RA120.014643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573272PMC
October 2020

Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets.

BMC Biol 2020 08 19;18(1):103. Epub 2020 Aug 19.

Department of Pathology and Immunology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA.

Background: The development of a safe, effective, reversible, non-hormonal contraceptive method for men has been an ongoing effort for the past few decades. However, despite significant progress on elucidating the function of key proteins involved in reproduction, understanding male reproductive physiology is limited by incomplete information on the genes expressed in reproductive tissues, and no contraceptive targets have so far reached clinical trials. To advance product development, further identification of novel reproductive tract-specific genes leading to potentially druggable protein targets is imperative.

Results: In this study, we expand on previous single tissue, single species studies by integrating analysis of publicly available human and mouse RNA-seq datasets whose initial published purpose was not focused on identifying male reproductive tract-specific targets. We also incorporate analysis of additional newly acquired human and mouse testis and epididymis samples to increase the number of targets identified. We detected a combined total of 1178 genes for which no previous evidence of male reproductive tract-specific expression was annotated, many of which are potentially druggable targets. Through RT-PCR, we confirmed the reproductive tract-specific expression of 51 novel orthologous human and mouse genes without a reported mouse model. Of these, we ablated four epididymis-specific genes (Spint3, Spint4, Spint5, and Ces5a) and two testis-specific genes (Pp2d1 and Saxo1) in individual or double knockout mice generated through the CRISPR/Cas9 system. Our results validate a functional requirement for Spint4/5 and Ces5a in male mouse fertility, while demonstrating that Spint3, Pp2d1, and Saxo1 are each individually dispensable for male mouse fertility.

Conclusions: Our work provides a plethora of novel testis- and epididymis-specific genes and elucidates the functional requirement of several of these genes, which is essential towards understanding the etiology of male infertility and the development of male contraceptives.
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http://dx.doi.org/10.1186/s12915-020-00826-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436996PMC
August 2020

PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis.

Cell Rep 2020 07;32(4):107950

Creative Research Initiatives Center for Epigenetic Code and Diseases, Seoul National University, Seoul 08826, South Korea; Department of Biological Sciences, Seoul National University, Seoul 08826, South Korea. Electronic address:

Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system.
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http://dx.doi.org/10.1016/j.celrep.2020.107950DOI Listing
July 2020

Diphtheria toxin-mediated transposon-driven poly (A)-trapping efficiently disrupts transcriptionally silent genes in embryonic stem cells.

Genesis 2020 09 9;58(9):e23386. Epub 2020 Jul 9.

Laboratory of Functional Genomics and Medicine, Division of Biological Science, Nara Institute of Science and Technology, Nara, Japan.

Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.
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http://dx.doi.org/10.1002/dvg.23386DOI Listing
September 2020

Protocadherin-7 contributes to maintenance of bone homeostasis through regulation of osteoclast multinucleation.

BMB Rep 2020 Sep;53(9):472-477

Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.

Osteoclasts are hematopoietic-derived cells that resorb bone. They are required to maintain proper bone homeostasis and skeletal strength. Although osteoclast differentiation depends on receptor activator of NF-κB ligand (RANKL) stimulation, additional molecules further contribute to osteoclast maturation. Here, we demonstrate that protocadherin-7 (Pcdh7) regulates formation of multinucleated osteoclasts and contributes to maintenance of bone homeostasis. We found that Pcdh7 expression is induced by RANKL stimulation, and that RNAi-mediated knockdown of Pcdh7 resulted in impaired formation of osteoclasts. We generated Pcdh7-deficient mice and found increased bone mass due to decreased bone resorption but without any defect in bone formation. Using an in vitro culture system, it was revealed that formation of multinucleated osteoclasts is impaired in Pcdh7-deficient cultures, while no apparent defects were observed in differentiation and function of Pcdh7-deficient osteoblasts. Taken together, these results reveal an osteoclast cell-intrinsic role for Pcdh7 in maintaining bone homeostasis. [BMB Reports 2020; 53(9): 472-477].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526982PMC
September 2020

RNA-binding protein Ptbp1 regulates alternative splicing and transcriptome in spermatogonia and maintains spermatogenesis in concert with Nanos3.

J Reprod Dev 2020 Oct 6;66(5):459-467. Epub 2020 Jul 6.

Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

PTBP1, a well-conserved RNA-binding protein, regulates cellular development by tuning posttranscriptional mRNA modification such as alternative splicing (AS) or mRNA stabilization. We previously revealed that the loss of Ptbp1 in spermatogonia causes the dysregulation of spermatogenesis, but the molecular mechanisms by which PTBP1 regulates spermatogonium homeostasis are unclear. In this study, changes of AS or transcriptome in Ptbp1-knockout (KO) germline stem cells (GSC), an in vitro model of proliferating spermatogonia, was determined by next generation sequencing. We identified more than 200 differentially expressed genes, as well as 85 genes with altered AS due to the loss of PTBP1. Surprisingly, no differentially expressed genes overlapped with different AS genes in Ptbp1-KO GSC. In addition, we observed that the mRNA expression of Nanos3, an essential gene for normal spermatogenesis, was significantly decreased in Ptbp1-KO spermatogonia. We also revealed that PTBP1 protein binds to Nanos3 mRNA in spermatogonia. Furthermore, Nanos3;Ptbp1 mice exhibited abnormal spermatogenesis, which resembled the effects of germ cell-specific Ptbp1 KO, whereas no significant abnormality was observed in mice heterozygous for either gene alone. These data implied that PTBP1 regulates alternative splicing and transcriptome in spermatogonia under different molecular pathways, and contributes spermatogenesis, at least in part, in concert with NANOS3.
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http://dx.doi.org/10.1262/jrd.2020-060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593632PMC
October 2020

Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility.

Am J Hum Genet 2020 08 2;107(2):330-341. Epub 2020 Jul 2.

Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413861PMC
August 2020

Knockout of family with sequence similarity 170 member A (Fam170a) causes male subfertility, while Fam170b is dispensable in mice†.

Biol Reprod 2020 08;103(2):205-222

Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA.

Families with sequence similarity 170 members A and B (FAM170A and FAM170B) are testis-specific, paralogous proteins that share 31% amino acid identity and are conserved throughout mammals. While previous in vitro experiments suggested that FAM170B, an acrosome-localized protein, plays a role in the mouse sperm acrosome reaction and fertilization, the role of FAM170A in the testis has not been explored. In this study, we used CRISPR/Cas9 to generate null alleles for each gene, and homozygous null (-/-) male mice were mated to wild-type females for 6 months to assess fertility. Fam170b-/- males were found to produce normal litter sizes and had normal sperm counts, motility, and sperm morphology. In contrast, mating experiments revealed significantly reduced litter sizes and a reduced pregnancy rate from Fam170a-/- males compared with controls. Fam170a-/-;Fam170b-/- double knockout males also produced markedly reduced litter sizes, although not significantly different from Fam170a-/- alone, suggesting that Fam170b does not compensate for the absence of Fam170a. Fam170a-/- males exhibited abnormal spermiation, abnormal head morphology, and reduced progressive sperm motility. Thus, FAM170A has an important role in male fertility, as the loss of the protein leads to subfertility, while FAM170B is expendable. The molecular functions of FAM170A in spermatogenesis are as yet unknown; however, the protein localizes to the nucleus of elongating spermatids and may mediate its effects on spermatid head shaping and spermiation by regulating the expression of other genes. This work provides the first described role of FAM170A in reproduction and has implications for improving human male infertility diagnoses.
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http://dx.doi.org/10.1093/biolre/ioaa082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401401PMC
August 2020

CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†.

Biol Reprod 2020 08;103(2):183-194

Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Developing a safe and effective male contraceptive remains a challenge in the field of medical science. Molecules that selectively target the male reproductive tract and whose targets are indispensable for male reproductive function serve among the best candidates for a novel non-hormonal male contraceptive method. To determine the function of these genes in vivo, mutant mice carrying disrupted testis- or epididymis-enriched genes were generated by zygote microinjection or electroporation of the CRISPR/Cas9 components. Male fecundity was determined by consecutively pairing knockout males with wild-type females and comparing the fecundity of wild-type controls. Phenotypic analyses of testis appearance and weight, testis and epididymis histology, and sperm movement were further carried out to examine any potential spermatogenic or sperm maturation defect in mutant males. In this study, we uncovered 13 testis- or epididymis-enriched evolutionarily conserved genes that are individually dispensable for male fertility in mice. Owing to their dispensable nature, it is not feasible to use these targets for the development of a male contraceptive.
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http://dx.doi.org/10.1093/biolre/ioaa083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401351PMC
August 2020

CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity.

Biol Reprod 2020 08;103(2):195-204

Graduate School of Medicine, Osaka University, Osaka, Japan.

As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.
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http://dx.doi.org/10.1093/biolre/ioaa084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401030PMC
August 2020

Analysis of the sperm flagellar axoneme using gene-modified mice.

Exp Anim 2020 11 18;69(4):374-381. Epub 2020 Jun 18.

Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.
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http://dx.doi.org/10.1538/expanim.20-0064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677079PMC
November 2020

Tmprss12 is required for sperm motility and uterotubal junction migration in mice†.

Biol Reprod 2020 08;103(2):254-263

Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes posttranslational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unknown. In this study, we focused on transmembrane serine protease 12 (Tmprss12). Based on multi-tissue expression analysis by PCR, Tmprss12 was specifically expressed in the testis, and its expression started on day 10 postpartum, corresponding to the stage of zygotene spermatocytes. TMPRSS12 was detected in the acrosomal region of spermatozoa by immunostaining. To reveal the physiological function of TMPRSS12, we generated two knockout (KO) mouse lines using the CRISPR/Cas9 system. Both indel and large deletion lines were male sterile showing that TMPRSS12 is essential for male fertility. Although KO males exhibited normal spermatogenesis and sperm morphology, ejaculated spermatozoa failed to migrate from the uterus to the oviduct. Further analysis revealed that a disintegrin and metalloprotease 3 (ADAM3), an essential protein on the sperm membrane surface that is required for sperm migration, was disrupted in KO spermatozoa. Moreover, we found that KO spermatozoa showed reduced sperm motility via computer-assisted sperm analysis, resulting in a low fertilization rate in vitro. Taken together, these data indicate that TMPRSS12 has dual functions in regulating sperm motility and ADAM3-related sperm migration to the oviduct. Because Tmprss12 is conserved among mammals, including humans, our results may explain some genetic cases of idiopathic male infertility, and TMPRSS12 and its downstream cascade may be novel targets for contraception.
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http://dx.doi.org/10.1093/biolre/ioaa060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401031PMC
August 2020

NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility.

Science 2020 06;368(6495):1132-1135

Immunology Frontier Research Center, Osaka University, Suita, Osaka 5650871, Japan.

The lumicrine system is a postulated signaling system in which testis-derived (upstream) secreted factors enter the male reproductive tract to regulate epididymal (downstream) pathways required for sperm maturation. Until now, no lumicrine factors have been identified. We demonstrate that a testicular germ-cell-secreted epidermal growth factor-like protein, neural epidermal growth factor-like-like 2 (NELL2), specifically binds to an orphan receptor tyrosine kinase, c-ros oncogene 1 (ROS1), and mediates the differentiation of the initial segment (IS) of the caput epididymis. Male mice in which had been knocked out were infertile. The IS-specific secreted proteases, ovochymase 2 (OVCH2) and A disintegrin and metallopeptidase 28 (ADAM28), were expressed upon IS maturation, and OVCH2 was required for processing of the sperm surface protein ADAM3, which is required for sperm fertilizing ability. This work identifies a lumicrine system essential for testis-epididymis-spermatozoa (NELL2-ROS1-OVCH2-ADAM3) signaling and male fertility.
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http://dx.doi.org/10.1126/science.aay5134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396227PMC
June 2020

Reduction in BDNF from Inefficient Precursor Conversion Influences Nest Building and Promotes Depressive-Like Behavior in Mice.

Int J Mol Sci 2020 Jun 1;21(11). Epub 2020 Jun 1.

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorioka, Ikeda, Osaka 563-8577, Japan.

We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor () was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.
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http://dx.doi.org/10.3390/ijms21113984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312902PMC
June 2020

CIB4 is essential for the haploid phase of spermatogenesis in mice†.

Biol Reprod 2020 08;103(2):235-243

Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Spermatogenesis is a complex developmental process that involves the proliferation of diploid cells, meiotic division, and haploid differentiation. Many genes are shown to be essential for male fertility using knockout (KO) mice; however, there still remain genes to be analyzed to elucidate their molecular mechanism and their roles in spermatogenesis. Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitously expressed protein that possesses three paralogs: CIB2, CIB3, and CIB4. It is reported that Cib1 KO male mice are sterile due to impaired haploid differentiation. In this study, we discovered that Cib4 is expressed strongly in mouse and human testis and begins expression during the haploid phase of spermatogenesis in mice. To analyze the function of CIB4 in vivo, we generated Cib4 KO mice using the CRISPR/Cas9 system. Cib4 KO male mice are sterile due to impaired haploid differentiation, phenocopying Cib1 KO male mice. Spermatogenic cells isolated from seminiferous tubules demonstrate an essential function of CIB4 in the formation of the apical region of the sperm head. Further analysis of CIB4 function may shed light on the etiology of male infertility caused by spermatogenesis defects, and CIB4 could be a target for male contraceptives because of its dominant expression in the testis.
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http://dx.doi.org/10.1093/biolre/ioaa059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401386PMC
August 2020

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

Proc Natl Acad Sci U S A 2020 05 11;117(21):11493-11502. Epub 2020 May 11.

Research Institute for Microbial Diseases, Osaka University, 565-0871 Osaka, Japan;

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 (//), transmembrane protein 95 (), and sperm acrosome associated 6 (), 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 , and rescued the sterility of , , and 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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http://dx.doi.org/10.1073/pnas.1922650117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261011PMC
May 2020

Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes globozoospermia and sterility in male mice†.

Biol Reprod 2020 08;103(2):244-253

Center for Drug Discovery, Baylor College of Medicine, Houston, TX.

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception.
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http://dx.doi.org/10.1093/biolre/ioaa040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401026PMC
August 2020

Prss55 but not Prss51 is required for male fertility in mice†.

Biol Reprod 2020 08;103(2):223-234

Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Mammalian spermatozoa are produced in the testis through spermatogenesis and matured in the epididymis to acquire fertilizing ability. Spermatozoa are ejaculated and migrate from the uterus to the oviducts to fuse with oocytes. Although over 2000 genes are expressed abundantly in mouse testes, the genes responsible for male fertility are not yet fully clarified. Here, we focused on two testis-enriched serine protease genes, Serine protease (Prss) 51 and Prss55, which overlap their gene loci partially in both mice and humans. To characterize their functions in male fertility, we first generated Prss51 and Prss55 double knockout (DKO) mice by CRISPR/Cas9 system and found that the DKO mice were sterile. DKO spermatozoa exhibit impaired migration from the uterus to the oviduct and impaired ability to bind the zona pellucida (ZP) of oocytes. Moreover, a sperm membrane protein, ADAM3 (a disintegrin and metalloprotease 3), which plays a role in sperm migration through uterotubal junction (UTJ) and sperm-ZP binding, disappeared in the DKO spermatozoa from the epididymis. We next generated single knockout (KO) mice lacking Prss51 and found that Prss51 KO mice are fertile. We also generated single KO mice lacking Prss55 and found that Prss55 KO mice phenocopy the DKO mice, demonstrating impaired sperm migration and sperm-ZP binding and a severe defect in fertility. We conclude that Prss55, but not Prss51, is required for male fertility in mice, by stabilizing ADAM3 protein for efficient sperm-UTJ migration and sperm-ZP binding. Our findings have implications for understanding additional genetic causes of the idiopathic male infertility and for the development of male or female contraceptives.
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http://dx.doi.org/10.1093/biolre/ioaa041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401375PMC
August 2020

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice.

Proc Natl Acad Sci U S A 2020 04 15;117(17):9393-9400. Epub 2020 Apr 15.

Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871 Osaka, Japan;

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.
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http://dx.doi.org/10.1073/pnas.1917060117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196805PMC
April 2020