年森 清隆

Aim: l-Carnitine, an essential cofactor for mitochondrial, β-oxidation of long-chain fatty acids, is known to play important roles in sperm maturation and metabolism when spermatozoa pass and acquire motility in the epididymis. We reported that obstructive azoospermia occurred in the epididymis in the juvenile visceral steatosis (JVS) mice, which are OCTN2 dysfunction mice caused by mutations in the gene encoding OCTN2, have been used for animal models of primary systemic carnitine deficiency. The aim of present study is to investigate the expression of OCTN2 protein in the mouse epididymis and its relation between the localization of OCTN2 and obstructive azoospermia in JVS mice as animal models for human male infertility. Methods: Animals used in this study were wild-type (C57BL/6 J) mice (n = 4) and JVS mice (n = 4). We made a specific polyclonal antibody against OCTN2 and examined immunohistochemically the localization of OCTN2 in the mouse epididymis. Results: OCTN2 was localized on the apical membrane of the principal cells of distal corpus and cauda epididymides. Immunocytochemistry demonstrated that OCTN2 was localized on the surface of microvillus upon the principal cells. In JVS mice, immunoreactivity started in a region immediately distal to where the sperm obstruction occurred. Conclusions: Our results suggest that OCTN2 functions as a carnitine transporter between the epithelium and the lumen in distal corpus and cauda epididymides and provides a clue as to why obstructive azoospermia is induced in distal parts of epididymis.

The immunoglobulin superfamily (IgSF) proteins are expressed on the plasma membrane between Sertoli cells and germ cells in the testis. IgSF proteins are specifically present at the apical Sertoli-germ cell junction, that is, ectoplasmic specialization and are involved in germ cell differentiation. Some IgSF proteins are present on the surface of germ cells and undergo further biochemical modifications during sperm maturation. These IgSF proteins undergo final modifications during capacitation and/or the acrosome reaction. The function and expression of IgSF proteins in the testis and spermatozoa, as they relate to spermatogenesis and sperm-egg interaction, are discussed. © 2006 The AuthorsJournal compilation © 2006 Japan Society for Reproductive Medicine.

Among the organic cation transporters, OCTN2 is identified as the most important carnitine transporter owing to the ability to transport carnitine. Although the OCTN2 is previously found in various tissues, there have been no reports showing the OCTN2 in the pancreas. In this study, we examined the expression and localization of OCTN2 in the mouse pancreas by the aid of an in situ hybridization technique and immunohistochemistry with anti-OCTN2 antibody. As a result, the OCTN2 expression was found in the A-cells for the first time. OCTN2 was not expressed in B-cells, notwithstanding that the metabolism of long-chain fatty acids, which are transported into the mitochondria with the help of carnitine, was expected for fatty acid-stimulated insulin secretion. Thus, this study suggests the possibility of carnitine uptake in the pancreatic A-cells through OCTN2 and implies the presence of carnitine transporter(s) other than OCTN2 in the B-cell. (c) 2005 Elsevier Ltd. All rights reserved.

We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by Martianov et al. (I. Martianov, S. Brancorsini, R. Catena, A. Gansmuller, N. Kotaja, M. Parvinen, P. Sassone-Corsi, and I. Davidson, Proc. Natl. Acad. Sci. USA 102:2808-2813, 2005) that reported similar results.

A conditionally immortalized epididymis caput cell line, MEPC5, was established by infecting primary cultured mouse epididymis caput cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and the cells grew continuously. However, the downregulation of T-antigen at a nonpermissive temperature of 39 degrees C and the upregulation of cell density at 33 degrees C were associated with growth arrest and the increased protein expression of p21(waf1), a cell cycle inhibitor. The cells expressed epididymal caput-expressed genes such as phosphatidylethanolamine binding protein, polyoma enhancer activator 3, ME1, sulfated glycoprotein-2 (SGP-2), androgen receptor, and retinoic acid receptor alpha. Interestingly, the expression levels of ME1 and SGP-2 were significantly elevated under the cell growth-restricted conditions. The established mouse epididymis caput epithelial cell line MEPC5 retains some characteristics of differentiated epididymis epithelial cells, and should prove an excellent model for studies of gene expression and the physiological functions of epididymis caput epithelial cells. (c) 2005 Elsevier Inc. All rights reserved.

We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1-13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1-13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis. gametogenesis, sperm maturation, spermatid, spermatogenesis, testis

The induction of null mutations by means of homologous recombination is a powerful technique for clarifying the biological activities of target genes. However, the problems of the genetic background and flanking genes should be borne in mind. Here we employed a breeding strategy to compare three lines of mice deficient in the basigin (Bsg)/CD147 gene. The first line was F2 from F1 hybrid offspring of the 129/SV chimera and C57BL/6J. The second one was from a C57BL/6J congenic line. Both lines showed high embryonic lethality, sterility, and blindness. The third one was 'reverse F2' from 'reverse F1' hybrid offspring of the C57BL/6J congenic line and 129/SV. Surprisingly, this line showed a normal birth rate, while sterility and blindness persisted. Our results clearly separate the effects of the induced null mutation from those of flanking genes and the genetic background, and provide a useful means of investigating the biological functions of Bsg. (C) 2004 Elsevier Inc. All rights reserved.

Cnot7 is a co-factor of transcription regulation, expressed in a variety of tissues including the lung, liver, thyroid gland, and testis. Our previous study (Nakamura et al., 2004) showed that deletion of the Cnot7 gene in mice caused almost no abnormal phenotypes except for male infertility, due to oligo-astheno-teratozoospermia. This study also showed that Cnot7-/- mouse germ cells transplanted as donors could colonize in recipient wild mouse testes to develop normal spermatogenesis by spermatogonial transplantation assay, suggesting that the abnormal spermatogenesis observed in the Cnot7-/- testes was induced by the impaired testicular microenvironment rather than a germ cell defect. In the present study, we have carried out reciprocal germ cell transplantation in which wild type germ cells were transplanted as donors into the recipient Cnot7-/- testes to evaluate the recipient microenvironment for supporting the spermatogenesis of donor cells. We noticed that donor cell colonization was less efficient in Cnot7-/- than in Cnot7-/-testes, and that the donor derived spermatids in the recipient Cnot7-/- testes showed severe deformities. These results support our previous report that Sertoli cell defects in the Cnot7-/- testes could induce oligo-astheno-teratozoospermia.

Deletion of the GOPC gene encoding mouse GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) causes infertile round-headed spermatozoa, which have acrosome-less round heads and deformed tails (Yao et al, 2002). This study investigated how GOPC deficient spermatids fail to assemble the peri-nuclear structures in round-headed spermatozoa during spermiogenesis in GOPC knockout mouse testes. In step 1-8 spermatids, Golgi-derived proacrosomal vesicles that are transported to the perinuclear region formed acrosome-like vesicles of various sizes, called pseudoacrosomes. The marginal ring of the acroplaxome, which is generally formed between the descending edge of a developing acrosome and nuclear envelope in a wild spermatid, was poorly formed between the pseudoacrosome and nuclear envelope. In step 9-11 elongating spermatids, a majority of pseudoacrosomes were detached from the nucleus and disappeared from the perinuclear region by spermiation. Concomitantly, several failures occurred on the nucleus, manchette, postacrosomal sheath (perinuclear theca), and posterior ring. Ectoplasmic specializations were poorly formed, and did not always associate with developing spermatids. Consequently, spermatid nuclear elongation to form round-headed spermatozoa developed was impaired. In addition to these sequential failures, the posterior ring deficiency was attributed to the tail deformation destined to occur during epididymal maturation as reported in an accompanying paper (Suzuki-Toyota et al., 2004 in this issue), its eventual phenotype being reminiscent of the round-headed spermatozoa of human infertile globozoospermia.

Male mice deleting the gene encoding GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) are infertile, showing globozoospermia with a coiled tail (Yao et al., 2002). We confirmed how and where tail anomalies were produced in spermatids and epididymal spermatozoa by light and electron microscopy. During spermiogenesis, tail formation occurred normally, but a defect was found at the posterior ring. Thereafter, remarkable sperm tail deformations were induced during epididymal passage. In the proximal caput epidiymidis, the tails remained normal and straight, but most of them coiled around the nucleus in the cauda epididymidis. Coiling is presumed to occur with the migration of the cytoplasmic droplet by the absence of the posterior ring. The connecting piece of the coiled tail was often dislocated or separated from the implantation fossa. Many mitochondria were separated from the outer dense fibers (ODFs) and formed a stratified mitochondrial sheath. Due to this, the distal part of the midpiece became bared of the mitochondrial sheath. The bared ODFs were often bent and disorganized. Tail deformities are attributed to weak or incomplete adhesion between the following structures: 1) plasma membrane and nuclear envelope at the posterior ring, 2) connecting piece and implantation fossa, and 3) mitochondria and ODFs. These defects result in a coiled tail, tail dislocation from the implantation fossa, and the stratified mitochondrial sheath accompanying bared ODFs in the midpiece, respectively. Thus the posterior ring is significant in preventing coiled tail formation. The GOPC-deficient spermatozoa provide a valuable model not only for head but also for tail anomalies.

Either a 20 or 200 μg/kg body weight/injection of bisphenol A (BPA) was subcutaneously administered to adult mice and rats for 6 days, and the effects on the testes were investigated by electron and light microscopy. Abnormalities were observed in the spermatids: acrosomal vesicles, acrosomal caps, acrosomes and nuclei of the spermatids were severely deformed. The ectoplasmic specialization between the Sertoli cell and spermatids were also affected: incomplete specialization, redundant ectopic specialization and aplasia were observed. Rats and mice responded similarly to BPA. There were no dose dependencies between the 20- and 200 μg/kg body weight/injection groups. The ectoplasmic specialization between adjoining Sertoli cells, or blood-testis barrier, was not affected. Since similar adverse effects were observed when adult mice were treated with β-estradiol 3-benzoate, the effects of BPA reported here seem to reflect the estrogenic effects on the testes. Animals kept for an additional two months after cessation of the administration were shown to be fertile and the testes showed normal histology, indicating that the adverse effects were transitory.

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(1). Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor(2), 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)(3). 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.

Hybrid breakdown is a type of reproductive failure that appears after the F2 generation of crosses between different species or subspecies. It is caused by incompatibility between interacting genes. Genetic analysis of hybrid breakdown, particularly in higher animals, has been hampered by its complex nature (i.e., it involves more than two genes, and the phenotype is recessive). We studied hybrid breakdown using a new consomic strain, C57BL/6J-XMSM, in which the X chromosome of C57BL/6J (derived mostly from Mus musculus domesticus) is substituted by the X chromosome of the MSM/Ms strain (M. m. molossinus). Males of this consomic strain are sterile, whereas F1 hybrids between C57BL/6J and MSM/Ms are completely fertile. The C57BL/6J-XMSM males showed reduced testis weight with variable defects in spermatogenesis and abnormal sperm head morphology. We conducted quantitative trait locus (QTL) analysis for these traits to map the X-linked genetic factors responsible for the sterility. This analysis successfully detected at least three distinct loci for the sperm head morphology and one for the testis weight. This study revealed that incompatibility of interactions of X-linked gene(s) with autosomal and/or Y-linked gene(s) causes the hybrid breakdown between the genetically distant C57BL/6J and MSM/Ms strains.

The formation and organization of a mammalian sperm head occurs through diverse cellular and molecular processes during spermiogenesis. Such cellular events include sequential changes in the nucleus and the acrosome-which is derived from the Golgi apparatus-in concert with prominent bundles of microtubules, the manchette. However, these complex processes are readily impaired by a variety of intrinsic and extrinsic factors, eventually causing various types of male infertility-such as teratozoospermia-which include the deformation of the acrosome and nucleus. In order to comprehend such idiopathic male infertility syndromes, it is important to clarify the mechanism involved in sperm head formation and organization. In addition to the manchette, two key structures in these events are the acroplaxome and the perinuclear theca. The acroplaxome forms the acrosome plate with periodic intermediate filament bundles of the marginal ring at the leading edge of the acrosome, and its nature has recently been characterized. The perinuclear theca, which is located in the perinuclear region in the sperm head, contains not only a cytoskeletal element to maintain the shape of the sperm head but also functional molecules leading to oocyte activation during fertilization. This review discusses recent developments regarding the formation and organization of the mammalian sperm head in relation to its relevant functions.

配偶子の形成から成熟に至るまでの過程は長い時間を要するが,受精過程は極めて迅速に進行し,初期発生につながる。その過程は形態学的,生化学的そして生理学的に興味ある現象を含む。歴史的には形態解析が先行し,近年,特異的なプローブを用いた分子細胞生物学的手法によって多くの関連分子が発見され,関連遺伝子も次第に判明してきている。臨床的には1992年以来,精子を直接卵子内に注入する補助生殖医療も行われている。しかし,不妊カップルが新生児を得るにはまだ問題も多い。

The mammalian acrosome is a highly specialized organelle overlying the anterior part of the sperm nucleus. and contains a variety of proteins, including hydrolytic enzymes and matrix molecules. Functionally, the anterior acrosome is involved in the acrosome reaction or sperm-zona pellucida interaction, while the equatorial segment (posterior acrosome) is involved in sperm-egg fusion. The acrosome is formed during spermiogenesis, during which, associated molecules are transported from the Golgi apparatus and organized. Many of the molecules thus arranged gradually become compartmentalized during sperm. passage through the epididymis. Some of them are further modified during the fertilization process. The findings indicate that acrosomal molecules are not only restricted to a specific. region (domain) of the acrosome but also undergo ongoing relocation in a stage-specific manner during sperm maturation in the testis and epididymis. Such maturation-associated modifications are considered essential for sperm molecules to reach the correct or final site before fertilization. This review focuses on the organization and modifications of the acrosomal molecules as well as their compartmentalization within the acrosome. (C) 2003 Wiley-Liss, Inc.

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece,region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the L-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida. Published 2003 Wiley-Liss, Inc.

Monoclonal antibody (mAb) MN13 labels mouse sperm head postacrosomal perinuclear theca (PT), which is possibly involved in oocyte activation during fertilization. The antigenic site is expressed after mild sonication followed by treatment with dithiothreitol (DTT) or heat (45degreesC), and is visible as a thick band in the postacrosomal region. The presence of protease inhibitors in the sonication medium suppresses the exposure of MN13 epitope (MN13p), suggesting the involvement of a proteolytic reaction in this process. Spermatozoa do not express MN13p after the induction of acrosome exocytosis by Ca2+ ionophore, zona binding, or during zona penetration, a strategy that ensures safe delivery of postacrosomal PT proteins to oocytes after fusion. MN13 labeling was not detectable during fertilization by zona-free in vitro fertilization, suggesting that the antigenic site does not react with proteolytic enzymes during sperm-oocyte fusion and the antibody does not recognize the nascent epitope. Microinjection of sperm heads prepared by sonication and DTT treatment led to the activation of metaphase II oocytes. The oocyte activating function of such sperm heads was significantly diminished after labeling with MN13 prior to intracytoplasmic sperm injection (ICSI), but labeling with antiequatorin antibody MN9 activated oocytes with a frequency similar to that of unlabeled sperm heads. The sperm heads in inactive oocytes formed premature chromosome condensations (PCCs), which were invested by independent metaphase-like spindles. These observations indicate that the postacrosomal PT recognized by mAb MN13 is involved in oocyte activation. MN13p is dissociated from sperm heads during the early stages of decondensation after ICSI. In activated oocytes, MN13-labeled fine granules were redistributed in the midzone spindle region, whereas in inactive oocytes they formed a ring around the polar regions of the metaphase II and PCC spindles.

A mouse homologue of the Drosophila melanogaster germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2beta (lamina-associated polypeptide 20). To elucidate the role of mgcl-1, we analyzed two mutant mouse lines that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that were probably due to impaired nuclear envelope integrity were observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities were observed in testis in which the highest expression of mgcl-1 was detected. Fertility was significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations showed insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, were aberrant. Considering that the first abnormality during the process of spermatogenesis was abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling.

Spergen-1, a recently identified molecule specifically expressed in haploid spermatids in testis, is a small protein of 154 amino acids with a mitochondria-targeting signal at the N terminus. To examine the localization of spergen-1 protein in germ cells, we performed immunocytochemistry with the anti-spergen-1 antibody on frozen sections of rat testis and purified spermatozoa. Immunolabeling for spergen-1 was detected in mitochondria of elongating spermatids and of the middle pieces of matured spermatozoa. Immunoelectron microscopy revealed that spergen-1 was localized to the surface of mitochondria in the middle piece of spermatozoa. To investigate the properties of spergen-1, COS-7 cells were transfected with vectors encoding various spergen-1 mutants. The transfection experiments showed that spergen-1 expressed in the cells tended to agglutinate mitochondria and assemble them into aggregations and that the C-terminal region of spergen-1 as well as the N-terminal mitochondrial targeting signal was requisite for induction of mitochondrial aggregation. These results suggest that spergen-1, a mitochondria-associated molecule in spermatozoa, has a property to induce mitochondrial aggregation at least in cultured cells. We hypothesize that spergen-1 might function as an adhesive molecule to assemble mitochondria into the mitochondrial sheath around the outer dense fibers during spermiogenesis. (C) 2002 Elsevier Science (USA).

The acrosome is a unique organelle that plays an important role at the site of sperm-zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia.