年森 清隆

Abstract Normal sperm flagellar shape and movement are essential for fertilization. The integral protein outer dense fiber 4 (ODF4) localizes to ODFs, but its function remains unclear. Adenylate kinase (AK) is a phosphotransferase that catalyzes the interconversion and controls the concentration equilibrium of adenine nucleotides. AK shuttles ATP to energy-consuming sites. Here, we report on the relationship of flagellar shape and movement with ODF4, AK1 and AK2 by using Odf4-deletion (Odf4^−/−) mice. Soluble ODF4 is coimmunoprecipitated with AK1 and AK2 in Odf4^+/+ spermatozoa. ODF4, AK1 and AK2 localize to whole flagella (plasmalemma, mitochondria, ODFs, and residual cytoplasmic droplets (CDs)), principal pieces, and midpieces, respectively. Odf4^−/− sperm flagella lose ODF4 and reduce AK1 and AK2 but produce ATP. The flagellum is bent (hairpin flagellum) with a large CD in the midpiece. There is no motility in the midpiece, but the principal piece is motile. Odf4^−/− spermatozoa progress backward and fail to ascend in the uterus. Thus, Odf4^−/− males are infertile owing to abnormal flagellar shape and movement caused mainly by the loss of ODF4 with AK1 and AK2. This study is supported by the rescue experiment; the abnormalities and male infertility caused by Odf4 deletion were reversed by Odf4 restoration.

Lysyl hydroxylase 2 (LH2) is an enzyme that catalyzes the hydroxylation of lysine (Lys) residues in fibrillar collagen telopeptides, a critical post-translational modification for the stability of intermolecular cross-links. Though abnormal LH2 activities have been implicated in various diseases including Bruck syndrome, the molecular basis of the pathologies is still not well understood. Since LH2 null mice die at early embryonic stage, we generated LH2 heterozygous (LH2+/-) mice in which LH2 level is significantly diminished, and characterized collagen and bone phenotypes using femurs. Compared to the wild-type (WT), LH2+/- collagen showed a significant decrease in the ratio of hydroxylysine (Hyl)- to the Lys-aldehyde-derived collagen cross-links without affecting the total number of aldehydes involved in cross-links. Mass spectrometric analysis revealed that, in LH2+/- type I collagen, the extent of hydroxylation of all telopeptidyl Lys residues was significantly decreased. In the helical domain, Lys hydroxylation at the cross-linking sites was either unaffected or slightly lower, but other sites were significantly diminished compared to WT. In LH2+/- femurs, mineral densities of cortical and cancellous bones were significantly decreased and the mechanical properties of cortical bones evaluated by nanoindentation analysis were compromised. When cultured, LH2+/- osteoblasts poorly produced mineralized nodules compared to WT osteoblasts. These data provide insight into the functionality of LH2 in collagen molecular phenotype and its critical role in bone matrix mineralization and mechanical properties.

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1-/- mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

<title>Abstract</title> The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

Outer dense fibre 2 (Odf2 or ODF2) is a cytoskeletal protein required for flagella (tail)-beating and stability to transport sperm cells from testes to the eggs. There are infertile males, including human patients, who have a high percentage of decapitated and decaudated spermatozoa (DDS), whose semen contains abnormal spermatozoa with tailless heads and headless tails due to head-neck separation. DDS is untreatable in reproductive medicine. We report for the first time a new type of Odf2-DDS in heterozygous mutant Odf2+/- mice. Odf2+/- males were infertile due to haploinsufficiency caused by heterozygous deletion of the Odf2 gene, encoding the Odf2 proteins. Odf2 haploinsufficiency induced sperm neck-midpiece separation, a new type of head-tail separation, leading to the generation of headneck sperm cells or headnecks composed of heads with necks and neckless tails composed of only the main parts of tails. The headnecks were immotile but alive and capable of producing offspring by intracytoplasmic headneck sperm injection (ICSI). The neckless tails were motile and could induce capacitation but had no significant forward motility. Further studies are necessary to show that ICSI in humans, using headneck sperm cells, is viable and could be an alternative for infertile patients suffering from Odf2-DDS.

To identify upregulated genes during the development of spermatozoa, we performed PCR-selected subtraction analysis of testes RNA samples from 10-day-old and 12-week-old shrews. A transcript, highly homologous to two mouse transcripts, Ms4a13-1 and Ms4a132, was differentially regulated. Ms4a13-2, but not Ms4a13-1, was shown to be primarily expressed in mouse testes in an agedependent manner. Ms4a13-2 cDNA contains an open-reading frame of 522 nucleotides, encoding a protein of 174 amino acids, with predicted molecular mass, 19,345 Da. MS4A13-2 protein was expressed along the periphery of nuclei of round and elongated spermatids (steps 3-16) in adult mouse testes, and in the equatorial region of the heads of fresh mature mouse spermatozoa. In addition, MS4A13-2 was found to localize to the outer acrosomal membrane in the equatorial region of heads in fresh spermatozoa. In acrosome-reacted spermatozoa, the MS4A13-2 expression extended to the entire sperm head including the postacrosomal region and acrosomal cap. MS4A family proteins are known to facilitate intracellular protein-protein interactions as ion channel/ adaptor proteins by oligomerization, and have important regulatory roles in cellular growth, survival and activation. We report that the MS4A family member, MS4A13-2, may form oligomers in sperm membranes, which may be involved in an interaction with the zona pellucida or cumulus during fertilization.

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.

Docosahexaenoic acid (DHA) is one of the essential omega-3 polyunsaturated fatty acids with a wide range of physiological roles important for human health. For example, DHA renders cell membranes more flexible and is therefore important for cellular function, but information on the mechanisms that control DHA levels in membranes is limited. Specifically, it is unclear which factors determine DHA incorporation into cell membranes and how DHA exerts biological effects. We found that lysophosphatidic acid acyltransferase 3 (LPAAT3) is required for producing DHA-containing phospholipids in various tissues, such as the testes and retina. In this study, we report that LPAAT3-KO mice display severe male infertility with abnormal sperm morphology. During germ cell differentiation, the expression of LPAAT3 was induced, and germ cells obtained more DHA-containing phospholipids. Loss of LPAAT3 caused drastic reduction of DHA-containing phospholipids in spermatids that led to excess cytoplasm around its head, which is normally removed by surrounding Sertoli cells via endocytosis at the final stage of spermatogenesis. In vitro liposome filtration assay raised the possibility that DHA in phospholipids promotes membrane deformation that is required for the rapid endocytosis. These data suggest that decreased membrane flexibility in LPAAT3KO sperm impaired the efficient removal of sperm content through endocytosis. We conclude that LPAAT3-mediated enrichment of cell membranes with DHA-containing phospholipids endows these membranes with physicochemical properties needed for normal cellular processes, as exemplified by spermatogenesis.

Objective: While type 1 programmed cell death (apoptosis) of T cells leads to immunosuppression in sepsis, a crosstalk between apoptosis and autophagy (type 2 programmed cell death) has not been shown. The aim of this study is to elucidate the details of the interaction between autophagy and immunosuppression. Design: Laboratory investigation in the murine sepsis model. Setting: University laboratory. Subjects: Six- to 8-week-old male mice. Interventions: We investigated the kinetics of autophagy in T cells from spleen in a cecal ligation and puncture model with green fluorescent protein-microtubule-associated protein light chain 3 transgenic mice. We analyzed apoptosis, mitochondrial homeostasis and cytokine production in T cells, and survival rate after cecal ligation and puncture using T cell-specific autophagy-deficient mice. Measurements and Main Results: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4 + T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4 + T cells from the cecal ligation and puncture-operated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. Conclusions: We demonstrated that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4 + T cells in the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis.

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/ cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14(-)/(-) cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14(-)/(-) spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14(-)/(-) sperm cells: the annulus, a ring-like structure at the mid-piece-principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.

Basigin is a member of the immunoglobulin superfamily and plays various important roles in biological events including spermatogenesis. To examine the basigin molecular variants during spermatogenesis and sperm maturation in the mouse, immunoprecipitated basigin samples from testis and epididymal spermatozoa were analyzed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results demonstrated that basigin molecules from the testis and spermatozoa were separable into two major bands and that the differences in the molecular sizes were possibly because of an endoproteolytic cleavage. Since basigin is known to be a chaperone for the monocarboxylate transporter 1 (MCT1), the localization of basigin, MCT1 and MCT2 was examined during postnatal testicular development. Immunohistochemical studies showed different expression patterns of MCT1 and MCT2. MCT1 was localized on the surface of spermatogonia, spermatocytes, and spermatids. In contrast, MCT2 appeared on the principal piece of spermatozoa in the testis, where basigin was also observed. In mature epididymal spermatozoa, MCT2 was located on the midpiece, where basigin co-localized with MCT2 but not with MCT1. Furthermore, MCT2 was immunoprecipitated with basigin in mouse testes and sperm. These results suggest that basigin has a functional role as a binding partner with MCT2 in testicular and epididymal spermatozoa.

Molecular biomarkers that can assess sperm acrosome status are very useful for evaluating sperm quality in the field of assisted reproductive technology. In this review, we introduce and discuss the localization and function of acrosomal proteins that have been well studied. Journal databases were searched using keywords, including "human acrosome", "localization", "fertilization-related protein", "acrosomal membrane", "acrosomal matrix", "acrosome reaction", "knockout mouse", and "acrosome marker".

The acrosome is a Golgi-derived sperm cell organelle enclosed by a continuous acrosomal membrane. The acrosomal membrane complexes with surrounding matrices containing molecules necessary for fertilization; however, the complex of acrosomal membrane and associating matrices (CAMAM) has not been visualized in detail under living conditions. Here, we analyzed the CAMAM at the nanometer level using super-resolution stimulated emission depletion (STED) fluorescence microscopy and equatorin-enhanced green fluorescent protein transgenic mice. The STED images were compared with the corresponding images taken by immunoelectron microscopy. Consequently, the substructure of CAMAM could be differentiated at nanometer-scale resolution by STED microscopy without the need for sectioning. The information obtained in this study will be beneficial not only for understanding the molecular mechanism of fertilization but also for cell imaging under living conditions.

Lactate represents a preferential energy substrate of germ cells rather than glucose. Testicular Sertoli cells are believed to produce lactate and pyruvate and to supply these to germ cells, particularly spermatocytes and spermatids. Monocarboxylate transporter (MCT), responsible for the transport of lactate and other monocarboxylates via the cell membrane, is abundant in the testes and sperm (MCT1, MCT2, and MCT4). For the uptake of glucose, germ cells within the seminiferous tubules and sperm have been known to intensely express GLUT3. The present study investigated expression profiles of MCTs and GLUTs and revealed their cellular and subcellular localization in the mouse and rat testis. An in situ hybridization analysis showed significant expressions of MCT1, MCT2, and GLUT3 mRNA in the testis. Immunohistochemically, spermatogonia, spermatocytes, and spermatids expressed MCT1 on their cell surfaces in a stage-dependent manner: in some seminiferous tubules, an intense expression of MCT1 was unique to the spermatogonia. MCT2 was restricted to the tails of elongated spermatids and sperm. An intense immunoreactivity for GLUT3 was shared by spermatocytes, spermatids, and sperm. Sertoli cells were devoid of any immunoreactivities for MCT1, MCT2, and GLUT3. The predominant energy source of germ cells may be lactate and other monocarboxylates-especially for spermatogonia, but glucose and other hexoses may be responsible for an energy supply to spermatocytes and spermatids.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6de1CACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

Oligo-astheno-teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol-binding protein in the oxysterol-binding protein (OSBP)-related protein family, causes male infertility due to severe OAT in mice. In ORP4-deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round-shaped head, curled back head or symplast. Spermatozoa derived from ORP4-deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4-deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

For normal fertilization in mammals, it is important that functionally mature sperm are motile and have a fully formed acrosome. The glycosyltransferase-like gene, human polypeptide N-acetylgalactosaminyl-transferase- like protein 5 (GALNTL5), belongs to the polypeptide N-acetylgalactosamine-transferase (pp-GalNAc-T) gene family because of its conserved glycosyltransferase domains, but it uniquely truncates the C-terminal domain and is expressed exclusively in human testis. However, glycosyltransferase activity of the human GALNTL5 protein has not been identified by in vitro assay thus far. Using mouse Galntl5 ortholog, we have examined whether GALNTL5 is a functional molecule in spermatogenesis. It was observed that mouse GALNTL5 localizes in the cytoplasm of round spermatids in the region around the acrosome of elongating spermatids, and finally in the neck region of spermatozoa. We attempted to establish Galntl5-deficient mutant mice to investigate the role of Galntl5 in spermiogenesis and found that the heterozygous mutation affected male fertility due to immotile sperm, which is diagnosed as asthenozoospermia, an infertility syndrome in humans. Furthermore, the heterozygous mutation of Galntl5 attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes, and caused aberrant localization of the ubiquitin-proteasome system. By comparing the protein compositions of sperm from infertile males, we found a deletion mutation of the exon of human GALNTL5 gene in a patient with asthenozoospermia. This strongly suggests that the genetic mutation of human GALNTL5 results in male infertility with the reduction of sperm motility and that GALNTL5 is a functional molecule essential for mammalian sperm formation.

The trans-Golgi network (TGN) functions as a hub organelle in the exocytosis of clathrin-coated membrane vesicles, and SMAP2 is an Arf GTPase-activating protein that binds to both clathrin and the clathrin assembly protein (CALM). In the present study, SMAP2 is detected on the TGN in the pachytene spermatocyte to the round spermatid stages of spermatogenesis. Gene targeting reveals that SMAP2-deficient male mice are healthy and survive to adulthood but are infertile and exhibit globozoospermia. In SMAP2-deficient spermatids, the diameter of proacrosomal vesicles budding from TGN increases, TGN structures are distorted, acrosome formation is severely impaired, and reorganization of the nucleus does not proceed properly. CALM functions to regulate vesicle sizes, and this study shows that CALM is not recruited to the TGN in the absence of SMAP2. Furthermore, syntaxin2, a component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is not properly concentrated at the site of acrosome formation. Thus this study reveals a link between SMAP2 and CALM/syntaxin2 in clathrin-coated vesicle formation from the TGN and subsequent acrosome formation. SMAP2-deficient mice provide a model for globozoospermia in humans.

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.

Galnt3 belongs to the GalNAc transferase gene family involved in the initiation of mucin-type O-glycosylation. Male Galnt3-deficient (Galnt3 (-/-)) mice were infertile, as previously reported by Ichikawa et al. (2009). To investigate the involvement of Galnt3 in spermatogenesis, we examined the differentiation of germ cells in Galnt3 (-/-) mice. Galnt3 mRNA was most highly expressed in testis, and Galnt3 protein was localized in the cis-medial parts of the Golgi stacks of spermatocytes and spermatids in the seminiferous tubules. Spermatozoa in Galnt3 (-/-) mice were rare and immotile, and most of them had deformed round heads. They exhibited abnormal acrosome and disturbed mitochondria arrangement in the flagella. At the cap phase, proacrosomal vesicles of various sizes, which had not coalesced to form a single acrosomal vesicle, were attached to the nucleus in Galnt3 (-/-) mice. TUNEL-positive cells were increased in the seminiferous tubules. The binding of VVA lectin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), in the acrosomal regions of spermatids and spermatozoa in Galnt3 (-/-) mice was drastically reduced. Equatorin is a N, O-sialoglycoprotein localized in the acrosomal membrane and is suggested to be involved in sperm-egg interaction. Immunohistochemical and Western blot analyses showed a drastic reduction in the reactivity with MN9 antibody, which recognizes the O-glycosylated moiety of equatorin and inhibits sperm-egg interaction. These findings indicate that deficiency of Galnt3 results in a severe reduction of mucin-type O-glycans in spermatids and causes impaired acrosome formation, leading to oligoasthenoteratozoospermia, and suggest that Galnt3 may also be involved in the process of fertilization through the O-glycosylation of equatorin.

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1-/- mice. Male Tysnd1-/- mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1-/- mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates. © 2013 Mizuno et al.

During the last decade, evidence-based medicine has been described as a paradigm shift in clinical practice, and as "the conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients". Appropriate statistical methods for analyzing data are critical for the correct interpretation of the results in proof of the evidence. However, in the medical literature, these statistical methods are often incorrectly interpreted or misinterpreted, leading to serious methodological errors and misinterpretations. This review highlights several important aspects related to the design and statistical analysis for evidence-based reproductive medicine. First, we clarify the distinction between ratios, proportions, and rates, and then provide a definition of pregnancy rate. Second, we focus on a special type of bias called 'confounding bias', which occurs when a factor is associated with both the exposure and the disease but is not part of the causal pathway. Finally, we present concerns regarding misuse of statistical software or application of inappropriate statistical methods, especially in medical research. © Japan Society for Reproductive Medicine 2011.

Mammalian sperm flagella have filament-forming Tektin proteins (Tektin 1-5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S-EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the perk axonemal component and not directly associated with axonemal tubulins. Resistance to S-EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre-embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome-related events, such as the acrosome reaction or sperm-egg fusion. Mol. Reprod. Dev. 78: 611-620, 2011. (C) 2011 Wiley-Liss, Inc.

Fertilization occurs when the sperm penetrates the egg, resulting in the combination of paternal and maternal genomes for the propagation of generations. To perform the task, the mammalian sperm membrane system, constructed during spermatogenesis, undergoes biochemical and cytological modifications. In this review, the following three points are discussed: (i) the nature of the acrosomal membrane disclosed by various types of microscopy, including transmission electron microscopy and the recently developed high-resolution fluorescence microscopy, (ii) the nascent acrosomal membrane dysfunction during acrosome biogenesis and (iii) the modification of the sperm membrane during sperm-egg interaction.

Objective: To describe the delivery of a healthy female infant after intracytoplasmic sperm injection (ICSI) using pentoxifylline-activated sperm from a patient with Kartagener's syndrome. Design: Case report. Setting: Private assisted reproductive technology clinic in Japan. Patient(s): A couple with male factor infertility due to Kartagener's syndrome. Intervention(s): Intracytoplasmic sperm injection using ejaculated sperm activated by pentoxifylline. Main Outcome Measure(s): Semen characteristics, sperm ultrastructure, fertilization, pregnancy, and birth after ICSI. Result(s): The fertilization rate was 7 of 12 (58.3%), and the blastocyst formation rate was 4 of 7 (57.1%); all blastocysts were vitrified. After a single blastcyst transfer, a pregnancy ensued and progressed to term; a healthy female infant was delivered. Conclusion(s): With ejaculated sperm, which was activated by pentoxifylline, successful fertilization was accomplished by ICSI; thus, fertilization, vitrification, pregnancy, and delivery are attainable with sperm obtained from men with Kartagener's syndrome. (Fertil Steril (R) 2011; 95: 2431. e9-e11. (C) 2011 by American Society for Reproductive Medicine.)

P&gt;RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175-/-) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175-/- mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175-/- testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175-/- testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.

Background: Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. Results: Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. Conclusions: These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin.

Purpose: Zona pellucida (ZP)-free eggs are often used for studies such as evaluating the interaction of sperm-oolemma. To acquire ZP-free eggs, the most commonly used methods employ acidified Tyrode's solution, enzymatic digestion with a trypsin-like enzyme, or mechanical methods using micropipettes. However, acidified Tyrode's solution and trypsin-like enzymes often damage the oolemma, especially when many eggs are treated at once for mass sample analyses. The mechanical method requires skill, and it is time-consuming to prepare many ZP-free eggs. Therefore, in this study, to establish an easy, reliable method for preparing ZP-free eggs, we examined the ZP digestion method originally reported by Zuccotti et al. (J Reprod Fertil 93:515-520, 1991) that uses collagenase. Methods: Mouse unfertilized eggs were treated with collagenase and acidified Tyrode's solution to compare the ZP-free rates, the effect on the oolemma, and the two-cell development rates of ZP-free eggs by in vitro fertilization. The effects on the oolemma were gauged by observing the polarity of the transmembrane protein localization of enhanced green fluorescence protein tagged CD9 protein (CD9-EGFP) and using differential interference contrast microscopy. Results: Collagenase removed the ZP and the cumulus cells from the cumulus oocyte complex. The collagenase method had no influence on the localization of CD9-EGFP, resulting in a high two-cell development rate. Additionally, the collagenase method could exclude low quality eggs with hardened ZP, since collagenase could not digest the hardened ZP. Conclusions: The one-step collagenase method is an easy preparation method for large numbers of high-quality ZP-free eggs. © 2011 Japan Society for Reproductive Medicine.

Recently we reported that an oocyte activation ability in human and mouse sperm is associated with head flatness or the presence of perinuclear theca (PT) substance, MN13, which is an oocyte activation-related protein localized on the post-acrosomal sheath (PAS). As such, we hypothesize that the appearance of oocyte activation ability is stage-specifically regulated and depends on the formation of the acrosome or PAS/PT in spermatids. We monitored the appearance and movement of MN13 as a PT-specific molecule during spermatogenesis and analysed how the MN13 localization is affected in mouse and human globozoospermic acrosomeless sperm. MN13 was first detected on the surface of acrosomic vesicles, i.e. on the nascent outer acrosomal membrane of step 5-6 round spermatids (Sb1 spermatids in human), and it was then translocated via the outer acrosomal membrane surface to the most distal region of the acrosome in step 7 round spermatids (Sb2 spermatids). As spermatids elongated, MN13 was translocated via the cytoplasmic space between the nuclear envelope and the overlying plasma membrane towards the post-acrosomal region, and it was organized on the top of the nascent PAS that was typically found in step 14 elongated spermatids (Sd1 spermatids). In contrast, MN13 was not found in any GOPC-deficient spermatids, which completely lack the acrosome but have manchettes (microtubule bundles), nor in mouse and human acrosomeless sperm. The MN13 appearance or the MN13-related PAS/PT formation is highly dependant on acrosome formation; the MN13-related oocyte activation factor/ability is stage-specifically acquired in elongating/elongated spermatids.

High-resolution microscopy has been used to investigate the mechanism of the migration of cytoplasmic droplets during epididymal maturation of guinea pig spermatozoa. On testicular spermatozoa, droplets are located at the neck and, after passage through the middle cauda epididymidis, migrate only as far as the center of the midpiece. Initially, the space between the plasma membrane and outer mitochondrial membranes outside the droplet is 30.8 +/- 11.0 nm, whereas on mature spermatozoa, it significantly (P &lt; 0.01) narrows to a more consistent 15.9 +/- 1.3 nm. This is accompanied by the appearance of thin filaments cross-linking the two membranes above and below the droplet. Changes also occur in the arrangement of intramembranous particles (IMPs) in the plasma membrane overlying the midpiece. At the spermatid stage, linear arrays of IMPs are absent but appear on immature spermatozoa, where they are short with an irregular orientation, in the epididymis. On mature spermatozoa, numerous parallel linear arrays are present at the region where the plasma membrane adheres to the mitochondria. The membrane adhesion process can thus be observed two-dimensionally. The initial migration of the droplet from the neck is probably attributable to diffusion, with the formation of cross-linking filaments between the two membranes in the proximal midpiece preventing any backward flow and squeezing the droplet distally until it is arrested at the central midpiece by the filaments formed in the distal midpiece. The filaments might also stabilize the flagellum against hypo-osmotic stress encountered during ejaculation and within the female tract.

A tetraspanin family protein, CD9, has not previously been identified in sperm cells. Here, we characterize sperm CD9 in the mouse, including its unique localization in sperm, appearance during spermatogenesis, and behavior and fate during mouse fertilization. In sperm, CD9 is an inner acrosomal membrane-associated protein, not a plasma membrane-associated protein. Its molecular weight is approximately 24 kDa throughout its processing, from testicular germ cells to acrosome-reacted sperm. A temporal difference was found between mRNA and protein expression; CD9 mRNA was detected in the stages from spermatogonia through round spermatids showing the strongest levels in midpachytene spermatocytes. CD9 protein was detected in the cytoplasm throughout the stages from spermatogonia to spermatocytes. While CD9 was weakly expressed in the spermatids from step 1 through step 14, the signals became clearly positive at the marginal region of the anterior acrosome in elongated spermatids. After the acrosome reaction, the majority of sperm CD9 was retained in the inner acrosomal membrane, but some quantity of CD9 was found on the plasma membrane covering the equatorial segment as detected by immunogold electron microscopy using anti-CD9 antibody. CD9 was maintained on the sperm head after reaching the perivitelline space of CD9-deficient eggs that were recovered after natural mating with wild males. Thus, this study characterizes CD9 in sperm development and fertilization.

To elucidate the molecular mechanisms involved in sperm maturation during epididymal transit, we intended to isolate secretory molecules that are region-specifically expressed along the epididymis and secreted into the lumen of epididymal ducts. By using differential display screening and DNA sequence analyses, we isolated a rat bactericidal/permeability-increasing protein (BPI) possessing a signal sequence at its N-terminal, which was expressed in the caput region of epididymis, but not in the caudal region. Reverse transcription polymerase chain reaction analysis and in situ hybridization showed that rat BPI messenger RNA (mRNA) was highly expressed in caput epididymal epithelium and that its expression level was developmentally up-regulated. Confocal laser scanning microscopy with the anti-BPI antibody revealed that in both rats and mice, BPI protein was detected on granulelike structures in the lumen of both caput and cauda epididymal ducts, as well as at the sperm surface covering the acrosome region in spermatozoa freshly isolated from epididymis. Acrosome reaction induced by calcium ionophore A23187 in vitro brought about the disappearance of BPI on mouse spermatozoa. These data suggested that BPI, which is synthesized in caput epididymis and secreted into the lumen, is associated with not only the granulelike structures, but also the sperm surface covering the acrosome region, and that BPI bound to the acrosome region is extinguished by acrosome reaction. Possibly BPI bound to the sperm surface covering the acrosome region in rodent spermatozoa is involved in sperm maturation or fertilization.

It is important to establish a reliable and progressive model of the acrosome reaction. Here, we present a progression model of the acrosome reaction centering around the acrosomal membrane-anchored protein equatorin (MN9), comparing the staining pattern traced by MN9 antibody immunofluorescence with that traced by Arachis hypogaea agglutinin (PNA)-FITC. Prior to the acrosome reaction, equatorin was present in both the anterior acrosome and the equatorial segment. Since sperm on zona pellucida showed various staining patterns, MN9-immunostaining patterns were classified into four stages: initial, early, advanced, and final. As the acrosome reaction progressed from the initial to the early stage, equatorin spread from the peripheral region of the anterior acrosome toward the center of the equatorial segment, gradually over the entire region of the equatorial segment during the advanced stage, and finally uniformly at the equatorial segment at the final stage. In contrast, the PNA-FITC signals spread more quickly from the peripheral region of the acrosome toward the entire equatorial segment, while decreasing in staining intensity, and finally became weak at the final stage. MN9-immunogold electron microscopy showed equatorin on the hybrid vesicles surrounded by amorphous substances at advanced stage of acrosome reaction. Equatorin decreased in molecular mass from 40-60 to 35 kDa, and the signal intensity of 35 kDa equatorin increased as the acrosome reaction progressed. Thus, the established equatorin-based progression model will be useful for analyzing not only the behavior of equatorin but also of other molecules of interest involved in the acrosome reaction. Reproduction (2010) 139 533-544

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. in addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.

Recent studies indicate that round-headed sperm cannot activate oocytes and lack the postacrosomal sheath (PAS) or perinuclear theca (PT), although normal flat-headed sperm can activate oocytes and do have PAS (PT). In this study, we investigated how oocyte activation ability correlates with sperm head morphology (round and flat) and the presence of PT, by studying MN13, a representative molecule of the PT. We analyzed sperm with flat and round heads from infertile patients with globozoospermia (n = 1) and teratozoospermia (n = 1), and also from GOPC(-/-) mice, an animal model of human globozoospermia. Differential interference contrast image analysis, immunocytochemistry with MN13 antibody, transmission electron microscopy and an oocyte activation assay (assessing pronucleus formation) with ICSI were used. Flat-headed (control) sperm from both a healthy fertile volunteer man and wild-type mice had MN13 and PAS (PT). Flat-headed sperm (&lt; 5% of the population) from GOPC(-/-) mice also had both MN13 and PAS (PT), and they showed high oocyte activation ability. In contrast, round-headed sperm from a globozoospermia patient (100%) and GOPC(-/-) mice (&gt; 95% of the population) had neither MN13, nor PAS (PT), nor oocyte activation ability. Oocyte activation was higher in flat- versus round-headed sperm from GOPC(-/-) mice (P &lt; 0.05). Oocyte activation ability may be related to sperm head flatness and presence of MN13 and PAS (PT) in human and mouse sperm. This information is a first step towards the possibility of selecting good-quality sperm with high oocyte activation ability for ICSI.

Mammalian mothers undergoing embryo implantation must specifically recognize the developing embryo in a species-restricted manner. We previously observed that immune cells derived from early pregnant mice could promote endometrial differentiation and embryo implantation in blastocyst-transferred pseudopregnant mice. Although the precise mechanism remains unknown, it is suggested that the maternal immune system undergoes functional changes after recognizing developing embryos from the very early stages of pregnancy. Since it is physically impossible for immune cells to directly interact with the developing embryo while it is surrounded by the zona pellucida (ZP), it is speculated that the embryo produces certain embryo- and species-specific soluble factor(s) in the oviduct before hatching. As a candidate for this factor, we have paid attention to the ZP that is normally protected from immunological attack during oogenesis in the ovarian follicle. ZP-specific glycoproteins are known to play important roles in the species- and oocyte-specific binding of sperm, and the ZP can also be considered an abundant store of oocyte- and species-specific glycoproteins. In contrast to unfertilized oocytes, developing embryos may degrade the ZP starting just after fertilization and proceeding until hatching using enzymes that are released from cortical granules or produced by the developing embryo. Accordingly, the developing embryo might provide ZP-degradation products including oligosaccharide chains to the immune system from the very early stages. Taken together, we propose here a novel hypothesis that these ZP-derivatives can act as an intrinsic signal from the developing embryo for maternal recognition by the immune system. (C) 2009 Elsevier Ireland Ltd. All rights reserved.