Cleavage of SPACA1 regulates assembly of sperm-egg membrane fusion machinery in mature spermatozoa†.

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.

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

Lysophosphatidic acid acyltransferase 3 tunes the membrane status of germ cells by incorporating docosahexaenoic acid during spermatogenesis.

Docosahexaenoic acid (DHA) is one of the essential ω-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 LPAAT3-KO 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.

Deletion of Eqtn in mice reduces male fertility and sperm-egg adhesion.

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn - / - ) mouse model and found that the EQTN-KO males have reduced fertility and sperm-egg adhesion, while the EQTN-KO females are fertile. Eqtn - / - sperm were normal in morphology and motility. Eqtn - / - -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn - / - with Eqtn +/+ -Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn - / - males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn - / - sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn - / - sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 - / - ) was more severe compared with that of Eqtn - / - males. Eqtn - / - -Tg (Eqtn) males, which were generated to rescue Eqtn - / - males, restored the reduced fertility.

A mutation of COX6A1 causes a recessive axonal or mixed form of Charcot-Marie-Tooth disease.

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_247-6delCACTC) 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.

Suppression of T Cell Autophagy Results in Decreased Viability and Function of T Cells Through Accelerated Apoptosis in a Murine Sepsis Model.

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.

Subcellular localization of MS4A13 isoform 2 in mouse spermatozoa.

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 Ms4a13-2, was differentially regulated. Ms4a13-2, but not Ms4a13-1, was shown to be primarily expressed in mouse testes in an age-dependent 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.

A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility.

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-acetylgalactosaminyltransferase-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.

Tysnd1 deficiency in mice interferes with the peroxisomal localization of PTS2 enzymes, causing lipid metabolic abnormalities and male infertility.

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 ß-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.

Oligo-astheno-teratozoospermia in mice lacking ORP4, a sterol-binding protein in the OSBP-related protein family.

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.

Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia.

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.

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy.

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.

Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

The association of ODF4 with AK1 and AK2 in mice is essential for fertility through its contribution to flagellar shape.

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.

Usefulness of statistics for establishing evidence-based reproductive medicine.

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.

Decrease of lysyl hydroxylase 2 activity causes abnormal collagen molecular phenotypes, defective mineralization and compromised mechanical properties of bone.

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.

Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage.

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.

GalNAcß1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with ß-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcß-nLc4Cer² sequence.

Characterization and subcellular localization of Tektin 3 in rat spermatozoa.

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 peri-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.

Dynamics of the mammalian sperm membrane modification leading to fertilization: a cytological study.

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.