Molecular Reproduction and Development, Oct 1, 1997
We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa tha... more We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, b-N-acetylglucosaminidase (GlcNAc8ase) and a-mannosidase (Man8ase), have been evidenced. They have an optimal pH of 6-6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of a-mannose and b-N-acetylglucosamine residues present on the egg shell at the site of sperm entry.
Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactio... more Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two b-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for b-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the a-subunit of the mammalian lysosomal b-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the b-subunit of all known b-N-acetylhexosaminidases, which we have named b 1 and b 2 , respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an ab 2 structure, and DmHEXB, with a b 1 b 2 structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding b-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional b-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.
beta-N-acetylglucosaminidase of sperm plasma membrane is crucial for egg fertilization in Drosoph... more beta-N-acetylglucosaminidase of sperm plasma membrane is crucial for egg fertilization in Drosophila melanogaster/ME Pasini, F. Cattaneo, F. Vernì, JHP Hackstein, ME Perotti.-In: Molecular Biology of the Cell.-ISSN 1059-1524.-10: Suppl.(1999).-p. 360A-...
Considerable evidence suggests that HIV infected macrophages and/or lymphocytes may mediate sexua... more Considerable evidence suggests that HIV infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. Our laboratory and other laboratories have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cells trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3 % PDR98-15 carrageenan (FMC Biopolymer, Rockland ME- PC-515), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When we inoculated supravitally stained mouse macrophages and T cells into the vagina of recipient mice, we observed that discrete numbers of donor’s cells migrated to the recipients ’ iliac and inguinal lymph nodes and to the spleen as well. When recipient mice were pre-inoculated with the carr...
Abstract Drosophila come modello per lo studio delle interazioni tra i gameti negli Insetti/ME Pa... more Abstract Drosophila come modello per lo studio delle interazioni tra i gameti negli Insetti/ME Pasini, F. Cattaneo, J. Intra, ME Perotti.-In: 65. Congresso nazionale dell'Unione zoologica italiana: riassunti dei contributi scientifici/[a cura di] NE Baldaccini.-[sl]: Università degli ...
Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immu... more Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fuc...
Come si incontrano spermatozoo e uovo in Drosophila? / ME Pasini, F. Cattaneo, F. Vernì, ME Perot... more Come si incontrano spermatozoo e uovo in Drosophila? / ME Pasini, F. Cattaneo, F. Vernì, ME Perotti. ((Intervento presentato al 62. convegno Congresso UZI tenutosi a Sanremo nel 2001. ... There are no files associated with this item. ... Items in AIR are protected by ...
Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coatin... more Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.
Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coatin... more Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.
The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER ... more The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER negative) form solid tumors within one week following inoculation into athymic nude mice. Tumor formation by MCF-7 and ZR 75-1 cells was dependent upon estrogen, whereas MDA-MB 231 cells formed tumors in ovariectomized mice with or without supplemental estrogen. Ultrastructural comparison of tumors formed by the three human breast carcinoma lines in athymic nude mice indicated that lactoperoxidase activity, milk protein and fat globule formation were virtually absent from all three tumors. The estrogen-dependent tumors (MCF-7, ZR 75-1), however, had more desmosomes, intermediate-sized microfilaments and collagen than the estrogen-independent tumor (MDA-MB 231). When the ultrastructure of the three human tumors was compared to the hormone-dependent, DMBA-induced rat mammary carcinoma and to the normal lactating rat mammocytes, the following observations were evident: a) the estrogen-depen...
ABSTRACT We have treated sea urchin gametes with quercetin to investigate the effects of this dru... more ABSTRACT We have treated sea urchin gametes with quercetin to investigate the effects of this drug on fertilization and egg activation. High concentrations of this drug gradually inhibited sperm motility, but pretreatment of sperm with the drug did not affect their fertility. Treatment of eggs with quercetin at micromolar concentrations completely blocked fertilization. The drug did not block the acrosome reaction of sperm, nor did it affect the binding of the acrosomal filament to the vitelline layer of the egg. However, fertilization was prevented. Inseminated quercetin-treated eggs could be activated by ionophore A23187, but still failed to incorporate sperm. Quercetin blocked fertilization if added after primary gamete binding, but 30 s before the beginning of the cortical reaction in experiments in which eggs were inseminated at low temperature. Drug added 10 s before the beginning of the cortical reaction was ineffective. Electron microscopy confirmed normal primary gamete binding and failure of fertilization. Vitelline layer removal by trypsin did not restore fertilizability to quercetin-treated eggs. Thus quercetin prevented fertilization by blocking gamete membrane fusion. These results indicate that quercetin can be a useful probe for the mechanism of membrane fusion in fertilization.
Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, ... more Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia
... Ill. Electron Microscopic Localization of [1 25 I] -hCG Binding to Sensitive and Desensitized... more ... Ill. Electron Microscopic Localization of [1 25 I] -hCG Binding to Sensitive and Desensitized Ovaries Seven Days after PMSG-hCG' WINSTON ANDERSON,2 YANG-HSU KANG,2 ME PEROTTI,2 THOMAS A. BRAMLEY3 and ROBERT J. RYAN3 ,4 ...
Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophoru... more Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophorus of hamsters and rats. Observations on cumuli prepared in a variety of ways including different chemical fixation techniques and cryofixation freeze substitution were compared. It was concluded that the cumulus mucus is not arranged in lamellae or granules as has previously been suggested but is composed of molecules which form very fine filaments when properly fixed. Morphometric analysis of cumuli fixed either in situ or after being explanted into medium revealed that the distance between neighboring cumulus cells was greater with increasing distance from the oocyte. Morphometry revealed that, when placed into medium, the cumulus expands possibly due to hydration. Thus physiological experiments carried out on cumuli should be performed very shortly after cumuli are isolated. From their ultrastructure cumulus cells appear to be actively involved in protein synthesis and secretion as we...
Molecular Reproduction and Development, Oct 1, 1997
We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa tha... more We investigated the presence of enzymes on the surface of Drosophila melanogaster spermatozoa that might bind to the carbohydrate residues of the egg shell. Spectrophotometric and fluorimetric studies were used on whole spermatozoa to assay galactosyltransferase and glycosidase activities. No galactosyltransferase is present on the sperm surface, whereas two glycosidases, b-N-acetylglucosaminidase (GlcNAc8ase) and a-mannosidase (Man8ase), have been evidenced. They have an optimal pH of 6-6.5 and 4, respectively. The same glycosidases were detected as soluble forms probably secreted by the seminal vesicle epithelium. We suggest that these enzymes might be involved in the recognition of a-mannose and b-N-acetylglucosamine residues present on the egg shell at the site of sperm entry.
Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactio... more Sperm surface b-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two b-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for b-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the a-subunit of the mammalian lysosomal b-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the b-subunit of all known b-N-acetylhexosaminidases, which we have named b 1 and b 2 , respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an ab 2 structure, and DmHEXB, with a b 1 b 2 structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding b-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional b-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.
beta-N-acetylglucosaminidase of sperm plasma membrane is crucial for egg fertilization in Drosoph... more beta-N-acetylglucosaminidase of sperm plasma membrane is crucial for egg fertilization in Drosophila melanogaster/ME Pasini, F. Cattaneo, F. Vernì, JHP Hackstein, ME Perotti.-In: Molecular Biology of the Cell.-ISSN 1059-1524.-10: Suppl.(1999).-p. 360A-...
Considerable evidence suggests that HIV infected macrophages and/or lymphocytes may mediate sexua... more Considerable evidence suggests that HIV infected macrophages and/or lymphocytes may mediate sexual transmission of HIV. Our laboratory and other laboratories have previously demonstrated that when vitally stained donor mouse lymphocytes or macrophages are placed in the vaginas of mice, some of the stained cells can later be found in the iliac lymph nodes. The aim of this study was to assess the extent of mononuclear cells trafficking from the vagina and to test the possibility that carrageenan formulation, a sulfated polysaccharide formulation containing 3 % PDR98-15 carrageenan (FMC Biopolymer, Rockland ME- PC-515), a vaginal microbicide, would prevent vaginal transmigration of macrophages. When we inoculated supravitally stained mouse macrophages and T cells into the vagina of recipient mice, we observed that discrete numbers of donor’s cells migrated to the recipients ’ iliac and inguinal lymph nodes and to the spleen as well. When recipient mice were pre-inoculated with the carr...
Abstract Drosophila come modello per lo studio delle interazioni tra i gameti negli Insetti/ME Pa... more Abstract Drosophila come modello per lo studio delle interazioni tra i gameti negli Insetti/ME Pasini, F. Cattaneo, J. Intra, ME Perotti.-In: 65. Congresso nazionale dell'Unione zoologica italiana: riassunti dei contributi scientifici/[a cura di] NE Baldaccini.-[sl]: Università degli ...
Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immu... more Fucosylated glycoconjugates play a role in a wide variety of biological processes, including immune responses, signal transduction, ontogenic events and pathogenesis of several human diseases. Alpha-L-fucosidases, which are responsible for their processing, have been demonstrated to be involved in lysosomal storage disease, inflammation, cystic fibrosis, cancer development and in the interactions between gametes in vertebrates as well as invertebrates. The sequence and comparative genomic analysis of these glycosyl hydrolases and the study of their evolutionary relationships appear therefore to be of considerable interest. In this work we carried out extensive similarity searches and comparative analyses to identify sequences encoding alpha-L-fucosidases. We have identified novel alpha-L-fucosidase coding sequences in worms, insects, sea urchin, ascidians, fish, chicken, amphibians, mammals and various bacteria resulting in a total of 39 alpha-L-fucosidase sequences. Two alpha-L-fuc...
Come si incontrano spermatozoo e uovo in Drosophila? / ME Pasini, F. Cattaneo, F. Vernì, ME Perot... more Come si incontrano spermatozoo e uovo in Drosophila? / ME Pasini, F. Cattaneo, F. Vernì, ME Perotti. ((Intervento presentato al 62. convegno Congresso UZI tenutosi a Sanremo nel 2001. ... There are no files associated with this item. ... Items in AIR are protected by ...
Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coatin... more Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.
Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coatin... more Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila.
The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER ... more The human breast cancer cell lines MCF-7 (ER positive), ZR 75-1 (ER positive) and MDA-MB 231 (ER negative) form solid tumors within one week following inoculation into athymic nude mice. Tumor formation by MCF-7 and ZR 75-1 cells was dependent upon estrogen, whereas MDA-MB 231 cells formed tumors in ovariectomized mice with or without supplemental estrogen. Ultrastructural comparison of tumors formed by the three human breast carcinoma lines in athymic nude mice indicated that lactoperoxidase activity, milk protein and fat globule formation were virtually absent from all three tumors. The estrogen-dependent tumors (MCF-7, ZR 75-1), however, had more desmosomes, intermediate-sized microfilaments and collagen than the estrogen-independent tumor (MDA-MB 231). When the ultrastructure of the three human tumors was compared to the hormone-dependent, DMBA-induced rat mammary carcinoma and to the normal lactating rat mammocytes, the following observations were evident: a) the estrogen-depen...
ABSTRACT We have treated sea urchin gametes with quercetin to investigate the effects of this dru... more ABSTRACT We have treated sea urchin gametes with quercetin to investigate the effects of this drug on fertilization and egg activation. High concentrations of this drug gradually inhibited sperm motility, but pretreatment of sperm with the drug did not affect their fertility. Treatment of eggs with quercetin at micromolar concentrations completely blocked fertilization. The drug did not block the acrosome reaction of sperm, nor did it affect the binding of the acrosomal filament to the vitelline layer of the egg. However, fertilization was prevented. Inseminated quercetin-treated eggs could be activated by ionophore A23187, but still failed to incorporate sperm. Quercetin blocked fertilization if added after primary gamete binding, but 30 s before the beginning of the cortical reaction in experiments in which eggs were inseminated at low temperature. Drug added 10 s before the beginning of the cortical reaction was ineffective. Electron microscopy confirmed normal primary gamete binding and failure of fertilization. Vitelline layer removal by trypsin did not restore fertilizability to quercetin-treated eggs. Thus quercetin prevented fertilization by blocking gamete membrane fusion. These results indicate that quercetin can be a useful probe for the mechanism of membrane fusion in fertilization.
Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, ... more Cytochrome oxidase activity via cytochrome c, as demonstrated by the diaminobenzidine procedure, has been employed in this electron microscope cytochemical study to determine the respiratory potency, integrity and fate of the Arbacia
... Ill. Electron Microscopic Localization of [1 25 I] -hCG Binding to Sensitive and Desensitized... more ... Ill. Electron Microscopic Localization of [1 25 I] -hCG Binding to Sensitive and Desensitized Ovaries Seven Days after PMSG-hCG' WINSTON ANDERSON,2 YANG-HSU KANG,2 ME PEROTTI,2 THOMAS A. BRAMLEY3 and ROBERT J. RYAN3 ,4 ...
Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophoru... more Ultrastructural and morphometric techniques were employed to examine the ovulated cumulus oophorus of hamsters and rats. Observations on cumuli prepared in a variety of ways including different chemical fixation techniques and cryofixation freeze substitution were compared. It was concluded that the cumulus mucus is not arranged in lamellae or granules as has previously been suggested but is composed of molecules which form very fine filaments when properly fixed. Morphometric analysis of cumuli fixed either in situ or after being explanted into medium revealed that the distance between neighboring cumulus cells was greater with increasing distance from the oocyte. Morphometry revealed that, when placed into medium, the cumulus expands possibly due to hydration. Thus physiological experiments carried out on cumuli should be performed very shortly after cumuli are isolated. From their ultrastructure cumulus cells appear to be actively involved in protein synthesis and secretion as we...
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