Etsuko Mori

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins.

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo-β-galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fucα1→2Galβ1→4(±SO−3→6)GlcNAc (type 2H), Galβ1→4(Fucα1→3)(±SO−3→6)GlcNAc(Lex) and Fucα1→3 or 4(±SO−3→6)GlcNAc, are expressed in the repeated outer chain moieties.

Expression of Fas-Fas ligand in murine testis.

Xu JP, Li X, Mori E, Guo MW, Matsuda I, Takaichi H, Amano T, Mori T. Expression of Fas‐Fas ligand in murine testis. AJRI 1999; 42:381–388

Expression of Class II Major Histocompatibility Complex Antigen on Mouse Sperm and Its Roles in Fertilization

ABSTRACT: The expression of class II major histocompatibility complex (MHC) antigen on the membrane of mouse sperm head was clearly demonstrated by indirect immunofluorescence (IIF) test, enzyme immunoassay (EIA), and mixed lymphocyte sperm reaction (MLSR) as well as the examination of its expression at transcriptional levels by nothern dot blotting. Furthermore, the roles of class II MHC antigen of sperm in fertilization were investigated with an in vitro fertilization (IVF) system. The successful ratio of IVF was significantly decreased by treatment of sperm with anticross‐reactive region of class II MHC antigen monoclonal antibody (MAb) but not with antiprivate region MAb. These results were not due to disturbances of sperm mobility by these MAbs. It was strongly suggested that the monomorphic or its related region of class II MHC antigen on sperm played an important role in the recognition between sperm and egg in fertilization.

Expression of Fas ligand in murine ovary.

Corresponding to the expression of Fas in the ovarian oocytes as previously reported (Guo et al., Biochem Biophys Res Commun 1994; 203:1438–1446; Mori et al., JSIR 1995; 9:49–50), the expression of Fas ligand (FasL) in the ovarian follicle was found to be restricted in the area of granulosa cells by the indirect immunofluorescence (IIF) test. Reverse transcriptase/polymerase chain reaction (RT/PCR) technique coupled with Southern blot hybridization analysis showed that the highest level of FasL mRNA was demonstrated in murine ovaries and granulosa cells 1 day after the administration of pregnant mares serum gonadotropin (PMSG), while the level of FasL mRNA became very weak on the day 5, respectively. The observed gradual decrease in FasL mRNA could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of constitutive expression of elongation factor 1α (EF‐1α) mRNA in murine ovaries and granulosa cells treated with PMSG. Furthermore, in situ hybridization analysis with a FasL‐specific probe confirmed that FasL was specifically localized in the granulosa cells of most follicles and its expression was regulated by PMSG administration. FasL localized in granulosa cells might possibly play an important role in the formation of the ovarian atretic follicles, most likely depending on PMSG administration.

Expression of Fas-Fas Ligand System Associated with Atresia through Apoptosis in Murine Ovary

We examined the contribution of Fas and its ligand (FasL) in the process of follicular atresia using murine intraovarian follicles and pregnant mares serum gonadotropin (PMSG)-hyperovulated eggs. Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in both intraovarian oocytes and hyperovulated eggs. In contrast, expression of FasL was only detected in granulosa cells. These findings were histologically confirmed by in situ hybridization using Fas- and FasL-specific probes. A time-course study showed that Fas mRNA was positive in atretic follicles through day 0 and day 2 of PMSG stimulation and negative thereafter. Levels of FasL mRNA were the highest on day 1 and tapered off toward day 5 of PMSG stimulation. Levels of elongation factor 1 alpha mRNA, a constitutive element, were constantly maintained throughout the experimental period. Coculture of ovulated eggs, intact and zona-free, and granulosa cells demonstrated positive TUNEL staining only in zona-free eggs. These findings indicate that follicular atresia is caused by apoptosis, and the apoptosis associated with internucleosomal DNA fragmentation is directly regulated by the Fas/FasL system.

BINDING OF FOREIGN DNA TO MOUSE SPERM MEDIATED BY ITS MHC CLASS II STRUCTURE

ABSTRACT: By means of radioimmunoassay, the expression of the major histocompatibility complex (MHC) class II molecules on murine sperm cells was clearly demonstrated as well as by our previous enzyme immunoassay (Mori T, et al. The expression of class II major histocompatibility antigen on mouse sperm and its role in fertilization. Am J Reprod Immunol. 1990; 24:9–14). The present study revealed that the site of sperm for binding foreign DNA was mediated by the complex structure of the MHC class II molecules localized at the posterior region of sperm head. This binding activity of sperm was time‐, temperature‐, and viability‐dependent and completely inhibited by the treatment of sperm cells with mouse anti Iak serum, but not with mouse normal serum. Scatchard analysis of this binding activity also showed a single receptor type on sperm cells. These results were directly confirmed morphologically by taking autoradiography of sperm cells binding foreign DNA.

Expression of CD4-like structure on murine egg vitelline membrane and its signal transductive roles through p56lck in fertilization.

ABSTRACT: Expression of CD4‐like molecule on vitelline membrane of murine eggs was demonstrated by indirect immunofluorescence (IIF) test and immunoprecipitation corresponding to the expression of major histocompatibility complex (MHC) class II molecule on murine sperm detected by immunoblotting. This molecule showed slightly larger size than that of the authentic CD4 molecule from T‐cells on SDS‐PAGE. This molecule was suggested to bind to MHC class II structure on sperm during fertilization because anti‐CD4 monoclonal antibody (mAb) blocked in vitro fertilization (IVF). In addtion, src‐related tyrosine protein kinase (p56lck) was demonstrated in the inner vitelline membrane of eggs by means of IIF with anti‐p56lck mAb and immune‐complex kinase assay. This molecule was suggested to be associated with CD4‐like molecule.

Expression of the signal transducing regions of CD4-like and lck genes in murine egg.

We previously demonstrated that the MHC class II molecule on murine sperm and CD4-like molecule on the egg vitelline membrane are involved in fertilization. In this study, the RNA transcripts in murine eggs corresponding to parts of the CD4 gene and lck gene in thymocytes were demonstrated by means of the reverse transcriptase (RT)/polymerase chain reaction (PCR) followed by the sequencing of the PCR products. The deduced sequences potentially encode the amino acid sequence of the transmembrane to the cytoplasmic domain of CD4 and that of the N-terminal domain of p56lck respectively. These findings indicate that a signal transducing complex similar to that in immune T cells is expressed at the transcriptional level in murine eggs.

Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines

Abstract Glycolipid antigen was examined by radioimmuno-thin-layer chromatography (RITLC), which is a combination of a thin-layer chromatography and radioimmunoassay. In this way Forssman antigen was studied in seven carcinoma cell lines. The usual Forssman antigen with a ceramide pentasaccharide structure was detected in cell lines of a gastric cancer and a breast cancer. In addition another glycolipid with slower mobility on thin-layer chromatography and with Forssman reactivity was found in cell lines of three gastric cancers and one lung cancer.

Molecular structure and function of CD4 on murine egg plasma membrane.

In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhesion was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.