Acrosin is essential for sperm penetration through the zona pellucida in hamsters.

During natural fertilization, mammalian spermatozoa must pass through the zona pellucida before reaching the plasma membrane of the oocyte. It is assumed that this step involves partial lysis of the zona by sperm acrosomal enzymes, but there has been no unequivocal evidence to support this view. Here we present evidence that acrosin, an acrosomal serine protease, plays an essential role in sperm penetration of the zona. We generated acrosin-knockout (KO) hamsters, using an in vivo transfection CRISPR/Cas9 system. Homozygous mutant males were completely sterile. Acrosin-KO spermatozoa ascended the female genital tract and reached ovulated oocytes in the oviduct ampulla, but never fertilized them. In vitro fertilization (IVF) experiments revealed that mutant spermatozoa attached to the zona, but failed to penetrate it. When the zona pellucida was removed before IVF, all oocytes were fertilized. This indicates that in hamsters, acrosin plays an indispensable role in allowing fertilizing spermatozoa to penetrate the zona. This study also suggests that the KO hamster system would be a useful model for identifying new gene functions or analyzing human and animal disorders because of its technical facility and reproducibility.

Functional characterization of testis-brain RNA-binding protein, TB-RBP/Translin, in translational regulation.

Testis-brain RNA-binding protein (TB-RBP/Translin) is known to contribute to the translational repression of a subset of haploid cell-specific mRNAs, including protamine 2 (Prm2) mRNA. Mutant mice lacking TB-RBP display abnormal spermatogenesis, despite normal male fertility. In this study, we carried out functional analysis of TB-RBP in mammalian cultured cells to understand the mechanism of translational repression by this RNA-binding protein. Although the amino acid sequence contained a eukaryotic translation initiation factor 4E (EIF4E)-recognition motif, TB-RBP failed to interact with EIF4E. In cultured cells, TB-RBP was unable to reduce the activity of luciferase encoded by a reporter mRNA carrying the 3'-untranslated region of Prm2. However, λΝ-BoxB tethering assay revealed that the complex of TB-RBP with its binding partner, Translin-associated factor X (TRAX), exhibits the ability to reduce the luciferase reporter activity by degrading the mRNA. These results suggest that TB-RBP may play a regulatory role in determining the sequence specificity of TRAX-catalyzed mRNA degradation.

Pseudomonas allokribbensis sp. nov. and Pseudomonas gozinkensis sp. nov., Two New Species Isolated from a Volcanic Island, Izu Oshima, Japan.

The genomes of two Pseudomonas strains, IzPS23T and IzPS32dT isolated from soil samples of Izu Oshima were compared to Pseudomonas type strains. Whole-genome sequence analysis revealed both belong to the Pseudomonas fluorescens lineage. The average nucleotide identity values of the whole-genome sequences of IzPS23T and IzPS32dT compared with other type strains showed high correlations with Pseudomonas kribbensis (93.1%) and Pseudomonas glycinae (93.5%), respectively. Genome-to-genome distances between the whole-genome sequences of IzPS23T and IzPS32dT showed correlations with Pseudomonas kribbensis (51.0%) and Pseudomonas glycinae (53.2%), respectively. Genotypic and phenotypic analysis indicated the two strains were novel species, and were named Pseudomonas allokribbensis (IzPS23T = CECT 9961T, = LMG 31525T) and Pseudomonas gozinkensis (IzPS32dT = CECT 9962T, = LMG 31526T), respectively.

SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

The glycosylphosphatidylinositol-anchored sperm hyaluronidases (Hyals), sperm adhesion molecule 1 (SPAM1) and HYAL5, have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we have shown that mouse sperm devoid of either Spam1 or Hyal5 are still capable of penetrating the COC and that the loss of either Spam1 or Hyal5 alone does not cause male infertility in mice. In the present study, we found that Spam1/Hyal5 double knockout (dKO) mice produced significantly fewer offspring compared with wild-type (WT) mice, and this was due to defective COC dispersal. A comparative analysis between WT and Spam1/Hyal5 dKO epididymal sperm revealed that the absence of these 2 sperm Hyals resulted in a marked accumulation of sperm on the outside of the COC. This impaired sperm activity is likely due to the deficiency in the sperm Hyals, even though other somatic Hyals are expressed normally in the dKO mice. The fertilization ability of the Spam1/Hyal5 dKO sperm was restored by adding purified human sperm Hyal to the in vitro fertilization medium. Our results suggest that Hyal deficiency in sperm may be a significant risk factor for male sterility.-Park, S., Kim, Y.-H., Jeong, P.-S., Park, C., Lee, J.-W., Kim, J.-S., Wee, G., Song, B.-S., Park, B.-J., Kim, S.-H., Sim, B.-W., Kim, S.-U., Triggs-Raine, B., Baba, T., Lee, S.-R., Kim, E. SPAM1/HYAL5 double deficiency in male mice leads to severe male subfertility caused by a cumulus-oocyte complex penetration defect.

Re-identification of strains deposited as Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida in GenBank based on whole genome sequences.

The taxonomic classification of Pseudomonas species has been revised and updated several times. This study utilized average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) cutoff values of 95 and 70 %, respectively, to re-identify the species of strains deposited in GenBank as P. aeruginosa, P. fluorescens and P. putida. Of the 264 deposited P. aeruginosa strains, 259 were correctly identified as P. aeruginosa, but the remaining five were not. All 28 deposited P. fluorescens strains had been incorrectly identified as P. fluorescens. Four of these strains were re-identified, including two as P. kilonensis and one each as P. aeruginosa and P. brassicacearum, but the remaining 24 could not be re-identified. Similarly, all 35 deposited P. putida strains had been incorrectly identified as P. putida. Nineteen of these strains were re-identified, including 12 as P. alloputida, four as P. asiatica and one each as P. juntendi, P. monteilii and P. mosselii. These results strongly suggest that Pseudomonas bacteria should be identified using ANI and dDDH analyses based on whole genome sequencing when Pseudomonas species are initially deposited in GenBank/DDBJ/EMBL databases.

Pseudomonas izuensis sp. nov., a novel species isolated from Izu Oshima, Japan.

An aerobic, Gram-stain-negative, rod-shaped bacterial strain, IzPS43_3003T, was isolated from Izu Oshima, an active volcanic island located 22 km east of the Izu Peninsula, Japan. The sequence of its 16S rRNA gene indicated that IzPS43_3003T belongs to the Pseudomonas fluorescens lineage, with its sequence being most similar to that of Pseudomonas vancouverensis DhA-51T (99.79 %). Phylogenetic analysis based on whole genome sequences showed that IzPS43_3003T was a member of the Pseudomonas jessenii subgroup. The average nucleotide identity values and genome-to genome distances between the whole genome sequences of IzPS43_3003T and other type strains showed that the highest correlations were with Pseudomonas moorei DSM 12647T (87.3 and 33.5% respectively). These genotypic and phenotypic analyses indicated that IzPS43_3003T belongs to a novel species, Pseudomonas izuensis sp. nov. Its type strain is IzPS43_3003T (=LMG 31527T,=CECT 9963T).

Protective activity of anti-lipoteichoic acid monoclonal antibody in single or combination therapies in methicillin-resistant Staphylococcus aureus-induced murine sepsis models.

Previously, we generated and screened a panel of monoclonal antibodies (mAbs) against methicillin-resistant Staphylococcus aureus (MRSA) to identify protective mAbs in mouse infection models. One of these mAbs, ZBIA3H, bound to lipoteichoic acid (LTA) and exerted protective effects in a mouse sepsis model. To reinforce the ability of the mAb to protect against infection, combination therapies with the mAb and antibiotics need to be examined. Therefore, herein, we studied the efficacy of ZBIA3H (in combination or alone) in a mouse sepsis model. ZBIA3H improved the survival rate in the mouse models of sepsis induced by highly virulent or refractory S. aureus (community-acquired MRSA strain MW2, vancomycin-intermediate S. aureus strain Mu3, or vancomycin-resistant S. aureus strain VRS1). Furthermore, ZBIA3H remarkably improved the survival rate in combination with antimicrobial agents (vancomycin, daptomycin, or linezolid) in mouse sepsis models. From these results we conclude that anti-LTA mAb ZBIA3H or its humanized form is a promising mAb individually, or in combination with antibiotics, against clinical refractory infection of S. aureus.

Pseudomonas atagosis sp. nov., and Pseudomonas akappagea sp. nov., New Soil Bacteria Isolated from Samples on the Volcanic Island Izu Oshima, Tokyo.

During the exploration of microbial natural resources, two strains of Pseudomonas, PS14T and PS24T, were isolated from samples taken from Izu Oshima, a volcanic island located 120 km southwest of central Tokyo. Phylogenetic analysis based on 16S rRNA gene sequences showed that PS14T was most similar to Pseudomonas baetica a390T (99.6%) and Pseudomonas helmanticensis OHA11T (99.5%), and that PS24T was most similar to Pseudomonas qingdaonensis JJ3T (98.8%) and Pseudomonas lutea OK2T (98.7%). The major fatty acids of these two strains were C16:0 and C17:0 cyclo, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and summed feature 8 (C18:1 ω7c and/or 18:1 ω6c). The phylogenetic analyses, DNA-DNA hybridization results and phenotypic traits indicated that PS14T and PS24T constitute two novel species, Pseudomonas atagosis sp. nov. (type strain PS14T = CECT 9940T, = LMG 31496T) and Pseudomonas akappagea sp. nov. (type strain PS24T = CECT 9941T, = LMG 31497T), respectively. The sequence data of the draft genomes of PS14T and PS24T were deposited in the GenBank database under accession numbers VXCA00000000 and VXCP00000000, respectively, and the sequence data of their 16S rRNA genes were deposited in the GenBank database under accession numbers MN396717 and MN382268, respectively.

Regional outbreak of community-associated methicillin-resistant Staphylococcus aureus ST834 in Japanese children.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection has recently become a challenging problem worldwide and in Japan. We experienced 10 pediatric patients infected with CA-MRSA and hospitalized from 2011 to 2014 in a tertiary care hospital in Saitama, Japan, and assessed the characteristic of the strains using a whole genome sequencing (WGS)-based approach. METHODS: CA-MRSA strains isolated from infected patients who required hospitalization for treatment were evaluated in this study. Antimicrobial susceptibility tests, molecular typing by PCR and pulse-field gel electrophoresis (PFGE) were performed to characterize MRSA strains. WGS was performed for detailed genetic analysis. RESULTS: A total of 582 MRSA strains (35.2%) were identified among 1625 S. aureus strains collected during the study period. Ten MRSA strains (1.7%) were defined as CA-MRSA clinically, and all were isolated from pediatric patients. All strains mainly caused purulent lymphadenitis, were susceptible to fluoroquinolone and tetracycline, exhibited sequence type (ST) 834 or its single-locus variants and contained staphylococcal cassette chromosome mec (SCCmec) type IVc. Phylogenic analysis by PFGE and WGS revealed close relatedness of all strains, with the number of single nucleotide polymorphisms ranging from 35 to 119 by WGS. Out of the ten strains, nine possessed the genomic island SaPISaitama2 containing tst, sec and sel genes. SaPISaitama2 comprises a mosaic of genomic islands SaPIm4 and SaPIm1 harbored by a hospital-associated MRSA strain Mu50. CONCLUSIONS: This study describes a regional outbreak of ST834-related CA-MRSA in children with a unique pathogenicity island in Japan. Pediatric patient tropism of this clone could be enhanced by susceptibility to fluoroquinolones and tetracyclines, which cannot be prescribed to children.

Behavior of ACRBP-deficient mouse sperm in the female reproductive tract.

Gene-knockout mice lacking ACRBP, a proacrosin-binding protein localized in the acrosome of sperm, have been shown to exhibit male subfertility, owing to abnormal formation of the acrosome. In this study, to elucidate the mechanism contributing to the subfertility phenotype, we examined the behavior of ACRBP-deficient mouse sperm in the female reproductive tract. When sperm that had migrated into the uterus and oviduct after mating were counted, the number of ACRBP-deficient sperm was noticeably smaller in the oviduct of mice post mating. However, ACRBP-deficient sperm recovered from the oviduct possessed morphologically normal head shape and retained normal motility. Importantly, ACRBP-deficient sperm displayed a marked reduction in the ability to successfully gain access to unfertilized oocytes. These data suggest that male subfertility of ACRBP-deficient mice may be attributed to incompleteness of the acrosome reaction rather than impairment in sperm migration from the uterus to the oviduct.

Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse.

Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis.

PAPOLB/TPAP regulates spermiogenesis independently of chromatoid body-associated factors.

Mutant mice lacking a testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, exhibit spermiogenesis arrest and male infertility. However, the mechanism by which PAPOLB regulates spermiogenesis remains unclear. In this study, we examined the relationships between PAPOLB and other spermiogenesis regulators present in the chromatoid body (CB). The loss of PAPOLB had no impact either on the abundance of CB components such as PIWIL1, TDRD6, YBX2, and piRNAs, or on retrotransposon expression. In addition, localization of CB proteins and CB architecture were both normal in PAPOLB-null mice. No interactions were observed between PAPOLB and PIWIL1 or YBX2. While PIWIL1 and YBX2 were associated with translationally inactive messenger ribonucleoproteins and translating polyribosomes, PAPOLB was present almost exclusively in the mRNA-free fractions of sucrose gradients. These results suggest that PAPOLB may regulate spermiogenesis through a pathway distinct from that mediated by CB-associated factors.

Selective stabilization of mammalian microRNAs by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2.

The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 3' ends of several miRNAs undergo post-dicing adenylation or uridylation. We isolated the liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 3'-terminal adenosine that is introduced after processing by Dicer. We identified GLD-2, which is a regulatory cytoplasmic poly(A) polymerase, as responsible for the 3'-terminal adenylation of miR-122 after unwinding of the miR-122/miR-122* duplex. In livers from GLD-2-null mice, the steady-state level of the mature form of miR-122 was specifically lower than in heterozygous mice, whereas no reduction of pre-miR-122 was observed, demonstrating that 3'-terminal adenylation by GLD-2 is required for the selective stabilization of miR-122 in the liver.

Surfing and Swimming of Ejaculated Sperm in the Mouse Oviduct.

To accomplish fertilization in the oviductal ampulla, ejaculated sperm are required to migrate through the female reproductive tract. However, this fundamental process largely remains unknown. In this study, we focused on the role of oviductal smooth muscle (myosalpinx) contractions in the sperm migration. Administration of prifinium bromide, padrin, to mice effectively suppressed myosalpinx contractions, resulting in a decreased rate of fertilization in a dose-dependent manner, and an abrogation of high-speed back-and-forth/shuttling flows of oviductal fluids around the isthmus. Regardless of padrin administration, no shuttling flows were found near the ampulla. In the isthmus, sperm formed a tight assemblage that was synchronized with the shuttling flows. The sperm assemblage was gradually loosened and then completely abolished near the ampulla. No sperm assemblage was formed in the isthmus when padrin was administrated. These results suggest that myosalpinx contractions play important roles in the formation of sperm assemblage in the isthmus, and in the transport of the assemblage to the middle region of the oviduct. It is also suggested that the motility of sperm is essential for the migration of sperm from the middle oviductal region to the ampulla.

Abnormal spermatogenesis and male infertility in testicular zinc finger protein Zfp318-knockout mice.

Zfp318, a mouse gene with a Cys2/His2 zinc finger motif, is mainly expressed in germ cells in the testis. It encodes two alternative transcripts, which regulate androgen receptor-mediated transcriptional activation or repression by overexpression of them. However, the role of Zfp318 is still obscure in vivo, especially in spermatogenesis. To elucidate the role of Zfp318 during gamete production, we established a knockout mouse line. Zfp318-null male mice exhibited infertility, whereas Zfp318-null female mice displayed normal fertility. ZFP318 was expressed during multiple stages of spermatogenesis, from spermatocytes to round spermatids. The nuclei of secondary spermatocytes showed high levels of expression. Histological analysis and quantitative analysis of DNA content showed decreased numbers of both spermatids in the seminiferous tubules and mature spermatozoa in the epididymides of Zfp318-null mice. These results suggest that Zfp318 is expressed as a functional protein in testicular germ cells and plays an important role in meiosis during spermatogenesis.

Functional compensation for the loss of testis-specific poly(A)-binding protein, PABPC2, during mouse spermatogenesis.

Mouse testes contain several isoforms of cytoplasmic poly(A)-binding proteins (PABPCs), including ubiquitous PABPC1 and testis-specific PABPC2/PABPt. PABPC2 is characterized by its absence from translationally active polyribosomes and elongating spermatids. To elucidate the function of PABPC2 in spermatogenesis, we produced mutant mice lacking PABPC2. The PABPC2-null mice showed normal fertility. The processes of spermatogenesis and sperm migration in the testes and epididymides, respectively, were normal in the mutant mice. When the involvement of PABPC2 in translational regulation of haploid-specific mRNAs was examined, these mRNAs were correctly transcribed in round spermatids and translated in elongating spermatids. Moreover, immunoblot analysis revealed low abundance of PABPC2 relative to PABPC1 in spermatogenic cells. These results suggest that PABPC2 may be either functionally redundant with other PABPCs (including PABPC1) or largely dispensable for translational regulation during spermiogenesis.

Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, is essential for spermatogenesis.

The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme's adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity.

Successful selection of an infection-protective anti-Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models.

Recent clinical trials to develop anti-methicillin-resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty-two anti-S. aureus IgG mAbs were obtained from mice that had been immunized with alkali-processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life-saving effects in mouse models of sepsis caused by community-acquired MRSA strain MW2 and vancomycin-resistant S. aureus strain VRS1. It also had a curative effect in a MW2-caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4-ß-linkage between N-acetylmuramic acid and N-acetyl-D-glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high-yield and/or high-affinity antibodies may not be the most suitable epitopes for developing anti-infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.

Possible role of p38 MAPK-MNK1-EMI2 cascade in metaphase-II arrest of mouse oocytes.

The Mos-MAPK signaling pathway involving the Mos-MEK1/2-ERK1/2-RSK1/2/3 or MSK1-EMI2 cascade is directly linked to metaphase-II arrest of vertebrate oocytes. In this study, we examined whether p38, a member of the MAPK subfamily, is regulated under the control of Mos and contributes to metaphase-II arrest in the mouse oocyte. Morpholino oligonucleotide-mediated depletion of Mos revealed a remarkable decrease in phosphorylation of p38. Simultaneous treatment of oocytes with two chemical inhibitors of p38 and MEK1/2 induced both release from metaphase II and degradation of cyclin B1, whereas the treatment with each of these two inhibitors had little effect. Moreover, phosphorylation of EMI2 was dramatically abolished by addition of the two inhibitors. Indeed, MNK1, a kinase downstream of p38, exhibited the ability to phosphorylate EMI2. These results suggest that in addition to the Mos-MEK1/2 pathway, the Mos-mediated p38 pathway may be implicated in metaphase-II arrest.

Beta-hemolysin promotes skin colonization by Staphylococcus aureus.

Colonization by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases and is often followed by epidermal damage and invasive infection. In this study, we investigated the mechanism of skin colonization by a virulent community-acquired methicillin-resistant S. aureus (CA-MRSA) strain, MW2, using a murine ear colonization model. MW2 does not produce a hemolytic toxin, beta-hemolysin (Hlb), due to integration of a prophage, Sa3mw, inside the toxin gene (hlb). However, we found that strain MW2 bacteria that had successfully colonized murine ears included derivatives that produced Hlb. Genome sequencing of the Hlb-producing colonies revealed that precise excision of prophage Sa3mw occurred, leading to reconstruction of the intact hlb gene in their chromosomes. To address the question of whether Hlb is involved in skin colonization, we constructed MW2-derivative strains with and without the Hlb gene and then subjected them to colonization tests. The colonization efficiency of the Hlb-producing mutant on murine ears was more than 50-fold greater than that of the mutant without hlb. Furthermore, we also showed that Hlb toxin had elevated cytotoxicity for human primary keratinocytes. Our results indicate that S. aureus Hlb plays an important role in skin colonization by damaging keratinocytes, in addition to its well-known hemolytic activity for erythrocytes.