年森 清隆

ADP ribosylation factors (ARFs) of small GTPase are molecular switches regulating various membrane dynamics. Among them, ARF6 has recently been highlighted because of its function to facilitate the interaction between the cytoskeleton and the plasma membrane. Each ARFs has its preferable or even specific guanine nucleotide exchange factors (GEFs) as its activators. According to our previous RT-PCR analysis, EFA6A, a guanine nucleotide exchange factor for ARF6, was restrictedly expressed in the brain, retina and testis. Different from previous studies on neurons, EFA6A, a guanine nucleotide exchange factor for ARF6, was first shown to be localized intensely in nuclei of spermatocytes of adult mouse testes in the present immunohistochemical study. This suggests a possible involvement of EFA6A-ARF6 signaling in the karyokinesis and cytokinesis.

Fluctuations in the intracellular calcium concentration ([Ca+2 i]) are a prerequisite for egg activation. The initial increase in [Ca+2i]is mainly due to Ca2+ release from the ER into the cytoplasm. The Ca2+ wave begins at the site of sperm-egg fusion, and this signal is then propagated throughout the cytoplasm. The repetitive increase in [Ca+2i] is termed "Ca2+ oscillation" and is maintained up to the time of pronucleus formation (Jones et al. 1995 Marangos et al. 2003). Two egg-activation pathways have been proposed (Fig. 9.1). The first pathway is via the sperm (cytoplasmic) factor in this case, the factor(s) can be directly inserted into the ooplasm. Among these factors, sperm-derived PLCzeta (ζ) is currently a candidate molecule. The other pathway is via the sperm receptor in this case, the receptor is expected to be present on the oolemma. © 2009 Springer-Verlag Berlin Heidelberg.

The differences in the behavior of the sperm-head components during sperm entry into the oocyte in the case of normal (natural) fertilization, IVF, ICSI, and round spermatid injection (ROSI) are listed in Table 10.1. © 2009 Springer-Verlag Berlin Heidelberg.

Proteins in the sperm plasma membrane are modified when the spermatozoa pass through the extracellular matrix surrounding the cumulus cells. Thus far, I have investigated the IgSF proteins basigin, CE9, and MC31 in this regard (Fig. 8.1). Mouse basigin (expressed in mice with the Okblood group) (Igakura et al. 1998 Kuno et al. 1998 Miyauchi et al. 1990) is homologous to the human extracellular matrix metalloproteinase inducer (EMMPRIN), HT-7, CD147, 5A11, M6 (leukocye- activation antigen), 5F7, tumor cell-derived collagenase factor (TCSF), gp42, neurothelin, and the MRC OX-47 T-cell-activation antigen. The rat homolog ofbasigin is CE9 (de la Luna et al. 1999 Petruszak et al. 1991), which is identical to MC31 (Toshimori et al. 1992a Wakayama et al. 2000). Mouse bsg is localized at locus 42.2 on chromosome 10, and human EMMPRIN is localized on chromosome 19p13.3. Molecular structure of BSG and CE9/MC31: Basigin and CE9 (MC31) are predicted to be single-pass type-1 glycoproteins that comprise 269 amino acids and contain three potential N-glycosylation sites. Basigin is composed of 269 amino acids, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analyses have revealed its molecular size to range from 35 to 60 kDa. The extracellular domain is composed of 187 amino acids and contains two Ig-related domains. The basigin-encoding gene BSG contains seven exons with a total length of 1.8 kb. Its molecular structure, as predicted on the basis of genetic analysis, is shown in Fig. 8.1. © 2009 Springer-Verlag Berlin Heidelberg.

The perinuclear theca contains many molecules such as MN13 (Manandhar and Toshimori 2003 Toshimori et al. 1991), PERF15 (Oko and Morales 1994), multiple-binding proteins (capping proteins α3 and β3) (von Bülow et al. 1997), actin-related protein (Arp)-T1 and Arp-T2 (Heid et al. 2002), the calmodulin-binding peptide (CaMBP) (Lécuyer et al. 2000), calicin (expressed in the postacrosomal calyx) (Leclerc and Goupil 2000), phospholipase C (PLC) zeta (Saunders et al. 2002 Yoda et al. 2004), SubH2B (Aul and Oko 2002), cysteine-rich PT1 (CYPT1) (Hansen et al. 2006), and postacrosomal sheath WW domain-binding protein (PAWP) (Wu et al. 2007). Ultrastructural observations reveal that electron-dense materials gradually accumulate in the nascent PAR of elongating spermatids as precursor substances for the biogenesis of the perinuclear theca (Bellvé et al. 1992 Kann et al. 1991 Longo and Cook 1991). This accumulation occurs actively in late elongating spermatids and gradually results in the formation of the paracrystalline sheet or the PAS. Interestingly, the cytoskeletal components of the paracrystalline sheet and PAS are not formed either in the round-headed spermatozoa produced by GOPC-/- mutant mice (Ito et al. 2004) or in the spermatozoa produced by humans with globozoospermia (Escalier 1990). These lines of evidence suggest that substances in the perinuclear theca regulate the shaping of the sperm head by keeping the developing nucleus bound to the acrosome and plasma membrane. Similar findings have been reported previously (Oko and Maravei 1995 Oko and Morales 1994). © 2009 Springer-Verlag Berlin Heidelberg.

Sperm DNA and the associated nuclear matrix are completely enclosed within the nuclear envelope and are not directly exposed to the cytoplasm. In contrast, the maternal genome is not enclosed within a nuclear envelope and is directly exposed to the cytoplasm. Despite being protected by a rigid enclosure, sperm chromatin DNA strands are likely to be damaged during the long process of spermatogenesis, in which apoptosis also causes DNA fragmentation. Further, sperm DNA fragmentation is also induced during the long journey of the sperm to reach the oocyte (Aitken and Krausz 2001). Such DNA fragmentation may lead to poorfertilization and embryonic development, reduced rates of implantation and successful pregnancy, and recurrent pregnancy losses (Chohan et al. 2006). During spermatogenesis, the sperm chromatin is essentially remodeled, and it condenses. The genomic DNA is transformed from a histone-associated nucleosome to a protamine-rich toroid (doughnut-like) structure (Fig. 7.1). Although these doughnut-like structures are highly resistant to external agents such as environmental toxins, genetic mutagens, oxidative stress, and smoking, the nuclear DNA is still considerably susceptible to oxidative stress (Aitken and Baker 2004 Bennetts and Aitken 2005 Lewis and Aitken 2005). Chromatin modification during the epididymal maturation of the sperm is important to ensure that the nucleus (genome) develops resistance to such external agents. During the entry of the sperm into the oocyte, the nuclear envelope and nuclear matrix proteins are gradually degraded by the action of the reducing agent glutathione, which is abundant in the ooplasm. It is interesting for researchers to evaluate the status of sperm DNA and to investigate the factors that induce DNA damage. In this regard, I describe the basic findings on the sperm nucleus and then proceed to discuss recently developed techniques for evaluating the degree of DNA damage in sperm. © 2009 Springer-Verlag Berlin Heidelberg.

The acrosome has several structural subdomains in which specific molecules are organized, depending on the species. Acrosomes are dynamically modified and mature during the life of the sperm. The events that bring about structural and biochemical modifications in the acrosomal membrane and matrix proteins are collectively known as the "post-testicular maturation of the acrosome." Here, I focus on this topic. © 2009 Springer-Verlag Berlin Heidelberg.

Various types of sperm-head anomalies can be observed in patients with teratozoospermia or oligozoospermia. The presence of such abnormalities is one of the primary causes of infertility in males. Here, I discuss these abnormalities in relation to spermatogenesis. Prior to their entry into the first meiosis, germ-line stem cells proliferate at the spermatogonial stage, undergoing self-renewal as type A spermatogonia (A1-A3 in the rat). Type A spermatogonia differentiate into type B spermatogonia, and these in turn differentiate into primary spermatocytes that subsequently undergo meiosis (Figs. 3.1 and 3.2). Prior to the first meiotic division, primary spermatocytes duplicate their DNA/chromosomes. A typical feature of the first meiosis is genetic recombination at this stage, a synaptonemal complex containing recombination nodules is formed in the pachytene-phase spermatocytes. Immediately after meiosis (within approximately 8 h in the case of humans), the secondary spermatocytes immediately develop into haploid spermatids. Thus, almost all of the molecules/proteins required for sperm-head formation are presumed to be prepared at the gene level in primary spermatocytes. The gene expression of spermatids is referred to as postmeiotic gene expression. These steps are followed by four major events of spermiogenesis: nuclear modification, acrosome biogenesis, cytoplasmic trimming, and flagellum formation. The first three events are involved in head formation. Sperm-head formation is triggered by the initiation of acrosome biogenesis during spermiogenesis. Head formation involves acrosome biogenesis, nuclear condensation and elongation (flattening), and the formation of cytoplasmic layers. Cytoplasmic molecules/substances gradually accumulate to form the perinuclear theca. Some of these essential molecules are transported along manchettes (a manchette is an organized microtubular structure that girdles the posterior region of the spermatid nucleus) and are organized into the various structural components. This organization occurs most actively in elongating spermatids. In particular, drastic changes occur from the mid-stage to the final step of spermiogenesis. The cytoplasm released by the germ cells is phagocytosed by the Sertoli cells before spermiation. Thus, germ cells produce proteins essential for fertilization, and these proteins are organized into the head and tail structures or their components in a time-sensitive manner. © 2009 Springer-Verlag Berlin Heidelberg.

Spermatozoa perform the function of carrying the paternal genome to the resting oocyte or the maternal genome. The activation of the resting oocyte is followed by fertilization, which leads to the embryonic development of the new individual (next generation). The factors involved in egg activation are expressed on the sperm head. The mammalian sperm head is formed by a highly programmed process and is further modified morphologically and functionally in the male and female reproductive tracts, after spermiation. These modifications are imperative for successful fusion between the sperm and the oocyte at the time of fertilization (i.e., when the genomes meet). Functionally primed mature spermatozoa that have undergone the necessary modifications are able to safely carry the paternal genome to the maternal genome. Each process involves dynamic modifications for the maturation of the sperm components, and these processes are governed by a large number of molecules. Genes control the expression of these molecules, and this expression is affected by both intrinsic and extrinsic factors. The paternal genome is constantly exposed to harmful environments, since spermatozoa are required to travel through sequentially changing microenvironments until the moment of gamete membrane fusion. To overcome obstacles presented by these changing environments, the sperm head and tail develop elaborate structures consisting of functional proteins, carbohydrates and lipids during spermatogenesis, and these structures are continuously modified and mature until fertilization/ embryonic development. Over the past two decades, powerful genetic, biophysical, biochemical, and molecular biology tools have been developed and have substantially improved our understanding of sperm biology. Since numerous molecules and genes involved in these processes have been identified thus far and are still under investigation, they cannot all be cited in this book. In the future, many studies undertaken worldwide will discover new molecules and features and will provide many invaluable insights into sperm biology, from spermatogenesis to egg activation. The progress of research in this manner will be useful for practical application in mammals, including humans, especially for clinical patients with infertility. © 2009 Springer-Verlag Berlin Heidelberg.

In general, post-testicular maturation events occurring in sperm are controlled by factors expressed in the luminal fluid within the epididymis. This indicates that the sperm interacts with the ductal epithelial cells and that these interactions are continuously maintained throughout the passage of the sperm along the male and female reproductive tracts until the time of fertilization. The sperm maturation-related molecules secreted in the epididymis have recently been designated as epididymosomes (Sullivan et al. 2007). The direct and indirect interactions between sperm and the epithelial cells help to protect and stabilize the sperm components, and they suppress early activation of sperm motility and membrane fusion. These interactions facilitate the functional maturation of the sperm, leading to capacitation and the acrosome reaction. Functional impairment of the epithelial cells lining the male and female reproductive tracts causes obstruction of the epididymal passage and restricts the migration ability of the sperm this has been demonstrated in cases of failed expression of the carnitine transporter (OCTN2) gene (Toshimori et al. 1999). © 2009 Springer-Verlag Berlin Heidelberg.

In mammals, spermatozoa complete meiosis before spermiation, while oocytes do not complete meiosis before ovulation. A mature spermatozoon weighs only picograms, and its head length is approximately 5 μm. On the other hand, a mature oocyte weighs 20-40 ng and is approximately 100 μm in width. Since spermatozoa have to cover a large distance to reach the oocyte, the sperm nucleus (containing the genome) must remain in a protected and safe condition in fact, it is thoroughly embedded in the nuclear matrix. Approximately 2.5 × 107 of such structurally complex spermatozoa are produced daily in the human testes in comparison, only a few oocytes (generally 1 cell per menstrual cycle in humans and about 10 per estrous cycle in rodents) develop in the ovaries and are ovulated into the fallopian tube. © 2009 Springer-Verlag Berlin Heidelberg.

Forkhead transcription factors are characterized by a winged helix DNA binding domain, and the members of this family are classified into 20 subclasses by phylogenetic analyses. Fkhl18 is structurally unique, and is classified into FoxS subfamily. We found Fkhl18 expression in periendothelial cells of the developing mouse fetal testis. In an attempt to clarify its function, we generated mice with Fkhl18 gene disruption. Although KO mice developed normally and were fertile in both sexes, we frequently noticed unusual blood accumulation in the fetal testis. Electron microscopic analysis demonstrated frequent gaps, measuring 100-400 nm, in endothelial cells of blood vessels. These gaps probably represented ectopic apoptosis of testicular periendothelial cells, identified by caspase-3 expression, in KO fetuses. No apoptosis of endothelial cells was noted. Fkhl18 suppressed the transcriptional activity of FoxO3a and FoxO4. Considering that Fas ligand gene expression is activated by Foxs, the elevated activity of Foxs in the absence of Fkhl18 probably explains the marked apoptosis of periendothelial cells in Fkhl18 KO mice.

Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

Aim: To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia. Methods: In order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization. Results: The actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy. Conclusion: Nectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the pectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

Meichroacidin (MCA) is a highly hydrophilic protein that contains the membrane occupation and recognition nexus motif. MCA is expressed during the stages of spermatogenesis from pachytene spermatocytes to mature sperm development and is localized in the male meiotic metaphase chromosome and sperm flagellum. MCA sequences are highly conserved in Ciona intestinalis, Cyprinus carpio, and mammals. To investigate the physiological role of MCA, we generated MCA-disrupted mutant mice; homozygous MCA mutant males were infertile, but females were not. Sperm was rarely observed in the caput epididymidis of MCA mutant males. However, little to no difference was seen in testis mass between wild-type and mutant mice. During sperm morphogenesis, elongated spermatids had retarded flagellum formation and might increase phagocytosis by Sertoli cells. Immunohistochemical analysis revealed that MCA interacts with proteins located on the outer dense fibers of the flagellum. The testicular sperm of MCA mutant mice was capable of fertilizing eggs successfully via intracytoplasmic sperm injection and generated healthy progeny. Our results suggest that MCA is essential for sperm flagellum formation and the production of functional sperm.

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha 1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBP beta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.

Human sperm has functional domains on the head and tail (flagellum). Since functional molecules are organized into the substructures of these domains, sperm with good appearance are expected to be fertile. Thus, it is of interest to see how sperm fine morphologies relate to fertility. The sperm head is divided into the acrosome and postacrosome regions. The acrosome region is further divided into the anterior acrosome for the acrosome reaction and posterior acrosome (equatorial segment) for gamete membrane fusion. The postacrosome region is involved in egg activation. In addition, the human sperm head has varieties of nuclear vacuoles. High-resolution light microscopy, which has recently been used in IMSI (intracytoplasmic morphologically selected sperm injection), is helpful in identifying vacuoles. However, IMSI is still insufficient for analysis of the contents of vacuoles electron microscopy and evaluation tests for sperm DNA damage are more helpful for profound analysis of the nature of nuclear vacuoles and DNA status, respectively. The neck region carries the paternal centrosome to the oocyte to evoke sperm aster, from which microtubules emanate. Organization failure of the midpiece and principal piece strongly affects motility. This paper discusses the relationship beween the normality/abnormality of sperm substructures and fertility and shows typical phenotypes found in gene knockout animal models. © 2008, JAPANESE SOCIETY OF OVA RESEARCH. All rights reserved.

The monoclonal antibody 6E2 raised against the embryonal carcinoma cell line NCR-G3 had been shown to also react with human germ cells. Thin-layer chromatography (TLC) immunostaining revealed that 6E2 specifically reacts with sialosylglobopentaosylceramide (sialylGb5), which carries an epitope of stage-specific embryonic antigen-4 (SSEA-4), known as an important cell surface marker of embryogenesis. The immunostaining of mouse preimplantation embryos without fixation showed that the binding of 6E2 caused the clustering and consequent accumulation of sialylGb5 at the interface between blastomeres. These results suggest that SSEA-4 actively moves on the cell surface and readily accumulates between blastomeres after binding of 6E2. (C) 2007 Elsevier Inc. All rights reserved.

Gopc (Golgi-associated PDZ- and coiled-coil motif-containing protein)(-'-) mice are infertile, showing globozoospermia, coiled tails, and a stratified mitochondrial sheath. Transmission electron microscope ITEM) images of the spermatozoa were studied quantitatively to analyze disorganization processes during epididymal passage. Factors maintaining straight tail and normal mitochondrial sheath were also studied by TEM and immunofluorescent microscopy. Sperm tails retained a normal appearance in the proximal caput epididymiclis. Tail disorganization started between the proximal and the middle caput epididymidis, and the latter is the major site for it. The tail moved up through the defective posterior ring and coiled around the nucleus to various degrees. Tail coiling occurred in the caput epididymidis suggesting it was triggered by cytoplasmic droplet migration. SPATA19/spergen-1, a candidate mitochondrial adhesion protein, remained on the stratified mitochondria, while GPX4/PHGPx, a major element of the mitochondrial capsule, was unevenly distributed on them. From these findings, we speculate GPX4 is necessary to maintain normal sheath structure, and SPATA19 prevents dispersal of mitochondria, resulting in a stratified mitochondrial sheath formation in Gopcl-spermatozoa. The epididymal epithelium was normal in structure and LRP8/apoER2 expression suggesting that tail abnormality is due to intrinsic sperm factors. Three cell structures are discussed as requisite factors for maintaining a straight tail during epididymal maturation: 1) a complete posterior ring to prevent invasion of the tail into the head compartment, 2) stable attachment of the connecting piece to the implantation fossa, and 3) a normal mitochondrial sheath supported by SPATA19 and supplied with sufficient and normally distributed GPX4.

Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) is a member of the Aatyk family of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several potential roles have been proposed for Brek and other Aatyk family members, the physiological functions of these kinases remain unclear. Here, we report that Brek(-/-) male mice are infertile, with azoospermia. Detailed histological analysis revealed that Brek(-/-) germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids. Testicular somatic cells appeared normal in these mice. Expression of Brek in testis was restricted to the germ cells, suggesting that the maturations of germ cells in Brek(-/-) mice are affected in a cell-autonomous manner. On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis. Further clarification of the mechanism by which Brek regulates spermatogenesis may help identify new targets for reproductive contraceptives and treatments against infertility.

Flutamide (FLUT) has potent anti-androgenic activity and is used in the medical field and in a screening test to detect endocrinologically active compounds. Our previous study demonstrated that FLUT induced histological deformation of spermatids and ultrastructural defects of the apical ectoplasmic specialization (ES) in the mouse testis. The apical ES is an actin-based junctional structure between the Sertoli cells and germ cells. Cortactin, an actin-binding protein, is found in the actin layer of ES. The protein level of cortactin was decreased in FLUT-treated testes as shown by Western blot analysis. The detailed analysis indicated that the protein level was drastically decreased in FLUT-treated seminiferous tubules of stages from VI to IX. Immunohistochemistry and immunoelectron microscopy showed that FLUT depressed cortactin expression in the apical ES. In addition, the effect of FLUT on cortactin localization appeared between 12 h and 8 days (about 180 h) after a one-day treatment. These results suggest that FLUT depressed the expression of cortactin in the apical ES with stage specificity. Therefore, the initial target of FLUT may be the cell-cell interactions between the Sertoli and germ cells. To our knowledge, this study is the first to document the decrease of cortactin expression in the abnormal apical ES following treatment with FLUT. (c) 2006 Elsevier Ltd. All rights reserved.

Our previous study revealed that the ectoplasmic specialization (ES) was deleted by the treatment of anti-estrogen, ICI 182.780 (ICI), and anti-androgen, flutamide (FLUT) in mouse testis. Also, expression of cortactin, an F-actin-binding protein, was decreased by the treatment of FLUT in mouse testis. Cortactin has been suggested to promote actin polymerizer at the ES in the testis, and also actin depolymerization is induced by tyrosine phosphorylation of cortactin. The present study revealed that exogenous treatment of ICI and FLUT caused the deletion of the cortactin in the apical ES and the increase of tyrosine phosphorylated cortactin in mouse testis. These results suggest that the sex hormone antagonists', ICI and FLUT, induced actin depolymerization and tyrosine phosphorylation of cortactin in the mouse testis. Also, the present study may be a key to elucidate the adverse affect exogenous compounds that affect spermiation. (c) 2006 Elsevier Inc. All rights reserved.

Previous reports have revealed that estrogen agonists or anti-androgenic chemicals induce abnormal spermiogenesis in rodents. In the seminiferous epithelium, the apical ectoplasmic specialization (ES) is an actin-based (cell-cell) junctional structure developing between the Sertoli cells and spermatids as is the basal ES also although it is located between adjoining Sertoli cells. In the apical and basal ES are several adhesion complex proteins that control the spermatid developing process. Cortactin, an actin-binding protein, is one of the ES adhesion proteins, combining with several cell-cell adhesions associating proteins. In the present study, 17 beta-estradiol (E2, 1.2,mu g/kg), bisphenol A (BPA, 2.4 mu g/kg), and diethylstilbestrol (DES, 2.5 mu g/kg) were subcutaneously injected in ICR 12-week-old male mice. Mice testes were observed for the expression of cortactin protein after E2, BPA, and DES treatments by Western blot analysis, immunohistochemical analysis, and immuno-electron microscopic analysis. Observations showed that the immunoreactivity of the treated testes was significantly decreased. The immunohistochemical reactivity of cortactin in the apical ES was decreased in the treated testis. In immunoelectron microscopic observations, ultrastructural immunolocalizations of cortactin protein in the apical ES by both E2 and BPA were decreased, and the immuno-gold particles of apical and basal ES by DES were much less than the control. In the toxicological field, cortactin may be considered to be one of the indicator proteins of abnormal spermiogenesis which is affected by exogenous chemicals, such as endocrine disrupting chemicals. In summary, this study helps toward understanding the cortactin protein expression underlying the histological abnormalities of spermatogenesis induced by exogenous hormonal chemical treatment.

Macrophage migration inhibitory factor (MIF) is a multifunctional protein that acts as a pro-inflammatory cytokine, pituitary hormone, immunoregulator and mitogen. The MIF gene knockout (MIFKO) mouse has been used to study the MIF response in many tissues, such as with skin injury and spinal cord injury; however, there is little information about the MIFKO mouse testis. This study reports the levels of serum and intratesticular estradiol and testosterone, the ultrastructure of the testis, and preliminary findings from in vitro fertilization. Our results revealed a decrease in estradiol and testosterone levels and deformation in spermiogenesis, in the MIFKO mouse. These initial findings study may lead to a better understanding of the role that MIF plays in the mouse testis. (c) 2005 Elsevier Inc. All rights reserved.

RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamilly with Ca(2+)-independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175(-/-)) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175(-/-) testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminall surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminall surface for timely release.

Mice with disrupted mammalian PcG (Polycomb group) genes commonly show skeletal transformation of anterior-posterior identities. Disruption of the murine M33 gene, a PcG member, displayed posterior transformation of the vertebral columns and sternal ribs. In addition, failure of T-cell expansion and hypoplasia and sex-reversal of the gonads, have been observed. In the present study, we identified defects in the splenic and adrenal formation of M33-knock-out (KO) mice on a C57BL/6 genetic background. The spleen in these animals was smaller than in the wild-type mice and was spotted red because of nonuniform distribution of blood cells. Histologic examination revealed disorganization of the vascular endothelium and its surrounding structures, and immunohistochemistry demonstrated disturbances in vascular formation and colonization of immature hematopoietic cells. These splenic phenotypes observed in the M33-KO mice were quite similar to those seen in Ad4BP/SF1 (Nr5a1) knock-outs. Moreover, the adrenal glands of M33-KO and Ad4BP/SF1 heterozygous KO mice were smaller than those of the wild-type mice. Western blot, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RTPCR) analyses of the M33 knock-outs all indicated significantly low expression of adrenal 4 binding protein/steroidogenic factor-1 (Ad4BP/SF-1), indicating that M33 is an essential upstream regulator of Ad4BP/SF1. In agreement with these observations, chromatin immunoprecipitation assays with adrenocortical Y-1 cells revealed direct binding of the M33-containing PcG to the Ad4BP/SF1 gene locus.

A mouse homologue of Drosophila germ cell-less, mouse germ cell-less-1 (mgcl-1),is highly expressed in the testis. Previous report revealed that the fertility of the mgcl-1(-/-) male mice is reduced significantly as a result of various morphological abnormalities in the sperm (Kimura et al., 2003). To elucidate the function of mgcl-l in spermatogenesis, the expression of mGCL-1 in the wild-type testis was examined. Immunohistochemical studies demonstrated that mGCL-1 first appeared in the nuclei of the pachytene spermatocytes at stage VI of the seminiferous epithelium, and existed in those of spermatids until step 8 during spermatogenesis. mGCL-1 was not detectable after step 9 spermatids. The testicular cells and epididymal sperm were further analyzed morphologically using mgcl-1(-/-) mice. In the testis, deformed nuclei first occurred in the pachytene spermatocytes at stage VI, which is consistent with the time of the first appearance of the mGCL-1 protein in the wild-type testis. Abnormal nuclei and acrosomes were found in spermatids after step 5, and nuclei of the spermatids and epididymal sperm were frequently invaginated. In addition, variously deformed sperm such as bent-neck, multi-headed or multi-nucleated sperm were observed in the mgcl-1(-/-) cauda epididymidis. However, several key structures such as the acroplaxome marginal ring (Kierszenbaum er al., 2003). postacrosonial sheath, and posterior ring apparently formed. In addition. MN7 and MN13. essential suhstances, for fertilization that are located in sperm heads. were detectable in the mgcl-1 null sperm. These observations provide important insights into the mechanisms regulating the nuclear architecture and causes of human infertility.

Background Studies showed that propentofylline enhances the action of adenosine and protects hippocampal neuronal damage against transient global cerebral ischaemia. Our study was to investigate the effect of propentofylline on hypoxic-ischaemic brain damage in neonatal rat. Methods Seven-day-old Wistar rats were subjected to unilateral common carotid artery ligation and hypoxia in oxygen 8 kPa for two hours at 37degreesC. Propentofylline (10 mg/kg) was administered intraperitoneally one hour after hypoxia-ischaemia (treated group). Control group rats were received an equivalent volume of saline. The effects of propentofylline were assessed by observing the body mass gain, behavioural alteration and neurohistological changes. The rats were sacrificed at 72 hours after hypoxia-ischaemia, and the brain sections were examined after haematoxylin and eosin staining. Results The propentofylline-treated rats had better body mass gain and better behavioural response than the paired saline-controls did. In the control group, the rats either lost body mass or had little mass gain after the insult, their average body mass gain was 97.3% at 24 h, 100.3% at 48 h, and 114.1% at 72 h of recovery. In propentofylline-treated group, there was a significant improvement of body mass gain at 24 h (100.2%, P < 0.05) and 48 h (110.3%, P < 0.01) of recovery; the percentage of rats that performed well on behavioural test was significantly higher from 48 h to 72 h of recovery (P < 0.05); the incidence of severe brain damage to the cerebral cortex and dentate gyrus was significantly reduced in propentofylline-treated rats (cortex, 93% - 70.8%, P < 0.01; dentate gyrus 95% -66.7%, P < 0.01) as compared with control rats. Conclusions Administration of propentofylline 1 hour after hypoxia-ischaemia significantly attenuates brain damage in both the cerebral cortex and dentate gyrus, and also improves the body mass gain as well as behavioural disturbance in 7-day-old rats.

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28 000-26 000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an antiMC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulusinvested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.

Background/Aims: Transforming growth factor-beta (TGF-beta) mediates the excess accumulation of extracellular matrix in the diabetic kidney. Smad family proteins have been identified as signal transducers for the TGF-beta superfamily. We sought to characterize the role of Smad proteins in mediating TGF-beta responses in the development of diabetic nephropathy. Methods: We evaluated the time course of TGF-beta(1) fibronectin, Smad2 and Smad3 protein expression and Smad3 activation in glomeruli from spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using immunohistochemistry and Western blot analysis. Results: The glomeruli of diabetic OLETF rats showed not only accelerated activation of Smad3, but also enhanced protein expression of Smad2 and Smad3, which occurred in parallel to the increased expression of TGF-beta and fibronectin compared with glomeruli of control, Long-Evans Tokushima Otsuka (LETO) rats at 30 weeks of age. No differences were found in TGF-beta(1) fibronectin, Smad2 and Smad3 protein expression and Smad3 activation in glomeruli between the two strains at 12 weeks of age when OLETF rats were not diabetic. Conclusions: The enhancement of Smad protein expression and activation may be involved in the TGF-beta signaling cascade that plays an important role in the development of diabetic nephropathy through progressive expansion of the mesangial matrix. Copyright (C) 2004 S. Karger AG, Basel.

Gambierol was isolated from Gambierdiscus toxicus, which causes ciguatera fish poisoning. The acute toxicological effects induced in mice by synthesized gambierol were studied. The lethal doses were about 80 mug/kg by i.p. and i.v., and 150 mug/kg by p.o. The main injury by this toxin was observed in the lung, and secondary in the heart, resulting in systemic congestion. Another toxic effect was seen in the stomach, inducing hypersecretion and ulceration. With survival from the severe stage during the initial 3 h, recovery was favorable, especially after 4 days. Additional effects were not evident during 1-week post-administration observation. (C) 2003 Elsevier Ltd. All rights reserved.

Differential display in combination with a cDNA cloning approach were used to isolate a novel gene, spergen-2, which has an open reading frame of 1500 nucleotides and encodes a protein of 500 amino acids that contains ankyrin repeat motifs and a putative nuclear localization signal. Expression of spergen-2 is developmentally upregulated in testis. In situ hybridization revealed that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1-6). Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spergen-2 protein is predominantly expressed in nuclei of late spermatocytes (stages IX-XIV) and spermatids (steps 1-11), indicating the restricted expression of spergen-2 during spermatogenesis. In nucleoplasm of spermatogenic cell nuclei, spergen-2 tends to localize in the interchromosome space with relatively low DNA density. These findings indicate a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.

Mammalian spermatozoa undergo morphological, biochemical, and physiological modifications initially in the testis (testicular maturation) and later in the epididymis (epididymal maturation). These maturational changes are commensurate with the functional events that occur in developing germ cells and maturing spermatozoa. This special issue reviews the recent, relevant topics dealing with spermatozoa maturation and focuses on the events that occur in internal components such as the nucleus, the acrosome, the perinuclear theca, the fibrous sheath, and the cytoplasmic droplet as well as the plasma membrane. These structures/elements and the constituent proteins of which they are comprised undergo a variety of sequential modifications starting from their origination in developing germ cells up to epididymal maturation. Several steps of the maturation processes on the sperm plasma membrane are mediated by external enzymes and secretions derived from the epithelium lining of the genital tract. Degradation of some of the constituent proteins and the elimination of defective spermatozoa are controlled by the degradation/recycling system, the ubiquitin system. These maturational modifications are necessary for spermatozoa to become fertilization-competent cells and to be stored safely in the male. (C) 2003 Wiley-Liss, Inc.

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.

We examined the issue of whether germ cell factors are required for testicular enlargement that occurs after recovery from neonatal hypothyroidism. Experiments were performed using W/W-v mutant mice (lacking germ cells) and normal mice (ICR). The pups in experimental group (neonatal hypothyroid) received 6 propyl 2-thio-uracil (PTU) treatment, administered by adding 0.1% (w/v) to the water provided to the mother from day 1 of birth through day 25 postpartum, while the pups of control group received drinking water only. Mice were sacrificed at the age of day 25, 50 and 90, in the case of ICR mice, or at day 25 and 90 in the case of W/W-v mutant mice. In both groups, early hypothyroidism caused a partial recoverable decrease in body growth and testicular development. Both ICR and W/W-v mutant mice, those recovered from neonatal hypothyroidism showed an increase in testis weights, the number of Sertoli cells, and the diameter of the semniferous tubules. This study demonstrates that neonatal hypothyroidism led recovery caused testicular enlargement not only in ICR mice but, also in germ cell depleted W/W-v mutant mice. Hence these findings deny direct involvement of the germ cell factors in the process of testicular enlargement in recovered mice even in vivo, and reaffirm the notion that thyroid hormone directly regulates the dynamics of Sertoli cell maturation.

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration th rough the zona pellucida a nd subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with antisyntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa. (C) 2000 Wiley-Liss, Inc.

In this study we examined the behaviour and role of an intra-acrosomal antigenic molecule, acrin 3, during mouse fertilisation in vitro by assessing the effect of its pertinent monoclonal antibody mMC101. Experiments were designed to assess the effect of mMC101 on sperm-zona pellucida binding, the acrosome reaction, zona pellucida penetration, sperm-egg fusion, and fertilisation in vitro. mMC101 did not affect sperm motility or primary and secondary binding to the zona pellucida, but significantly inhibited fertilisation of zona-pellucida-intact oocytes in a dose-dependent manner. In the presence of mMC101 at 100 μg/ml concentration in TYH medium, none of the oocytes developed to pronuclear stage by 5 h after co-incubation of the gametes, but the pronucleus formation rate recovered to some extent (45.3%) after 8 h, indicating a delay of early embryonic development. mMC101 also delayed and significantly suppressed zona pellucida penetration by sperm. Acrin 3 dispersed and did not remain on completely acrosome-reacted sperm. Although mMC101 did not influence the zona-pellucida-induced acrosome reaction, it significantly inhibited fertilisation when acrosome-reacted sperm in the presence of mMC101 inseminated zona-pellucida-free oocytes. However, fertilisation remained unaffected when acrosome-reacted sperm in the absence of mMC101 inseminated zona-pellucida-free oocytes even in its presence. Thus, acrin 3 appears to facilitate zona pellucida penetration and is also likely to be involved in sperm-oocyte fusion by modifying the sperm plasma membrane during the acrosome reaction.

We have recently shown that a 90-kDa glycoprotein, acrin1 (MN7), is exclusively localized in the dorsal region of the acrosomal apical segment of mature guinea pig sperm, and that its location changes during epididymal maturation. The present study examined the process of transport and organization of this protein in the acrosome during spermatogenesis in the guinea pig testis. Immunoperoxidase electron microscopy showed stage-specific localization of acrin1 within the developing acrosome, as follows: acrin1 first appeared in the proacrosomic vesicles of the early Golgi phase spermatids, and it was then localized in the electron-lucent matrix region of the acrosomic vesicles of the late Golgi phase spermatids. During the cap phase, acrin1 was abundant in the electron-lucent matrix of the acrosomal apical segment and in the head-cap region (principal segment). acrin1 became more restricted to the peripheral region of the electron-lucent matrix of acrosome phase spermatids and it was localized in the electron-lucent dorsal matrix region of maturation phase spermatids. In the final step of spermiogenesis, acrin1 disappeared from the equatorial and principal segments, and it was finally confined to the dorsal matrix region of the acrosomal apical segment. In addition, Western blot analysis showed that acrin1 of testes and epididymal sperm was of the identical size, indicating that acrin1 is not proteolytically modified during epididymal sperm maturation. These results indicate that acrosome morphogenesis is closely associated with the rearrangement of acrosomal proteins. (C) 2000 Wiley-Liss, Inc.