Yoshitaka Fujihara's research while affiliated with National Cerebral and Cardiovascular Center and other places
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Publications (102)
Spermatozoa need to undergo an exocytotic event called the acrosome reaction before fusing with eggs. Although calcium ion (Ca2+) is essential for the acrosome reaction, its molecular mechanism remains unknown. Ferlin is a single transmembrane protein with multiple Ca2+-binding C2 domains, and there are six ferlins, dysferlin (DYSF), otoferlin (OTO...
Background:
Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 f...
Background:
Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa.
Objective:
We generated phosducin-like 2 (PDCL2) knockout (-/-) mice to investigate t...
Background:
A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due...
Background
Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin–proteasome pathway. E2 and E3 enzymes con...
Purpose:
Tulp2 (tubby-like protein 2) is a member of the tubby protein family and expressed predominantly in mouse testis. Recently, it was reported that Tulp2 knockout (KO) mice exhibited disrupted sperm tail morphology; however, it remains to be determined how TULP2 deletion causes abnormal tail formation.
Methods:
The authors analyzed male fe...
The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. m...
Glycosylphosphatidylinositol (GPI) is a post-translational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of G...
Significance
We show that Bouncer’s homolog in mammals, SPACA4, is required for efficient fertilization in mice. In contrast to fish, in which Bouncer is required for female fertility, SPACA4 is expressed exclusively in the sperm and is required for male fertility. SPACA4 and Bouncer present an intriguing example of homologous proteins that both pl...
Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to disce...
Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in non-mammalian species do not have obvious...
The process of sperm-egg fusion is critical for successful fertilization, yet the underpinning mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D....
Osteoclasts are multinucleated, giant cells derived from myeloid progenitors. While receptor activator of NF-κB ligand (RANKL) stimulation is the primary driver of osteoclast differentiation, additional signaling further contributes to osteoclast maturation. Here, we demonstrate that immunoglobulin superfamily member 11 (IgSF11), whose expression i...
Background:
Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored....
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 t...
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...
Local control of sperm maturation
Newly produced spermatozoa within the testis do not have fertilizing ability but become fully functional when they mature in the epididymis. The development of the epididymis itself is dependent on testicular factors arriving via luminal flow. Improper signaling between the testis and epididymis is hypothesized to...
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 se...
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 CRISP...
Spermatozoa are produced in the testis but gain their fertilizing ability during epididymal migration. This necessary step in sperm maturation includes post-translational modification of sperm membrane proteins that includes protein processing by proteases. However, the molecular mechanism underpinning this epididymal sperm maturation remains unkno...
Significance
As the human body is composed of 60 trillion cells that originate from a fertilized egg, sperm–oocyte fusion is the initial event of our life. Few sperm–oocyte fusion factors have been unveiled to date, and only IZUMO1 has been identified as a sperm-specific fusion-mediating protein. Here, we identified the testis-specific 4930451I11Ri...
There are over 200 genes that are predicted to be solely expressed in the oocyte and ovary, and thousands more that have expression patterns in the female reproductive tract. Unfortunately, many of their physiological functions, such as their roles in oogenesis or fertilization, have yet to be elucidated. Previous knockout (KO) mice studies have pr...
Diacylglycerol kinase γ (DGKγ) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKγ directly interacts with PKCγ and is phosphorylated by PKCγ, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for moto...
In mammals, more than 2,000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the CXC motif containing metallothionein family. La...
Significance
While the emergence of gene modification technologies has produced major discoveries in biomedical sciences, the recent development of the CRISPR/Cas9 system has dramatically altered the trajectory of phenotypic analysis in animal models. In this study, we identified male-specific gene clusters ( Cst and Pate ) and family genes ( Gdpd...
Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chr...
More than 1,000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically-related idiopathi...
Irie and colleagues identified a "toxic conformer", which possesses a turn structure at positions 22-23, among various conformations of Aβ and have been reporting its potent oligomeric capacity and neurotoxicity. This toxic conformer was detected in the brains of AD patients and AD model mice (Tg2576 line), and passive immunization targeting this c...
Seminal vesicle secretions (SVSs), together with spermatozoa, are ejaculated into the female reproductive tract. SVS7, also known as PATE4, is one of the major SVS proteins found in the seminal vesicle, copulatory plug, and uterine fluid after copulation. Here, we generated Pate4 knockout (-/-) mice and examined the detailed function of PATE4 on ma...
Author summary
Paternal genetic information is transmitted to the offspring via sperm. The unique cell morphology of the sperm plays essential roles in sperm transport through the female reproductive tract and in fertilization with oocytes. Sertoli cells are somatic cells located in the seminiferous tubules of the testis and are known to contribute...
F-actin dynamics observed with SDSRM.
EGFP-LifeAct was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 2 s. EGFP, enhanced green fluorescent protein; F-actin, filamentous actin; SDSRM, spinning disk superresolution microscopy; WT, wild-type.
(MOV)
mDia3 expression in the seminiferous tubules throughout the spermatogenic cycles.
(A) Immunohistochemistry staining for mDia3 (green) and phalloidin staining (magenta) of WT testis sections. Arrowheads indicate mDia3 staining at the basal ectoplasmic junction and arrows indicate mDia3 staining at the apical ectoplasmic junction. (B) Immunohistochem...
Increased apoptotic cells in mDia1/3 DKO seminiferous tubule.
(A) Apoptotic cells (green) in the mDia1/3 DKO seminiferous tubules. Nuclei (magenta) were stained with Hoechst. Scale bar, 100 μm. (B) Quantification of the number of apoptotic cells per seminiferous tubule. Data represented mean ± SEM (91 seminiferous tubules from four WT mice and 99 s...
Reduced F-actin staining of mDia1/3 DKO primary cultured Sertoli cell.
(A) Confocal images of actin filaments of WT (left) and mDia1/3 DKO (right) primary cultured Sertoli cells. The lines (magenta and green) were used to quantify the fluorescence intensity by line scan, and the fluorescence intensity profiles along these lines are shown in the rig...
Model of the molecular mechanism of mDia1/3 function in spermatogenesis through the regulation of F-actin and cell adhesions between Sertoli cell and germ cell.
F-actin, filamentous actin; mDia1/3, mammalian diaphanous homolog1/3.
(TIF)
Single-molecule observation of fast movement of EGFP-mDia3 in a primary cultured Sertoli cell with TIRF microscopy.
EGFP-mDia3 was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 200 ms. EGFP, enhanced green fluorescent protein; mDia3, mammalian diaphanous homolog3; TIRF, total internal reflection; WT, wild-typ...
Fast polymerization of cortical actin filament meshwork observed with SDSRM.
EGFP-LifeAct was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 2 s. EGFP, enhanced green fluorescent protein; SDSRM, spinning disk superresolution microscopy; WT, wild-type.
(MOV)
Reduced number, abnormal morphology, and impaired motility of mDia1/3 DKO sperm.
(A) HE-stained epididymal cross sections from adult WT or mDia1/3 DKO mice. Scale bar, 25 μm. (B) Total number of sperm per epididymis of WT or mDia1/3 DKO mice. Data represented mean ± SEM. n = 3 and 4 for WT and mDia1/3 DKO, respectively. **P < 0.01 (P = 0.0062, Stud...
Intracellular molecular movement of EGFP-mDia3 underneath the cell cortex, observed with TIRF microscopy.
EGFP-mDia3 was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 200 ms. EGFP, enhanced green fluorescent protein; mDia3, mammalian diaphanous homolog3; TIRF, total internal reflection; WT, wild-type.
(MOV)
Reduced cortical F-actin meshwork in mDia1/3 DKO Sertoli cells was rescued by expression of EGFP-mDia3.
(A) mDia1/3 DKO primary cultured Sertoli cells transfected with pEGFP-mDia3 (green) were stained with phalloidin (magenta). The cell on the right is EGFP-mDia3 positive. The magenta and green dotted line was used to quantify the fluorescence inte...
Male infertility of mDia1/3 DKO mice.
(A and B) Micrographs (A) and fertilization rate (B) of IVF of ZP-intact oocytes with sperms from WT or mDia1/3 DKO mice. Scale bar, 50 μm. Data represented mean ± SEM. Data are the average of three independent experiments. ***P < 0.001 (Student t test). DKO, double knockout; IVF, in vitro fertilization; mDia1/...
F-actin architecture of Sertoli cells in intact seminiferous tubules.
(A) Transduction of LifeAct-EGFP expressing lentivirus in the testis of WT mouse. Seminiferous tubule was microinjected with LifeAct-EGFP expressing lentivirus and analyzed at 1 wk after injection under fluorescence stereomicroscope. (B) Observation strategy of LifeAct-EGFP expre...
Specificity of anti-mDia1 and anti-mDia3 antibodies for immunohistochemistry.
(A) Immunohistochemistry staining for mDia1 (green) and vimentin (magenta) as a marker for Sertoli cells in testis sections from WT and mDia1 KO adult mice. Positive mDia1 signals at the vimentin-positive Sertoli cells observed in WT mice were abolished in mDia1 KO mice....
Immunohistochemistry of espin1 together with phalloidin staining in seminiferous tubule throughout spermatogenic cycles.
(A) Immunohistochemistry staining for espin1 (green) and phalloidin staining (magenta) of testis sections from WT mice. Espin1 signals were observed at the apical ES junction of elongated spermatid from stages II–V seminiferous t...
Nectin-2 expression in the seminiferous tubules throughout the spermatogenic cycles.
(A) Immunohistochemistry staining for nectin-2 (green) and Hoechst staining (magenta) of testis sections from WT mice. Strong nectin-2 signals were observed at the apical ectoplasmic specialization junction. In addition, nectin-2 signals were observed at the adhere...
Three-dimensional reconstruction of deconvolution spinning disk confocal images of Sertoli cell F-actin in intact seminiferous tubule.
Z-series images of Sertoli cell transduced by LifeAct-EGFP expressing lentivirus in intact seminiferous tubule were acquired by spinning disk confocal microscopy and processed with a deconvolution algorithm. Three-d...
Chemokines are signaling proteins that are secreted to induce chemotaxis during immunological events. However, the functions of transmembrane-type chemokine-like factor (CKLF) and the protein family CMTM (CKLF-like MARVEL transmembrane domain containing) remain to be determined. In this study, we focused on the testis-specific mouse Cmtm gene clust...
Abnormalities in limbic neural circuits have been implicated in the onset of anxiety disorders. However, the molecular pathogenesis underlying anxiety disorders remains poorly elucidated. Here, we demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) regulates amygdala circuitry to control the activity of the hypothalamic...
Mammalian fertilization is comprised of many steps including sperm survival in the uterus, sperm migration in the female reproductive tract, physiological and morphological changes to the spermatozoa, and sperm-egg interaction in the oviduct. In vitro studies have revealed essential factors for these fertilization steps for over half a century. How...
Significance
Immune cell activation is accompanied by dynamic changes in expression of genes related to inflammation. Concomitantly, immune reactions are tightly controlled to prevent harmful pathologies due to sustained inflammation. Gene expression is controlled at multiple checkpoints. Among these, the post-transcriptional regulation of the bala...
Background & Aims
The transient receptor potential ankyrin 1 (TRPA1) channel is highly expressed in the intestinal lamina propria, but its contribution to gut physiology/pathophysiology is unclear. Here, we evaluated the function of myofibroblast TRPA1 channels in intestinal remodeling.
Methods
An intestinal myofibroblast cell line (InMyoFibs) was...
A recent genetic analysis of infertile globozoospermic patients identified causative mutations in three genes: a protein interacting with C kinase 1 (PICK1), dpy 19-like 2 (DPY19L2), and spermatogenesis associated 16 (SPATA16). Although mouse models have clarified the physiological functions of Pick1 and Dpy19l2 during spermatogenesis, Spata16 rema...
shRNA sequences.
CD4⁺ T-helper cells producing interleukin-17 (IL-17), known as T-helper 17 (TH17) cells, comprise heterogeneous subsets that exhibit distinct pathogenicity. Although pathogenic and non-pathogenic TH17 subsets share a common RORγt-dependent TH17 transcriptional programme, transcriptional regulatory mechanisms specific to each of these subsets are mo...
Supplementary Figures and Supplementary Table 1
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) can be shed from the cell membrane by GPI cleavage. In this study, we report a novel GPI-processing enzyme, termed post-glycosylphosphatidylinositol attachment to proteins 6 (PGAP6), which is a GPI-specific phospholipase A2 mainly localized at the cell surface. CRIPTO, a GPI-AP, which plays c...
Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenoty...
Pluripotent stem cells can be classified into two distinct states, naïve and primed, which show different degrees of potency. One difficulty in stem cell research is the inability to distinguish these states in live cells. Studies on female mice have shown that reactivation of inactive X chromosomes occurs in the naïve state, while one of the X chr...
Sperm-egg fusion is the critical step in mammalian fertilization, and requires the interaction between IZUMO1 on the sperm surface and JUNO (also known as folate receptor (FR) 4 or IZUMO1R) on the egg surface. Whereas other FRs bind and uptake folates, JUNO binds IZUMO1 and establishes the cell-cell adhesion. However, the mechanism of IZUMO1 recogn...
Supplementary Figures 1-5
Significance
In the mouse genome, thousands of genes are predominantly expressed in the testis, where these genes are thought to play important roles in spermatogenesis and fertilization. However, in this study, we report that 54 evolutionarily conserved and testis-enriched genes are not essential individually for male mouse fertility. Because the...
Ca(2+)influx triggers sperm capacitation; however, the underlying regulatory mechanisms remain incompletely understood. Here, we show that CNNM4, a Mg(2+)transporter, is required for Ca(2+)influx during capacitation. We find thatCnnm4-deficient male mice are almost infertile because of sperm dysfunction. Motion analyses show that hyperactivation, a...
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) contain a covalently linked GPI anchor located on outer cell membranes. GPI-APs are ubiquitously conserved from protozoa to vertebrates and are critical for physiological events such as development, immunity, and neurogenesis in vertebrates. Both membrane-anchored and soluble GPI-APs play a r...
The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of over-expressed Plac1 o...
Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on
male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains
a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mic...
