bluuebunny/crossref_metadata_2025_split huggingface dataset:
Zona pellucida modifications in the mouse in the absence of oocyte activation
Authors: Vincent C. , Turner K. , Pickering S. J. , Johnson M. H. 
Published: February 2005

AbstractMouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168–175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313–324, 1987). This paper shows that dimethylsulfoxide cause proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.
Preimplantation embryo biopsy: Detection of trisomy in a single cell biopsied from a four‐cell mouse embryo
Authors: Kola Ismail , Wilton Leeanda 
Published: February 2005

AbstractThis paper describes an efficient technique for the production of metaphase spreads from single blastomeres biopsied from four‐cell preimplantation mouse embryos. The karyotype obtained by chromosomal analysis of single biopsied cells is shown to be fully predictive of subsequent fetal karyotype. The data in this study also demonstrate that the entire process of embryo biopsy and karyotypic analysis of biopsied blastomeres does not adversely affect the ability of biopsied embryos to form fetuses after transfer into pseudopregnant recipients. This study has potential clinical relevance in that it demonstrates that chromosomally defective embryos can be accurately identified before implantation. In addition, the techniques developed in this study may facilitate more efficient procedures for the genesis of animal models for human disorders such as Down syndrome and Alzheimers disease.
Follicle somatic cells influence pig oocyte penetrability and cortical granule distribution
Authors: Galeati G. , Modina S. , Lauria A. , Mattioli M. 
Published: February 2005

AbstractThe influence of somatic cells on oocyte penetrability was studied during in vitro maturation. Four experiments were carried out. In the first, pig oocytes were fertilized in vitro immediately after collection (immature oocytes) or after being cultured for 44 hr with cumulus cells connected to the whole wall of the extroverted follicle (follicle oocytes) or without cumulus cells (denuded oocytes) (Mattioli et al.: Gamete Res 20:177–183, 1988a). In follicle and immature oocytes, 12 hr after insemination, the sperm were equally distributed between zona and ooplasm; in denuded oocytes, the majority (90.5%) of the sperm were located in the zona. In the second experiment, zonae pellucidae of follicle and denuded oocytes, obtained after 44 hr of culture, were incubated with a sperm suspension for 2 hr at 39°C to evaluate the zona binding. The different number of sperm found on the zona pellucida of follicle or denuded oocytes (49.3 ± 2.97 vs. 37.8 ± 1.77) did not achieve statistical significance (P > 0.05). In the third experiment, follicle and denuded oocytes were denuded of their zonae after maturation and then fertilized in vitro. The number of sperm recorded in the ooplasm of zona‐free follicle oocytes was significantly higher than that recorded in zona‐free denuded oocytes (9.2 ± 0.93 vs 1.19 ± 0.28) (P<0.01). In the last experiment the influence of somatic cells on the distribution of cortical granules was evaluated. Pig oocytes were denuded at different stages of culture and, after completion of maturation, processed for electron microscopy. The removal of somatic cells at 5–20 hr of culture resulted in a premature migration of cortical granules underneath the oolemma. An increase of exocytotic events was also recorded. These data demonstrate that the presence of cumulus cells during maturation is fundamental for the pig oocyte to maintain its penetrability. The persistence of the interaction between cumulus cells and oocyte stabilizes the distribution of cortical granules, and the interruption of this coupling induces cortical granule exocytosis and may be responsible for the reduced penetrability of oocytes matured without cumulus cells.
Nucleocytoplasmic translocation of ribosomal RNA in the rabbit blastocyst: Participation of sulfhydryl groups
Authors: Manes Cole 
Published: February 2005

AbstractThe release of ribosomes from the nucleus in the rabbit blastocyst was investigated by pulse‐labeling embryos to within 5 min of the earliest appearance of radiolabeled ribosomal RNA (rRNA) in the cytoplasmic fraction. The accumulation of radiolabeled 4.7 and 1.9 kilobase mature rRNA species in the cytoplasm was then followed during a 2 hour chase period, using polyacrylamide gel electrophoresis to identify the rRNAs. Colchicine, cytochalasin B, KCN, and EDTA were found to have no effect on the release of radiolabeled rRNA from the blastocyst nucleus during the 2 hour chase. Oligomycin, a known inhibitor of the nuclear envelope nucleoside triphosphatase, and the protein synthesis inhibitors puromycin and cycloheximide blocked rRNA release after a short delay. In contrast, actinomycin D and the sulfhydryl‐reactive agents N‐ethylmaleimide and diamide produced an abrupt and complete block to further rRNA release. The results indicate that ribosomes leave the nuclear compartment by an energy‐dependent process. They further underscore the importance of reduced sulfhydryl groups in a rapidly growing blastocyst with a high level of oxidative metabolism.
Analysis of proteins expressed at the time of murine organogenesis
Authors: Van Keuren Margaret L. , Iacob Ruxandra A. , Kurnit David M. 
Published: February 2005

AbstractTwo‐dimensional electrophoretograms were prepared from wild‐type C57BL/6J embryos from day 7.5 through day 9.0 of development. This time period encompasses a critical window of development as the embryo traverses from an egg cylinder through major organogenesis. Consequently, we term this resource MOPED (for mouse organogenesis protein electrophoresis database). By resolving and analyzing the behavior of approximately 1,000 polypeptides per time point, we were able to track many of these polypeptides through this time period in development. Of special note was a burst of induced protein synthesis that was observed on day 8.5 of development. Polypeptides observed in mouse embryos that match those identified previously in mouse fibroblasts were noted. Two of them (the intermediate filament‐associated protein and tropomyosin‐4) were significantly altered in 8.5 day embryos. As more polypeptides are designated, it will be possible to expand the known proteins in the database. MOPED establishes the patterns of synthesis of a large number of polypeptides during a crucial period of development. Thus MOPED is designed to analyze proteins relevant to mouse embryogenesis in the future.
Guinea pig proacrosin is synthesized principally by round spermatids and contains O‐linked as well as N‐linked oligosaccharide side chains
Authors: Anakwe Onyeama O. , Sharma Sunita , Hardy Daniel M. , Gerton George L. 
Published: February 2005

AbstractProacrosin is the zymogen precursor of acrosin, a sperm protease believed to play an essential role in fertilization. In this study, we used primary cultures of guinea pig spermatogenic cells to examine the temporal appearance and mechanisms of synthesis and processing of proacrosin during acrosome development. Following [35S]methionine incorporation and immunoprecipitation, cultured spermatogenic cells were found to synthesize two forms of proacrosin (Mr 54,000 and 57,000). Proacrosin was synthesized mainly by round spermatids. By immunoblotting, proacrosin became very prominent in round spermatids and persisted throughout spermiogenesis. Pluse‐chase experiments demonstrated that the Mr 54,000 form of proacrosin was converted to the Mr 57,000 form, presumably reflecting posttranslational processing of carbohydrate side chains. When spermatogenic cells were cultured in the presence of tunicamycin, the synthesized proacrosin had an Mr of 54,000. However, in vitro translation of mRNA extracted from guinea pig testis followed by immunoprecipitation indicated that the core polypeptide of procrosin has an Mr of 44,000. Guinea pig spermatogenic cells incorporated glucosamine and fucose into the oligosaccharides of proacrosin. Treatment of guinea pig testis proacrosin with N‐glycosidase or O‐glycosidase reduced the Mr by 3–7%. These results indicate that proacrosin is synthesized by postmeiotic cells and the enzyme contains N‐ and O‐linked oligosaccharides.
Complement component C1q and its receptor are involved in the interaction of human sperm with zona‐free hamster eggs
Authors: Fusi F. , Bronson R. A. , Hong Y. , Ghebrehiwet B. 
Published: February 2005

AbstractC1q is a component of the classical complement pathway that can react with the Fc‐fragment of immunoglobulins and with other proteins, such as fibronectin, laminin, and a specific C1q receptor present on several cell types. Given its role in many adhesion systems, mainly related to phagocytosis, we tested the effects of C1q on the interaction between human spermatozoa and zona‐free hamster eggs. The presence of C1q in the medium used for gamete coincubation resulted in promotion of sperm‐oolemma adhesion and an inhibition of penetration. The number of adherent sperm per egg at 5 μg/ml concentration was 90 ± 35 vs. 29 ± 7 for the control (P < 0.001). At 1 μg/ml, the lower concentration at which C1q had an effect, the number of penetrating sperm/egg was 0.6 vs. 1.7 for the control without C1q (P < 0.01), and the percent of penetrated eggs was 28% vs. 85%. At 50 μg/ml, the percent of penetrated eggs was 7%, with a penetration index of 0.07. The addition of C1q to the medium resulted in sperm agglutination, which varied between sperm donors. The presence of C1q receptors, as detected by anti‐C1qR monoclonal antibodies (Mabs), was demonstrated both on zona‐free hamster eggs by immunobead rosetting and on human spermatozoa by immunobead binding and indirect immunofluorescence. Mabs directed against different epitopes of C1qR had different effects on gamete interaction, with a partial inhibition of penetration mediated by some of them. The binding of C1q to antibody‐free human spermatozoa was also demonstrated both by means of indirect immunofluorescence and utilizing 125I‐C1q. Cur results suggest that C1q and its receptor could be involved in fertilization and provide additional support for the hypothesis that sperm incorporation by the oocyte utilized a family of cell membrane receptors or binding sites similar to those involved in phagocytosis.
Immunoelectron microscopic localization of small nuclear ribonucleoproteins during bovine early embryogenesis
Authors: Kopečný V. , Fakan S. , Pavlok A. , Pivko J. , Grafenau P. , Biggiogera M. , Leser G. , Martin T. E. 
Published: February 2005

AbstractSmall nuclear ribonucleoproteins (snRNPs) were localized using human autoimmune or monoclonal anti‐snRNP antibodies and ultrastructural immunocytochemistry in early preimplantation bovine embryos before and at transcription onset. Bovine cleavage stages up to 16‐cell embryos were obtained either by culture of in vitro‐fertilized ovarian oocytes or by isolation at slaughter of embryos fertilized and developed in vivo. Before transcription onset, up to the four‐cell stage, diffuse labeling of nucleoplasm was detected, whereas cytoplasm labeling remained low. At the transcription onset, labeling of all eight‐cell embryo nuclei was markedly concentrated at the borderline of already formed, condensed chromatin aggregates, where it was associated mainly with perichromatin fibrils. The condensed chromatin blocks revealed by several different staining methods were more prominent than is the case in most somatic cells. The degree of chromatin condensation and the pattern of its distribution differed between in vivo‐ and in vitro‐produced eight‐cell embryos: the in vitro embryos showed a higher degree of chromatin condensation and a peripheral distribution of chromatin blocks. A relation of this observation to the developmental potential of cow embryos is suggested. In two‐ and four‐cell embryos, intensive labeling was seen in interchromatin granules, which, in their turn, were often seen in close proximity to the nucleous precursor bodies, or in the four‐cell stage were interconnected to them. No labeling was ever seen, using antibodies specific for the snRNP Sm antigen, in nucleolar precursor bodies during embryonic nucleologenesis nor in the resulting nucleoli. There was some incidental labeling of the large central vacuole appearing at the beginning of the nucleolus precursor body transformation, testifying the nucleoplasmic origin of this entity.
Cyclic appearance of cytoplasmic NOR–silver‐stained particles in sea urchin embryos
Authors: Amikura Reiko M. , Ihnuma Morio , Aikawa Eizo , Yamada Hisafumi , Nagano Hiroshi 
Published: February 2005

AbstractWhen sea urchin embryos were subjected to nucleolar organizer region (NOR)‐silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell‐cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine‐treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) showed that 95‐kDa and 38‐kDa proteins were the NOR–silver‐staining proteins in sea urchin embroys. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95‐kDa and 38‐kDa proteins are the major NOR–silver‐staining proteins in sea urchin embryos.
Mouse blastocysts respond metabolically to short‐term stimulation by insulin and IGF‐1 through the insulin receptor
Authors: Harvey Mark B. , Kaye Peter L. 
Published: February 2005

AbstractInsulin specifically stimulates protein synthesis in compacted mouse embryos on days 3 and 4 after fertilization, with an EC50 of 0.5 pM (Harvey and Kaye, 1988). The identity of the receptor mediating this short‐term effect of insulin was further examined by dose–response studies with IFG‐1 and by using a specific anti‐insulin receptor antiserum that has no appreciable cross‐reaction with IGF‐1 receptors. IGF‐1 caused a maximum 40% stimulation of protein synthesis after 4 h exposure (similar to the response to insulin) with an EC50 of 150 pM IGF‐1. The insulin receptor‐specific antiserum, or IgGs isolated from it, also stimulated protein synthesis at dilutions as high as 1:1,000 to the same degree as insulin (∼40%). This agonistic action of the insulin receptor antiserum, the EC50 of 150 pM for IGF‐1, and the previously established EC50 of 0.5 pM for insulin, all with similar maximal stimulation, strongly support the conclusion that the short‐term metabolic stimulation of mouse blastocysts by insulin is mediated by insulin receptors. Immunosurgical isolation of inner cell masses before and after exposure to 1.7 pM insulin (sufficient to stimulate only the insulin receptor) showed that insulin stimulates protein synthesis in these cells as well as in the trophectoderm cells of the blastocyst. This finding suggests that in intact blastocysts, insulin may travel across the trophectoderm to the inner cell mass, acting anabolically on both tissues. Analysis of the agonistic effect of the B‐10 antiserum showed there was no evidence of an unresponsive subpopulation of embryos.
Sperm analysis by flow cytometry using the fluorescent thiol labeling agent monobromobimane
Authors: Seligman J. , Shalgi R. , Oschry Y. , Kosower N. S. 
Published: February 2005

AbstractThe fluorescent labeling agent monobromobimane (mBBr) was used to label thiols and disulfides (after reduction of sperm disulfides by dithiothreitol) in intact spermatozoa. Bimane‐labeled sperm of several mammalian species were analyzed by flow cytometry (FCM) and examined by fluorescent microscopy. FCM analysis showed sperm thiol oxidation to disulfides during epididymal maturation. FCM of labeled mature spermatozoa showed differences among species in the sperm thiol content. Heterogeneity in thiol content of sperm within individual samples was also observed. In addition, FCM patterns showed heterogeneity among and within samples in the content of disulfides and their resistance to reduction. FCM analysis reflected the microscopic appearance of the labeled spermatozoa. FCM analysis of bimanelabeled spermatozoa offers a convenient method for the study of sperm thiol–disulfide status and permits detection of sperm subpopulations within an individual sample. FCM analysis of mBBr‐labeled spermatozoa may serve as a test to evaluate sperm quality.
Characterisation of 24‐kD proteins from rat testes using polyclonal sera reactive to human sperm antigens
Authors: Shaha Chandrima , Seshadri T. , Suri A. , Talwar G. P. 
Published: February 2005

AbstractA group of antigens of 24‐kD Mr from rat testes were characterised biochemically. These antigens were part of a larger molecule of ∼200 kD. On treatment with disulfide bond reducing agent, the 200‐kD molecule was reduced to subunits. Immunoreactivity was confined to a doublet of ∼24 kD and a single band of ∼50 kD Mr after the reduction. Glycoprotein in nature, this antigen shared immunoreactive epitopes with a 40‐kD antigen on human spermatozoa. Antiserum raised in rabbits against the 24‐kD antigen from rat testes reacted with antigens on the acrosome of human spermatozoa. Agglutination of sperm could be induced by the antiserum. The carbohydrate residue could be removed by mannosidase digestion. Chemical deglycosylation studies showed a slight decrease in molecular weight. Immunoreactivity was however not completely lost after chemical deglycosylation. Isoelectric focusing of the antigen identified nine isoelectric species. Two relatively minor species showed immunoreactivity. Acrosome‐reacted spermatozoa showed loss of antigens from acrosome.
Developmental regulation of a serum response element binding activity in amphibian embryos
Authors: Varley Joel , Brennan Sean 
Published: February 2005

AbstractAs part of our studies of transcriptional control during early development in vertebrates, we have examined embryos of the amphibian Xenopus laevis for the presence of sequence‐specific DNA‐binding proteins, using gel electrophoresis mobility‐shift assays. Our analysis has focused on sequence elements in the cytoskeletal actin gene, whose embryonic transcription is initially activated at the gastrula stage, approximately 16 hours after fertilization. We detect activites capable of specific binding to two known transcriptional regulatory elements, the serum response element and the GC‐box, located in the 5′‐flanking region of the cytoskeletal actin gene. Binding activity specific for a region downstream of the transcriptional startsite is also detected, in a region which may be involved in controlling developmental activation of this gene. Serum response element‐binding activity, as well as the downstream binding activity, is enriched in extracts from gastrula and neurula stage embryos, compared to egg extracts, suggesting that increased levels of one or both of these activities might play a role in developmentally timed transcriptional activation of the cytoskeletal actin gene in the embryo.
Vitrification of mouse oocytes: Improved rates of survival, fertilization, and development to blastocysts
Authors: Shaw P. W. , Fuller B. J. , Bernard A. , Shaw R. W. 
Published: February 2005

AbstractRall and Fahy's (1985) vitrification procedure for the cryopreservation of 8‐cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1‐exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5–8°C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.
Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization
Authors: Schwerin Manfred , Blottner Steffen , Thomsen Preben D. , Roschlau Daniela , Brockmann Gudrun 
Published: February 2005

AbstractAn easy assay for quantification of Y chromosome‐bearing sperm (Y‐sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male‐specific DNA fragment was tested for identification of Y‐sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y‐sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.