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

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 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik 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.

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.

CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

Single-cell RNA sequencing of mycosis fungoides reveals a cluster of actively proliferating lymphocytes.

A 70-year-old man's chronic erythematous skin lesion in the extremity had developed into a tumour one year before his first visit at our hospital. A biopsy showed atypical lymphocyte-like cells, and immunostaining identified atypical cells as CD3+, CD4+, CD5+ and FOXP3+. Single-cell RNA sequencing (scRNA-seq) analysis using BD Rhapsody revealed the higher expression of CD3, CD4, CD5 and FOXP3 genes in a group of cells that highly expressed genes, such as PCNA, in the S/M phase, which is in agreement with immunofluorescence staining results. The use of scRNA-seq analysis data is expected to promote personalised medicine for cutaneous lymphoma.

Co-expression of sperm membrane proteins CMTM2A and CMTM2B is essential for ADAM3 localization and male fertility in mice.

Chemokines are signaling proteins that are secreted to induce chemotaxis during an immunological response. However, the functions of transmembrane-type chemokine-like factor (CKLF) and the CMTM (CKLF-like MARVEL transmembrane domain containing) protein family remain to be determined. In this study, we focused on the testis-specific mouse CMTM gene cluster (Cmtm1, Cmtm2a and Cmtm2b) and generated CRISPR/Cas9-mediated mutant mice to examine their physiological functions. Although Cmtm1 mutant mice were fertile, Cmtm2a and Cmtm2b double mutant mice had defects in male fertility due to impaired sperm function. We found that co-expression of sperm membrane proteins CMTM2A and CMTM2B is required for male fertility and affects the localization of the sperm membrane protein ADAM3 in regulating sperm fertilizing ability.

MVH in piRNA processing and gene silencing of retrotransposons.

VASA is an evolutionarily conserved RNA helicase essential for germ cell development. The mouse PIWI family proteins MILI and MIWI2 are involved in production of Piwi-interacting RNAs (piRNAs) in fetal male germ cells through a ping-pong amplification cycle. Expression of retrotransposons is elevated in MILI- and MIWI2-deficient male germ cells due to defective de novo DNA methylation, which is presumably caused by impaired piRNA expression. Here, we report that essentially the same abnormalities are observed in MVH (mouse VASA homolog)-deficient mice. Comprehensive analysis of piRNAs in MVH-deficient fetal male germ cells showed that MVH plays crucial roles in the early phase of the ping-pong amplification cycle.

Human Globozoospermia-Related Gene Spata16 Is Required for Sperm Formation Revealed by CRISPR/Cas9-Mediated Mouse Models.

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 remains to be determined. Globozoospermic patients carried a homozygous point mutation in SPATA16 at 848G→A/R283Q. We generated CRISPR/Cas9-mediated mutant mice with the same amino acid substitution in the fourth exon of Spata16 to analyze the mutation site at R284Q, which corresponded with R283Q of mutated human SPATA16. We found that the point mutation in Spata16 was not essential for male fertility; however, deletion of the fourth exon of Spata16 resulted in infertile male mice due to spermiogenic arrest but not globozoospermia. This study demonstrates that Spata16 is indispensable for male fertility in mice, as well as in humans, as revealed by CRISPR/Cas9-mediated mouse models.

Insulinoma-Associated Protein 1 Is a Crucial Regulator of Neuroendocrine Differentiation in Lung Cancer.

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway.

GPAT2, a mitochondrial outer membrane protein, in piRNA biogenesis in germline stem cells.

piRNA (PIWI-interacting RNA) is a germ cell-specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.

Successful treatment of ulceration in hidradenitis suppurativa with topical bucladesine: A case report.

We report a case of hidradenitis suppurativa (HS) with skin ulceration in a 19-year-old man. He was successfully treated with topical bucladesine ointment treatment, resulting in a hypertrophic scar 2 months after the treatment. Bucladesine can be an alternative treatment option for ulceration in HS.

Tsukushi controls the hair cycle by regulating TGF-ß1 signaling.

The hair follicle contains stem/progenitor cells that supply progeny for skin development and the hair cycle. Several signaling molecules belonging to the Wnt, BMP, shh, and transforming growth factor ß (TGF-ß) signaling cascades are involved in the normal hair follicle cycle. However, the systemic mechanism of how these humoral factors are controlled remains largely unknown. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions extracellularly as a key coordinator of multiple signaling networks. Here, we show that TSK is expressed at the restricted areas of hair follicle during the morphogenesis and the hair cycle. Targeted disruption of the TSK gene causes the hair cycle to be delayed with low levels of TGF-ß1 and phosphorylated Smad2/3 (pSmad2/3) expression. Biochemical analysis indicates that TSK directly binds to TGF-ß1. Our data suggest that TSK controls the hair cycle by regulating TGF-ß1 signaling.

The pathological significance of Notch1 in oral squamous cell carcinoma.

Notch signaling has been reported to be involved in several types of malignant tumors; however, the role and activation mechanism of Notch signaling in oral squamous cell carcinoma (OSCC) remains poorly characterized. The purpose of this study was to elucidate the pathological significance of Notch signaling and its activation mechanism in the development and progression of OSCC. In this study, we showed that the expression of Notch1 and intracellular Notch domain (NICD) are upregulated in OSCCs. In addition, Notch1 and NICD were found to be characteristically localized at the invasive tumor front. TNF-α, a major inflammatory cytokine, significantly activated Notch signaling in vitro. In a clinicopathological analysis, Notch1 expression correlated with both the T-stage and the clinical stage. Furthermore, loss of Notch1 expression correlated with the inhibition of cell proliferation and TNF-α-dependent invasiveness in an OSCC cell line. In addition, γ-secretase inhibitor (GSI) prevented cell proliferation and TNF-α-dependent invasion of OSCC cells in vitro. These results indicate that altered expression of Notch1 is associated with increased cancer progression and that Notch1 regulates the steps involved in cell metastasis in OSCC. Moreover, inactivating Notch signaling with GSI could therefore be a useful approach for treating patients with OSCC.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

BACKGROUND: Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. OBJECTIVES: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. MATERIALS AND METHODS: Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. RESULTS: Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. DISCUSSION AND CONCLUSION: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Vitamin E derivative ETS-GS reduces liver ischemia-reperfusion injury in rats.

BACKGROUND: Ischemic liver injury is often the result of surgical procedures such as liver transplantation and hepatic resection. Liver damage occurs after reperfusion, leading to increased systemic inflammation. Recent studies have reported that vitamin E and glutathione can ameliorate ischemia-reperfusion (I/R) injury. In the present study, we evaluated the ability of a new vitamin E derivative, ETS-GS, to improve liver I/R injury. MATERIALS AND METHODS: Male Wistar received a subcutaneous injection of ETS-GS (10 mg/kg) or saline before experimentally-induced liver I/R injury or sham treatment. The rats were sacrificed after the 60-min ischemia and 24-h reperfusion. Histology and serum levels of cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-6, and high-mobility group box 1 (HMGB1) protein] and liver enzymes were determined to evaluate the protective effects of ETS-GS. RESULTS: We found that ETS-GS treatment attenuated I/R-induced histologic alterations, reduced levels of liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). In addition, ETS-GS treatment decreased serum cytokine levels. CONCLUSIONS: Taken together, our results demonstrate that ETS-GS attenuates I/R injury in a rat model and suggests that ETS-GS may exert anti-inflammatory effects. Accordingly, ETS-GS may have therapeutic potential to treat various clinical conditions involving I/R injury.

Prurigo pigmentosa clinically and immunologically mimicking autoimmune bullous disease: A case report.

A 15-year-old Japanese male noticed brown macules on his back 9 months ago. Initial examination revealed reticulated infiltrative erythema and pigmentation with blisters on the erythema of the back. Histopathology showed blisters with eosinophil infiltration in the epidermis, and direct immunofluorescence showed negative results for immunoglobulin (Ig) G, Ig A, Ig M, and C3 in the epidermal basement membrane zone. Immuno-serological tests revealed the presence of IgG antibodies against BP180, linear IgA disease antigen 1 (LAD-1), and laminin α3. The autoimmune bullous disease was suspected, and prednisolone at a concentration of 20 mg/day (0.3 mg/kg/day) was started. When the prednisolone dose was reduced to 10 mg/day, erythema and blisters recurred. The patient was diagnosed with prurigo pigmentosa based on clinical features and was treated successfully with oral doxycycline hydrochloride hydrate and topical tacrolimus ointment. This is the first case of prurigo pigmentosa with blisters in which autoantibodies to the epidermal basement membrane zone were found, which might be secondary non-pathogenic antibodies.

Nucleosome assembly protein 1-like 4, a new therapeutic target for proliferation and invasion of melanoma cells.

BACKGROUND: Melanoma is one of the deadliest skin cancers. The treatment of advanced melanoma has been dramatically improved by immune checkpoint inhibitors and targeted therapies. However, many patients still do not respond to these therapies. OBJECTIVE: To investigate whether NAP1L4 can be a new therapeutic target for melanoma. METHODS: Immunohistochemical analysis of human nevus and melanoma tissues was performed. Real-time RT-PCR and immunoblotting were performed using human samples and melanoma cell lines. Next, we examined the effect of NAP1L4 knockdown in melanoma cell lines using cell migration and invasion assays. To investigate the molecular mechanism related to these results, immunoblotting of p21 and Slug was examined. MMP-2 and MMP-9 activity assays were also performed. Further, pathway analysis between NAP1L4 and MMP-2 was performed. Finally, the effects of NAP1L4 knockdown on cell proliferation, apoptosis, and cell cycle were analyzed. RESULTS: NAP1L4 was overexpressed in melanoma tissues compared to the nevus tissue. NAP1L4 knockdown reduced melanoma cell migration and invasion. NAP1L4 knockdown upregulated p21 and downregulated Slug expression in melanoma cells. NAP1L4 knockdown decreased the active levels of MMP-2 in the supernatant from melanoma cells. NAP1L4 knockdown inhibited apoptosis in camptothecin-induced DNA damage, induced cell cycle arrest at the G1/S phase, and inhibited cell proliferation. CONCLUSIONS: NAP1L4 may play a role in cell migration and invasion in melanoma cells through the regulation of Slug. We propose that NAP1L4 can be a new therapeutic target for proliferation and invasion of melanoma cells.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells.

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

Methyl-CpG binding domain protein 3: a new diagnostic marker and potential therapeutic target of melanoma.

Methyl-CpG binding domain protein 3 (MBD3) belongs to the methyl-CpG binding protein family. MBD3 facilitates the initiation of neural stem cell reprogramming. Melanoma originates in melanocytes derived from neural crest stem cells; therefore, we investigated the role of MBD3 in melanoma. MBD3 was overexpressed in melanoma compared with pigmented nevi. MBD3 knockdown had no effect on the proliferation of melanoma cells (A375 and A2058 cells). Contrarily, it significantly reduced the migration and invasion of A375 cells, but had no significant effect on A2058 cells. Furthermore, MBD3 knockdown reduced N-cadherin protein levels and matrix metalloproteinase-2 (MMP-2) activity in A375 cells, but had no significant effect on A2058 cells. Based on these results, the MBD3 expression level may be a useful biomarker for the diagnosis of melanoma. Thus, MBD3 has potential as a novel therapeutic target for some melanoma patients.

Matrin-3 plays an important role in cell cycle and apoptosis for survival in malignant melanoma.

BACKGROUND: A previous study revealed that matrin-3 is an essential component in maintaining fibroblast growth factor 2 (FGF2)-mediated undifferentiation of neural stem cells (NSCs) using a proteomics approach. Malignant melanoma (MM) arises from melanocytes that originate from neural crest stem cells during development. Additionally, it has been reported that the expression of FGF2 is positively correlated with the progression of MM. OBJECTIVE: We expected that matrin-3, as a downstream component of FGF2, might be associated with the aggressiveness or differentiation of MM. METHODS: Matrin-3 expression was measured in human melanoma patient tissues and human MM cell lines. We analyzed the effect of matrin-3 siRNA on the proliferation of human MM cell lines and focused on cell cycle progression and apoptosis. We carried out in vivo xenograft tumor experiments by implanting A375 cells transfected with matrin-3 shRNA. RESULTS: Matrin-3 was highly expressed in MM, and matrin-3 knockdown inhibited the proliferation of melanoma cellsin vivo and in vitro. Furthermore, we found that matrin-3 knockdown led to an accumulation of cells in the G1 phase and an increase in apoptotic cell number. CONCLUSION: Our results suggest that matrin-3 could be a new therapeutic target for the treatment of MM.

Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells.

To investigate the mechanisms underlying the maintenance of neural stem cells, we performed two-dimensional fluorescence-difference gel electrophoresis (2D-DIGE) targeting the nuclear phosphorylated proteins. Nuclear phosphorylated protein Matrin-3 was identified in neural stem cells (NSCs) after stimulation using fibroblast growth factor 2 (FGF2). Matrin-3 was expressed in the mouse embryonic subventricular and ventricular zones. Small interfering RNA (siRNA)-mediated knockdown of Matrin-3 caused neuronal differentiation of NSCs in vitro, and altered the cerebral layer structure of foetal brain in vivo. Transfection of Matrin-3 plasmids in which the serine 208 residue was point-mutated to alanine (Ser208Ala mutant Matrin3) and inhibition of Ataxia telangiectasia mutated kinase (ATM kinase), which phosphorylates Matrin-3 Ser208 residue, caused neuronal differentiation and decreased the proliferation of neurosphere-forming stem cells. Thus, our proteomic approach revealed that Matrin-3 phosphorylation was essential for FGF2-dependent maintenance of NSCs in vitro and in vivo.

Evidence for phosphorylation of rat liver glucose-regulated protein 58, GRP58/ERp57/ER-60, induced by fasting and leptin.

Glucose-regulated protein 58 (GRP58)-like immunoreactivity in rat liver obtained in the evening or after fasting underwent an electrophoretic band-shift, which disappeared after phosphatase-treatment. Since mass spectrometric analysis raised a possibility that Ser150 of GRP58 is phosphorylated, an antibody against the phosphoserine150 GRP58 was generated. Immunoreactivity to this antibody was increased in the evening and after fasting. Since GRP58 was shown to interact with signal transducer and activator of transduction 3 (STAT3), a leptin-related protein, the effect of leptin was examined. Immunoreactivity to the anti-phosphoGRP58 antibody was markedly elevated after the leptin injection, indicating that Ser150 of GRP58 is phosphorylated after fasting and leptin-treatment.