RESUMEN
Microbial communities found on the surface of overwintering plants may be exposed to low temperatures as well as multiple freeze-thaw events. To explore the adaptive mechanisms of these epiphytes, with the objective of identifying products for freeze-protection, enrichment libraries were made from frost-exposed leaves. Of 15 identified bacteria from 60 individual clones, approximately half had ice-association activities, with the great majority showing high freeze-thaw resistance. Isolates with ice nucleation activity and ice recrystallization inhibition activity were recovered. Of the latter, two (Erwinia billingiae J10, and Sphingobacterium kitahiroshimense Y2) showed culture and electron microscopic evidence of motility and/or biofilm production. Mass spectrometric characterization of the E. billingiae extracellular polymeric substance (EPS) identified the major proteins as 35 kDa outer membrane protein A and F, supporting its biofilm character. The addition of the EPS preparation increased the freeze-thaw survival of the more susceptible bacteria 1000-10000 times, and protection was at least partially dependent on the protein component.
Asunto(s)
Proteínas Bacterianas/química , Biopelículas/efectos de los fármacos , Erwinia/fisiología , Consorcios Microbianos/fisiología , Sphingobacterium/fisiología , Adaptación Fisiológica , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Biopelículas/crecimiento & desarrollo , Chrysanthemum/microbiología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Congelación , Hielo , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica , Hojas de la Planta/microbiología , Pseudomonas syringae/efectos de los fármacos , Pseudomonas syringae/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , SimbiosisRESUMEN
We have investigated the toxic effects of trichloroethylene (TCE) on the epididymis and epididymal sperm in mice. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for 1 to 4 weeks. Segments of the epididymis (caput, corpus and cauda) were examined by light and electron microscopy. At the light microscopic level, degeneration and sloughing of epithelial cells were evident as early as 1 week after TCE exposure, and were most pronounced after 4 weeks. Such epithelial damage was observed in the caput, corpus and cauda regions of the epididymis. Ultrastructural observations revealed vesiculation in the cytoplasm, disintegration of basolateral cell membranes, and sloughing of epithelial cells. Sperm were found in situ in the cytoplasm of degenerated epididymal cells. Additionally, a large number of sperm in the epididymal lumen exhibited abnormalities including malformation of head and tail components. Our results demonstrated that exposure to TCE by inhalation causes damage to the epididymal epithelium and sperm.
Asunto(s)
Epidídimo/citología , Células Epiteliales/efectos de los fármacos , Solventes/toxicidad , Espermatozoides/efectos de los fármacos , Tricloroetileno/toxicidad , Administración por Inhalación , Animales , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Masculino , Ratones , Ratones Endogámicos , Solventes/administración & dosificación , Espermatozoides/patología , Espermatozoides/ultraestructura , Factores de Tiempo , Tricloroetileno/administración & dosificaciónRESUMEN
Lectins were used as probes in conjunction with quantitative analysis to investigate the distribution of different carbohydrate residues in hamster zona pellucida and their possible modification patterns after in vivo fertilization and in vitro egg activation. Several lectins including HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), DBA, AAA and MAA were used to label the zona pellucida of both unfertilized and fertilized eggs. With the exception of PNA and BSAIB(4), the same lectins were also used to label the zona pellucida of oocytes activated in vitro. A multicomparison quantitative analysis of the density of labelling in the inner and outer regions of the zona pellucida before and after fertilization in vivo, as well as after in vitro egg activation, was performed. Of all the lectins studied, preferential localization of labelling by RCA-I and DSA to the inner zona pellucida of unfertilized eggs was observed. After in vivo fertilization, there was an increase in labelling in the inner region of the zona pellucida when thin sections of fertilized oocytes were incubated with HPA, BSAIB(4) and AAA. Although increased labelling by RCA-I was observed, a significant decrease in labelling intensity was obtained with WGA and the sequence Neu-WGA in both the inner and outer zona pellucida of fertilized oocytes. A significant increase in the density of labelling with WGA was also observed after digestion with neuraminidase. In parallel, when hamster oocytes activated in vitro were compared with those fertilized in vivo, a difference in lectin-gold labelling was observed in both the inner and outer region of the zona pellucida. Labelling with HPA, WGA, DSA and MAA increased significantly in both the inner and outer regions of the zona pellucida, whereas labelling by DBA significantly decreased in the inner portion of the zona pellucida. After neuraminidase treatment, a significant increase in labelling density was observed when thin sections of in vitro-activated oocytes were incubated with WGA. These results demonstrate: (i) the post-fertilization modifications of major saccharidic determinants that may play a role in the sperm-egg interaction process of fertilization in vivo; and (ii) that the modified properties of zonae pellucidae of fertilized and in vitro-activated eggs resulting from the action of hydrolytic enzymes, as well as glycoproteins released through exocytosis of cortical granules, are not identical.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Fertilización In Vitro , Zona Pelúcida/metabolismo , Animales , Calcimicina/farmacología , Carbohidratos/análisis , Cricetinae , Femenino , Ionóforos/farmacología , Lectinas , Microscopía Inmunoelectrónica , Neuraminidasa/farmacología , Oocitos/química , Oocitos/metabolismo , Zona Pelúcida/químicaRESUMEN
In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohydrate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin-gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progressively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB(4) was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analysis was also performed on labeling by lectin-gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization.
Asunto(s)
Glicósidos/metabolismo , Lectinas/metabolismo , Oocitos/metabolismo , Oocitos/ultraestructura , Ovulación/metabolismo , Zona Pelúcida/metabolismo , Animales , Cricetinae , Femenino , Oro Coloide , Microscopía Electrónica , Oocitos/química , Oocitos/crecimiento & desarrollo , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Folículo Ovárico/ultraestructura , Superovulación , Zona Pelúcida/ultraestructuraRESUMEN
In the present study, lectin-gold cytochemistry and antibodies against ZP2 and ZP3 glycoproteins were used to investigate the oligosaccharide content of mouse ovarian zona pellucida (ZP) during follicular development. The entire thickness of the ZP and several organelles of the oocyte (cortical granules, Golgi apparatus, and vesicular aggregates) were reactive to RCA-I, DSA, AAA, WGA, MAA, and LFA throughout follicular development. HPA labeling was not detected at the earliest stages of follicular folliculogenesis. HPA reactivity was first observed in the ZP, Golgi apparatus, and the vesicles of oocytes at the trilaminar primary follicle stage. HPA labeling in the ZP was always restricted to the inner region of the zona matrix. After neuraminidase treatment, HPA reacted with the entire ZP in ovarian follicles at different stages of development. Immunolabeling with specific antibodies showed that, although ZP2 and ZP3 glycoproteins were uniformly distributed in the zona matrix of ovarian oocytes, there was a progressive increase in thickness of the ZP in parallel with the proliferation of follicular cells. ZP3 glycoprotein was also localized to the Golgi apparatus and vesicular aggregate. The present results suggest: (1) a difference in composition of carbohydrate content between the inner and outer region of the fully developed ZP generated probably by a modification in the biosynthetic pathway of oligosaccharides in the oocyte during folliculogenesis, (2) that newly synthesized ZP glycoproteins displace previously synthesized ZP components in a direction toward the follicular cells and, therefore, no redistribution of the ZP matrix occurs during folliculogenesis, and (3) that the vesicular aggregates in the ooplasm constitute an intermediate step in the secretory pathway of ZP glycoproteins.
Asunto(s)
Proteínas del Huevo/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligosacáridos/metabolismo , Folículo Ovárico/fisiología , Receptores de Superficie Celular , Zona Pelúcida/metabolismo , Animales , Femenino , Lectinas/metabolismo , Ratones , Microscopía Inmunoelectrónica , Neuraminidasa/farmacología , Folículo Ovárico/ultraestructura , Zona Pelúcida/efectos de los fármacos , Zona Pelúcida/ultraestructura , Glicoproteínas de la Zona PelúcidaRESUMEN
We have shown that sperm sulfolipidimmobilizing protein 1 (SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid (SGG)-binding protein, is significant in sperm-zona pellucida (ZP) interaction. The objective of this study was to localize SLIP1 on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1 Fab before coincubation with sperm, showed a significant, dose-dependent decrease in sperm-egg plasma membrane binding. Similar results were obtained when affinity-purified antiSLIP1 IgG was used for egg pretreatment. The significance of egg SLIP1 in sperm-egg plasma membrane binding was further demonstrated by a decrease (36-52%) in in vitro fertilization when zona-intact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has been shown to bind SGG in vitro, we investigated the possibility that sperm SGG may participate in sperm-egg plasma membrane binding through egg SLIP1. Pretreatment of sperm with antiSGG Fab prior to coincubation with zona-free eggs resulted in a dose-dependent decrease in sperm-egg plasma membrane binding. Collectively, these findings strongly suggest a role for egg SLIP1 in sperm-egg plasma membrane interaction, which may be through its binding to sperm SGG.
Asunto(s)
Proteínas Portadoras/fisiología , Interacciones Espermatozoide-Óvulo , Animales , Anticuerpos/farmacología , Proteínas Portadoras/análisis , Proteínas Portadoras/antagonistas & inhibidores , Membrana Celular/química , Membrana Celular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Directa , Immunoblotting , Masculino , Ratones , Microscopía Inmunoelectrónica , Óvulo/química , Óvulo/fisiología , Óvulo/ultraestructura , Proteínas de Unión al ARN , Espermatozoides/fisiología , Espermatozoides/ultraestructuraRESUMEN
The epithelium of mammalian oviducts consists mainly of ciliated and non-ciliated secretory cells. In some mammals, secretory products originating from oviductal secretory cells have been shown to bind to the surface of, or accumulate within, ovulated eggs and/or developing embryos. These findings suggest that the secretions of the oviductal epithelial cells may play an important role in reproductive and developmental events that occur in the oviduct. In the present study, ultrastructural and cytochemical features of secretory cells in the hamster ampullary epithelium were shown by routine electron microscopy, lectin-gold cytochemistry and both conventional freeze-fracture and rapid-freezing techniques with special reference to the organizational aspects of their secretory granules. The use of ferrocyanide-reduced osmium tetroxide as a post-fixative in the Epon embedment of ampullary tissue samples also proved to be advantageous especially in revealing the carbohydrate contents of certain cellular compartments. The most conspicuous characteristic of the secretory cells, based on their staining property, was the presence of two types of secretory granules: those with a homogeneous electron-dense matrix and those with an electron-lucent matrix. Under favorable conditions, distinct features of the organizational arrangement of a crystalline lattice inside the secretory granules were also revealed. This well organized crystalline lattice shown in sections of Epon-embedded oviductal tissue was confirmed by examination of replicas of freeze-fractured oviducts prepared by the rapid-freezing technique. We also demonstrated with high resolution lectin-gold cytochemistry the intracellular distribution of lectin-binding glycoconjugates in the secretory cells of the hamster oviductal ampulla often in a linear array following the crystalline lattice. The results obtained in this study, taken together, provide insight into a possible link of the internal topographical features of oviductal secretory granules along with the cytochemical properties of their contents to the anticipated regulatory mechanism underlying their process of secretions.
Asunto(s)
Gránulos Citoplasmáticos/ultraestructura , Células Epiteliales/ultraestructura , Trompas Uterinas/ultraestructura , Animales , Cricetinae , Gránulos Citoplasmáticos/metabolismo , Células Epiteliales/metabolismo , Trompas Uterinas/metabolismo , Femenino , Técnica de Fractura por Congelación , Glicoconjugados/metabolismo , Oro Coloide , Lectinas/metabolismo , Mesocricetus , Microscopía Electrónica de RastreoRESUMEN
High resolution lectin-gold cytochemistry was used to quantitatively analyze the distribution of glycoconjugates in the hamster oviductal ampulla during the five stages of the estrous cycle. Lectins binding to N-acetyl-D-galactosamine-, D-galactose-, and sialic acid-associated glycoconjugates in the secretory granules of ampullary epithelial secretory cells showed staining of equal intensity throughout the five different stages of the estrous cycle. In contrast, the labeling intensity of glycoconjugates which contain N-acetylglucosamine as terminal sugar residues reached its maximum around the time of ovulation, i.e., at proestrus. Glycoconjugates which carry fucose and mannose as terminal sugar residues appeared to be totally absent from the secretory granules of the oviductal ampulla during the estrous cycle. Together, electron microscopic observations combined with quantitative results indicate that N-acetyl-D-galactosamine-, D-galactose-, and sialic acid-associated glycoconjugates may be secreted into the ampullary lumen irrespective of the stage of the estrous cycle, whereas the secretion of certain N-acetylglucosamine-associated glycoconjugates is stage specific and reaches its peak at the time of ovulation. These findings suggest that, at the time of ovulation, the ampullary epithelium changes its secretory activity and contributes its secretory products to the zona pellucida of oocytes freshly released from the ovary.
Asunto(s)
Gránulos Citoplasmáticos/química , Estro , Trompas Uterinas/ultraestructura , Glicoconjugados/análisis , Lectinas , Animales , Cricetinae , Femenino , Glicoconjugados/metabolismo , Histocitoquímica , Lectinas/metabolismo , Mesocricetus , Microscopía ElectrónicaRESUMEN
Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcbeta1,4Galbeta1,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcbeta1,4Galbeta1,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcbeta1,4Galbeta1,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcbeta1,4Galbeta1,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcbeta1,4Galbeta1,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.
Asunto(s)
Disacáridos/metabolismo , Zona Pelúcida/metabolismo , Zona Pelúcida/ultraestructura , Animales , Disacáridos/análisis , Femenino , Histocitoquímica , Ratones , Microscopía Electrónica , N-Acetilgalactosaminiltransferasas/análisis , N-Acetilgalactosaminiltransferasas/metabolismoRESUMEN
In the present study, we examined by immunohistochemistry the cell-specific distribution of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in the mouse uterus during the oestrous cycle and throughout the first 7 days of pregnancy. Paraffin-embedded tissue samples were immunostained using the avidin-biotin peroxidase technique and then examined by light microscopy. Our results showed that immunostaining for EGF was detected in the stroma but not in the luminal or glandular epithelium. A high concentration of EGF was detected in the stroma around the time of embryo implantation at days 3, 4 and 5 of pregnancy. The implanted embryo at day 7 of gestation showed immunostaining for EGF between the ectoderm and endoderm layers. The cell distribution pattern for PDGF was found to be different from that observed with EGF. Luminal and glandular epithelia displayed PDGF immunostaining throughout the first 7 days of pregnancy, with the highest intensity at days 4 and 5 of gestation. In contrast, no immunostaining was observed in the luminal and glandular epithelia at post-oestrus, dioestrus and pro-oestrus stages. However, a weak reaction started to appear at oestrus. The embryo at the blastocyst stage displayed a strong immunoreaction for antibody against PDGF. In addition, the decidual boundary zone surrounding the implanted embryo at days 5, 6 and 7 of gestation also showed an immunostaining for PDGF. The present observations demonstrate clearly the presence of EGF and PDGF in the mouse uterus in high concentrations at the peri-implantation period. Thus, our results, together with what is known about the effect of EGF and PDGF in controlling the growth, differentiation and activation of a variety of cell types, suggest a possible role for these growth factors during the preparation of the endometrium for implantation in controlling the proliferation activity of stromal and/or epithelial cells.
Asunto(s)
Factor de Crecimiento Epidérmico/análisis , Estro/metabolismo , Factor de Crecimiento Derivado de Plaquetas/análisis , Preñez , Útero/química , Animales , Femenino , Edad Gestacional , Inmunohistoquímica , Ratones , Embarazo , Glándulas Salivales/química , Factores de Tiempo , Distribución TisularRESUMEN
Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas del Huevo/metabolismo , Fertilización/fisiología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular , Zona Pelúcida/metabolismo , Animales , Anticuerpos Monoclonales/química , Carbohidratos/química , Proteínas del Huevo/química , Femenino , Galactosa Oxidasa/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Indicadores y Reactivos , Lectinas , Glicoproteínas de Membrana/química , Ratones , Neuraminidasa/química , Oocitos/química , Oocitos/metabolismo , Embarazo , Glicoproteínas de la Zona PelúcidaRESUMEN
Sulfoglycolipid immobilizing protein 1 (SLIP 1) is an evolutionally conserved sperm head plasma membrane protein (M(r) = 68 kDa) that binds to sulfogalactosylglycerolipid (SGG), the major sulfoglycolipid present in mammalian sperm. The purpose of this study was to characterize the initial localization and the immunoaggregated relocalization of SLIP1 on the mouse sperm head. Direct immunofluorescence (DF) of live sperm using FITC-antiSLIP1 Fab fragments and FITC-antiSLIP1 IgG indicated that SLIP1 was present in the postacrosomal region of the sperm head, although the intensity of immunostaining by FITC-antiSLIP1 IgG was greatest at the border between the postacrosomal region and the acrosome. Unlike that observed with FITC-antiSLIP1 Fab, DF using FITC-antiSLIP1 IgG indicated that SLIP1 was also present in the anterior tip of the sperm head convex ridge. Results from electron microscopic studies, using antiSLIP1 IgG followed by protein A-gold on live mouse sperm, were similar to the DF findings. In contrast, indirect immunofluorescence (IIF) of live mouse sperm using antiSLIP1 IgG and FITC-secondary antibody IgG detected SLIP1 in the sperm head convex ridge only. The IIF and DF results strongly suggest that these bivalent antibodies could induce the sperm antigen relocalization on live sperm heads. SLIP1 redistribution may be dependent on availability of excess SGG, the SLIP1 binding ligand, based on the observation that purified exogenous biotinylated SLIP1 bound to live mouse sperm at both the postacrosomal and convex ridge regions of the mouse sperm head. Immunoaggregation induced by the primary antiSLIP1 IgG or antiSLIP1 Fab with secondary antibody IgG did not cause the acrosome reaction, suggesting that SLIP1 is not involved in sperm signal transduction. Furthermore, postacrosomal SLIP1 was shown to be involved in zona binding, since sperm pretreated with antiSLIP1 Fab fragments (100 micrograms/ml) bound to the egg zona pellucida in vitro at approximately 35% of control levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Cabeza del Espermatozoide/metabolismo , Animales , Proteínas Portadoras/inmunología , Femenino , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Inmunoelectrónica , Proteínas de Unión al ARN , Cabeza del Espermatozoide/ultraestructura , Interacciones Espermatozoide-Óvulo/fisiologíaRESUMEN
Oviductins are a family of glycoproteins which are synthesized and secreted by oviductal secretory cells and which, upon their secretion in the lumen of the oviduct, become associated with postovulatory oocytes and developing embryos. Recently, we showed that hamster oviductin is maximally secreted in the oviduct at the time of ovulation and is later associated with a certain population of uterine epithelial cells, where it is subsequently endocytosed and degraded. In light of these results, this study was conducted to follow the fate of hamster oviductin in the oviduct and uterus during early gestation. Using a monoclonal antibody against hamster oviductin, immunofluorescence and immunogold labeling revealed that during early gestation, immunoreactivity to oviductin in the uterus gradually diminished to an almost total disappearance at time of implantation. However, the strong labeling intensity remained unchanged in the oviduct. Biochemical analyses demonstrated that a degradation of oviductin occurs in the uterus, and a loss of immunoreactivity was also observed as gestation progressed, so that by the time of implantation, immunoreactivity to oviductin was barely detectable. The decrease of oviductin along the uterine epithelium at the time of blastocyst attachment and its final disappearance at implantation suggest that this glycoprotein could be a potential modulator of uterine receptivity.
Asunto(s)
Trompas Uterinas/enzimología , Serina Endopeptidasas/metabolismo , Útero/enzimología , Animales , Western Blotting , Cricetinae , Electroforesis en Gel de Poliacrilamida , Femenino , Edad Gestacional , InmunohistoquímicaRESUMEN
Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.
Asunto(s)
Colesterol/análisis , Lípidos de la Membrana/análisis , Fosfolípidos/análisis , Capacitación Espermática , Espermatozoides/ultraestructura , Animales , Filipina , Técnica de Fractura por Congelación , Oro Coloide , Histocitoquímica , Masculino , Ratones , Fosfolipasas A , Fosfolipasas A2 , Espermatozoides/fisiologíaRESUMEN
We used fracture-label and label-fracture cytochemistry in conjunction with the phospholipase A2-colloidal gold (PLA2-CG) technique to study the distribution of phospholipids in ejaculated boar spermatozoa. These techniques provide visualization of the topographical distribution of phospholipids in freeze-fractured sperm membranes in a three-dimensional view. In various freeze-fractured boar sperm membranes and crossfractured cytoplasmic structures, quantitative analysis revealed that the nuclear envelope membranes and the nuclear content possessed the highest labeling density of PLA2-CG. Moderate labeling was detected over acrosomal membranes, especially the inner acrosomal membrane. Replicas of both protoplasmic and exoplasmic fracture faces of the plasma membrane of boar sperm head showed a relatively low density of PLA2-CG labeling. Moreover, a differential distribution of phospholipids was seen over the protoplasmic face of the plasma membrane domains of the sperm head, which showed the highest concentration of gold particles in the postacrosomal region, followed by the equatorial segment and the anterior acrosome region. The PLA2-CG labeling densities over the post-acrosomal region and the equatorial segment were significantly higher than that over the anterior acrosome region. In the flagellum, an intense labeling was also seen over crossfractured mitochondria, dense fibers, and fibrous sheath. The protoplasmic fracture face of the plasma membrane over the middle piece, the annulus, and the principal piece was moderately labeled by PLA2-CG. No significant difference in mean labeling density of PLA2-CG was detected among the three membrane domains. In label-fracture preparations, exoplasmic halves of the plasma membrane of the head and the middle piece of the tail were uniformly labeled with PLA2-CG. However, the annulus and principal piece were devoid of PLA2-CG binding sites. These results indicate that differential distribution of phospholipids associated with the boar sperm membranes may reflect phospholipid composition of membrane domains characteristic of special physiological functions.
Asunto(s)
Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Espermatozoides/metabolismo , Animales , Membrana Celular/metabolismo , Técnica de Fractura por Congelación , Histocitoquímica , Membranas Intracelulares/metabolismo , Masculino , Espermatozoides/ultraestructura , PorcinosRESUMEN
Smooth endoplasmic reticulum assembly was studied in a cell-free system using thin-section and freeze-fracture electron microscopy. Incubation of rat hepatocyte rough and smooth microsomes in the presence of ATP, GTP, cytosol (Xenopus egg) and an ATP-regenerating system led to assembly of membrane networks comprising a central core of interconnecting smooth tubules continuous with peripherally located rough membrane cisternae. Glucose-6-phosphatase cytochemistry confirmed the endoplasmic reticulum origin of the reconstituted membranes. When both ATP and GTP were omitted from the incubation medium, or when GTP was replaced by a variety of nucleotide analogues, including GTP gamma S, membrane aggregates contained only unfused microsomes. The presence of GTP alone stimulated assembly of rough membrane cisternae but had no effect on smooth membranes. Smooth tubule formation occurred independent of cytosol and an ATP-regenerating system, but did require GTP and ATP. Omission of ATP, or replacement of this nucleotide with a variety of analogues, including ATP gamma S, prevented tubule formation but did not affect the assembly of the rough membrane cisternae. Morphometric studies revealed sequential formation of rough membrane cisternae (0-60 minutes) followed by appearance of interconnecting smooth tubules (> 60 minutes). The amount of rough membrane cisternae per membrane network diminished with time after 60 minutes; that of smooth tubules increased. Thus GTP is required for reconstitution of rough membrane cisternae, both GTP and ATP are required for smooth tubule formation, and assembly of smooth tubules occurs as an outgrowth (i.e. via tubulation) from rough membranes.
Asunto(s)
Retículo Endoplásmico Rugoso/ultraestructura , Retículo Endoplásmico Liso/ultraestructura , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Sistema Libre de Células , Citosol/metabolismo , Retículo Endoplásmico Rugoso/efectos de los fármacos , Retículo Endoplásmico Rugoso/metabolismo , Retículo Endoplásmico Liso/efectos de los fármacos , Retículo Endoplásmico Liso/metabolismo , Técnica de Fractura por Congelación , Glucosa-6-Fosfatasa/metabolismo , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacología , Técnicas In Vitro , Cinética , Microscopía Electrónica , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/ultraestructura , Nucleótidos/farmacología , RatasRESUMEN
Oviductal non-ciliated secretory epithelial cells, under hormonal stimulation, synthesize and secrete a family of glycoproteins referred to as oviductins. These glycoproteins are found in oviductal fluid in several mammalian species, and have been localized in the oviduct, and in the zona pellucida of ovulated oocytes. In the golden hamster, this glycoprotein is named hamster oviductin-I. Recently, an immunofluorescent study on hamster uterine tissue has revealed the presence of the glycoprotein in luminal epithelial cells in a heterogeneous labelling pattern during the estrous cycle. The mechanism of endometrial epithelial cell receptivity to hamster oviductin-1 is not known. In this study, immunohistochemical studies were performed using a monoclonal antibody against the oviductin in conjunction with silver enhancement technique, in an attempt to determine further the factors playing a role in uterine receptivity to oviductin-1. Paraffin sections of hamster uterus obtained from different stages of the estrous cycle and from days 1-6 of gestation, and paraffin sections of hamster oviduct obtained from days 1-6 of gestation were used in this study. The results we obtained using the silver enhancement technique show that hamster uterus luminal epithelial cells exhibit a homogeneous, high intensity immunolabelling pattern throughout the estrous cycle, whereas, during gestation, labelling intensity decreases as the period for blastocyst implantation approaches. Oviduct epithelial cells revealed no definite fluctuating pattern in immunolabelling intensities during gestation, indicating no change in synthesis and secretion of the glycoprotein during this period. It is speculated that receptors for hamster oviductin-1 are present at the apical cell surface of endometrial cells and that implantation of the developing blastocyst into the uterine wall is possible only following downregulation of these receptors. The use of the silver enhancement technique proves to be an effective tool in immunohistochemical studies at the light microscope level, as seen through this study.
Asunto(s)
Estro/fisiología , Inmunohistoquímica/métodos , Preñez/fisiología , Serina Endopeptidasas/análisis , Útero/química , Animales , Cricetinae , Endometrio/química , Endometrio/metabolismo , Trompas Uterinas/química , Trompas Uterinas/metabolismo , Femenino , Mesocricetus , Embarazo , Serina Endopeptidasas/inmunología , Útero/metabolismoRESUMEN
In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins that become associated with the zona pellucida of the ovulated oocyte. These glycoproteins are collectively designated as oviductins. A monoclonal antibody directed against hamster oviductin was used to study the ontogeny of this glycoprotein. Indirect immunofluorescence experiments performed on sections of hamster oviduct revealed that the glycoprotein begins to be secreted in 10-day-old females and that all of the oviductal secretory cells showed fluorescent staining by day 14. The intensity of the immunofluorescence reaction reached a maximum in the 28-day-old females. The oviducts of the 7-day-old hamster incorporated [35S]methionine in vitro into several proteins; however, the production and secretion of detectable amounts of radiolabeled oviductin only began at 14 days of age and reached a maximum at day 28 of age. It appears that the ontogeny of oviductin parallels the hormone dependent changes leading to sexual maturation and that its maximum secretion is already established at the time of the first ovulatory cycle. These results are substantiated by the fact that the production of oviductin is induced in estradiol-treated, but not progesterone or non-treated prepubertal animals, as determined by indirect immunofluorescence experiments.
Asunto(s)
Estradiol/farmacología , Trompas Uterinas/metabolismo , Serina Endopeptidasas/biosíntesis , Envejecimiento/fisiología , Animales , Anticuerpos Monoclonales , Autorradiografía , Western Blotting , Cricetinae , Trompas Uterinas/efectos de los fármacos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Mesocricetus , Técnicas de Cultivo de Órganos , Progesterona/farmacología , Zona Pelúcida/metabolismoRESUMEN
The hamster oviduct secretes a high molecular weight antigen that belongs to the family of glycoproteins known as oviductins. In the present study, using immuno-electron microscopy, we examined the location of this hamster oviductin-1 (Hm Ov-1) in hamster oviductal oocytes and early embryos up to the blastocyst stage. The immunoreactive pattern of Hm Ov-1 changes markedly during the embryo development. In oviductal oocytes prior to fertilization, Hm Ov-1 was associated exclusively with the zona pellucida. Following fertilization, immunolabeling was detected in the perivitelline space and over the plasma membrane of 2-cell, 4-cell, and 8-cell embryos as well as young blastocysts. The change of the immunoreactive pattern was accompanied by the formation of an abundant number of coated pits, endocytic vesicles, multivesicular bodies, and lysosomal-like structures which were strongly labeled by gold particles. These immunogold-labeled cytoplasmic organelles characteristic of the endosomal-lysosomal apparatus were particularly evident in 2-cell, 4-cell, and 8-cell embryos and showed a decrease in number in the blastocysts. The close resemblance between the labeled flocculent material detected in the perivitelline space and that found in the zona matrix of early embryos and blastocysts suggested that the Hm Ov-1-associated electron-dense, flocculent material in the perivitelline space originated from the zona pellucida and was later endocytosed by the blastomeres through coated pits and endocytic vesicles. The detection of Hm Ov-1 in numerous multivesicular bodies and lysosomal structures indicated that the oviductin is eventually degraded. Although the exact functional role of Hm Ov-1 is not known, the presence of a copious amount of Hm Ov-1 in early hamster embryos may be ascribed to a special relationship between this particular oviductin and embryo development.
Asunto(s)
Blastocisto/química , Desarrollo Embrionario y Fetal/fisiología , Trompas Uterinas/embriología , Oocitos/química , Serina Endopeptidasas/análisis , Animales , Anticuerpos Monoclonales , Blastocisto/ultraestructura , Cricetinae , Epitelio/embriología , Epitelio/ultraestructura , Trompas Uterinas/ultraestructura , Femenino , Inmunohistoquímica , Mesocricetus , Microscopía Inmunoelectrónica , Oocitos/ultraestructura , Orgánulos/química , Serina Endopeptidasas/ultraestructura , Zona Pelúcida/química , Zona Pelúcida/ultraestructuraRESUMEN
The fracture-label technique was used in conjunction with a monoclonal antibody to actin and the phospholipase A2-colloidal gold (PLA2-CG) method to examine the spatial distribution of actin filaments in relation to the three-dimensional arrangement of tight junctional strands in rat testes and exocrine pancreatic acinar cells. The intimate association of actin filaments with tight junctional strands in the pancreas and testis was also illustrated by a double-labeling experiment in which freeze-fractured pancreas or testis was labeled with monoclonal antibody-protein A-gold (30 nm gold size) followed by incubation with a PLA2-CG complex (11 nm gold size). Freeze-fracture-exposed tight junctional strands in both testicular and exocrine pancreatic cells labeled by PLA2-CG complex, indicated the presence of phospholipids in these cylindrical membranous structures. Immunolabeling of freeze-fractured testes with a monoclonal antibody to actin revealed a narrow band of gold particles juxtaposed to the cytoplasmic aspect of the protoplasmic membrane halves decorated with parallel linear arrays of cylindrical tight junctional strands. Many of the gold particles representing actin antigenic sites were in direct contact with the cross-fractured tight junctional strands. Fracture-label preparations of exocrine pancreas labeled with the monoclonal anti-actin antibody also exhibited a similar labeling pattern at the apex of acinars cells where the tight junction complex is located. Double-labeling experiments revealed the simultaneous labeling of actin and phospholipids in the same fracture-label preparations. Digestion of testicular and pancreatic tissue samples in a free PLA2 solution prior to labeling with the monoclonal antibody or PLA2-CG complex removed not only the gold labeling previously seen over the tight junctional strands but also reduced drastically the immunolabeling for actin that was previously seen associated with the tight junction complex. Taken together, results of the present study showed that actin filaments are structural components of the tight junction strands and are connected to the cytoplasmic aspect of the latter structures. The interaction between this particular cytoskeletal element and the tight junction may be through the binding of a special domain of the actin filament to the phospholipids that partially make up the tight junctional complex.