bluuebunny/crossref_metadata_2025_split huggingface dataset:
Different cytoskeletal organization in two maturation stages of Discoglossus pictus (anura) oocytes: Thickness and stability of actin microfilaments and tropomyosin immunolocalization
Authors: Campanella Chiara , Chaponnier Christine , Quaglia Lucia , Gualtieri Roberto , Gabbiani Giulio 
Published: February 2005

AbstractIn Discoglossus pictus oocytes, the germinative area (GA) contains long and irregular microvilli where actin microfilaments are located. In the egg, the funnel‐shaped dimple that originates by invagination of the GA is present. In the dimple both microvilli and microfilament bundles have a very orderly appearance. This report extends previous observations (Campanella and Gabbiani, Gamete Res 3:99–114, 1980) and shows that GA microfilaments are thinner (36 Å average) than dimple microfilaments (60 Å average), as measured in ultrathin section. Moreover, the interfilament distance is smaller in GA bundles than in the dimple bundles. To get an insight into actin‐depolymerizing factor (ADF) in which cryostat sections were incubated prior to immunofluorescent staining with antiactin antibodies. The microfilaments of the GA microvilli and partially of the oocyte cortex are resistant to ADF when compared to those in the dimple and the rest of the egg cortex. We also investigated immunocytochemically the presence of tropomyosin and found that this protein is localized in the dimple and in the cortex of oocytes and eggs but is absent in the GA.
Bovine oviductal fluid components and their potential role in sperm cholesterol efflux
Authors: Ehrenwald Eduardo , Foote Robert H. , Parks John E. 
Published: February 2005

AbstractBovine oviductal fluid (OF) was collected and analyzed throughout the estrous cycle, and the capacity of the protein and lipoprotein components to support cholesterol efflux from bovine sperm was evaluated. Blood was collected and assayed for progesterone (P4) to monitor the estrous cycle. Protein and lipoprotein separation was achieved by density gradient centrifugation. Two major bands were identified. The first (1.056 < ∂20 < 1.140 g/ml) corresponded to bovine and rabbit plasma high‐density lipoprotein (HDL) based on distribution in the density gradient and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The second band (1.235 < ∂20<1.243 g/ml) consisted predominantly of oviductal fluid albumin (OFA). Oviductal fluid protein concentration increased as serum P4 decreased around the time of estrus. Mean OF protein concentration was 21.3 mg/ml when serum P4 was lower than 0.5 ng/ml and 6.9 mg/ml when serum P4 was greater than 0.5 ng/ml. An inverse log relationship was found between HDL protein concentration and serum P4. Unesterified cholesterol (UC), cholesteryl ester, and phospholipid (PL) content of HDL for HDL protein concentrations of 3–56.1 μg/ml were 1.35–46.2 μg/ml, 1.91–44.48 μg/ml, and 1.69–59.8 μg/ml, respectively. Phosphatidylcholine and ‐ethanolamine were the major PLs present in the HDL fraction and their molar ratio (4:1 mol/mol) was relatively constant through the estrous cycle. The OFA fraction of the same samples accounted for more than 90% of total protein and for most of the variation in OF protein. To determine the ability of OF components to serve as sperm cholesterol acceptors, OF samples were incubated 1:1 (v/v) with and without 4 × 108 bovine sperm in 1.0 ml of modified Tyrode's solution and OF for 2 hr at 39°C. After incubation, HDL and OFA fractions were isolated and analyzed for changes in protein and lipid content. After OF, samples were incubated with sperm, an increase in UC was found in the HDL fractions. UC in HDL increased by 12.1 ± 1.0 μg/ml (X ± SE) when serum P4 was ≤0.5 ng/ml. For samples corresponding to higher serum P4, the increase in UC was 3.60 ± 0.89 μg/ml. Values for UC in HDL were corrected for the contribution of UC from OFA of OF samples. Cholesterol efflux from sperm has been implicated in the process of sperm capacitation. These results indicate that HDL from OF is elevated during the follicular phase of the estrous cycle and can serve as an acceptor for bovine sperm cholesterol.
Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine
Authors: Liu Ming‐Sun , Chan Kaimin , Lau Yun‐Fai , Lee Chi‐Yu Gregory 
Published: February 2005

AbstractA monoclonal antibody, HS‐63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA‐63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA‐63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA‐63. Using the established cDNA clone as a probe, the gene encoding for MSA‐63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA‐63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS‐63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm‐specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen‐based immunocontraceptive vaccines.
Immunohistochemical localization of prostaglandin H synthase in the embryo and uterus of the mouse from ovulation through implantation
Authors: Marshburn P. B. , Shabanowitz R. B. , Clark M. R. 
Published: February 2005

AbstractProstaglandins (PGs) in the embryo and endometrium are involved in processes that are important for implantation. Although the presence of PGs (PGE2, PGF2α, PGI2) in decidualized endometrium has been widely reported, less is known about the capacity of the pre‐implantation embryo to synthesize PGs. Prostaglandin H (PGH) synthase is necessary for the production of PGs. Using an immunohistochemical method, PGH synthase was localized in the mouse embryo and uterus from superovulation through embryo implantation. No PGH synthase was detected in oocytes at the time of ovulation or in single‐cell embryos 1 day post‐fertilization (PF). Circular areds of immunostaining became evident in the cytoplasm of blastomeres at the morula stage (day 3 PF). After implantation (day 5 PF), a low level of PGH synthase reactivity was observed in embryonic cells; no PGH synthase was detected in the embryo by day 7 PF. The endometrial glands exhibited maximal immunostaining by day 3 PF, and after implantation, PGH synthase appeared in decidual cells along the border of placentation. Low levels of PGH synthase reactivity were detected in myometrial cells during the period after superovulation through day 7 PF. This is the first demonstration of PGH synthase in the mouse embryo prior to apposition with glandular endometrial epithelium, supporting the hypothesis that the embryo has the potential to produce PGs that may mediate autocrine and/or paracrine responses at the time of nidation.
Cytological effects of the microinjection of antibody to ras p21 in early cleavage Xenopus embryos
Authors: Miron Marie‐José , Lanoix Joël , Paiement Jacques 
Published: February 2005

AbstractThe presence of two ras‐related proteins (22 and 23 kDa) was demonstrated in Xenopus embryonic extracts by selective immunoprecipitation using anti‐ras monoclonal antibodies 142‐24EO5 and Y13‐259. We further describe the cytological effects of the microinijection of anti‐ras monoclonal antibody Y13‐259 into early cleavage blastomeres of Xenopus embryos. Injection of the antibody into a blastomere at the two‐, four‐, or eight‐cell stage caused cleavage arrest in the descendants of the injected blastomere. Light microscopy (LM) of cleavage‐arrested cells revealed extensive deformation of the cells as well as heterogeneity of distribution of yolk platelets and pigment granules. LM analysis of serial sections of cleavage‐arrested cells revealed the presence of multiple nuclei. Although the nuclei expressed similar morphological properties, indicating that they were probably in the same stage of the nuclear cycle, they revealed highly variable chromatin densities. Electron microscope (EM) analysis of the cytoplasm of cleavage‐arrested cells revealed the accumulation of vesicles and large membranous elements coincident with cleavage arrest. Further‐more, endoplasmic reticulum (ER) existed in two forms, as closed, circular profiles and as long, linear arrays. Mitochondria were characteristically aligned in single file on both sides of the two types of ER cisternae. EM analysis of nuclei confirmed variations in chromatin organization and suggested the occurrence of unique nuclear envelope fusion among micronuclei in cleavage‐arrested cells. Cleavage arrest and changes in cytological features were not observed in the cytoplasm of cells microinjected with normal rat lgG. Thus the immunochemical data and microinjection experiments suggest that ras‐like or ras antigenicity exists within rapidly replicating Xenopus blastomeres and may be involved in the organization of a number of its cytoplasmic elements.
Modulation of mammalian sperm motility by quercetin
Authors: Nass‐Arden Lea , Breitbart Haim 
Published: February 2005

AbstractThe flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3–4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548–11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin‐treated cells show high motility, which continues for additional 2–3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin‐treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration. It is also concluded that lipid peroxidation activity has a major role in loss of sperm motility during time of incubation.
Lectin‐induced abnormalities of mouse blastocyst hatching and outgrowth in vitro
Authors: Dealtry Gillian Barbara , Sellens Martin Howard 
Published: February 2005

AbstractWe have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachement, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL‐1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin‐1 (LTA‐1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded balstocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their enviroment.
Development of cortical vesicles in Sicyonia ingentis ova: Their heterogeneity and role in elaboration of the hatching envelope
Authors: Pillai Muralidharan C. , Clark Wallis H. 
Published: February 2005

AbstractIn the marine shrimp Sicyonia ingentis, ova lack cortical vesicles at spawning. Previous ultrastructural studies suggested that two different populations of cortical vesicles (dense vesicles and the ring vesicles) appear within 30 min post‐spawning. These vesicles undergo sequential exocytosis (exocytosis of the dense vesicles followed by exocytosis of the ring vesicles) that leads to the formation of a hatching envelope around the ovum (see Pillai and Clark: Tissue & Cell 20:941–52, 1988). In the present study, lectins were used as molecular probes to study the development of cortical vesicles subsequent to spawning and the role of these vesicles in formation and elaboration of the hatching envelope. Isolated envelopes were screened with 11 different lectins to determine what group(s) were specific to the envelope glycoconjugates; Concanavalin A (Con A), Griffonia simplicifolia (GS II), Lens culinaris (LCA), and wheat germ agglutinin (WGA) bound to the envelopes. FITC‐lectin studies of sectioned ova (fixed at various time points after spawning) utilizing WGA and LCA showed different labelling patterns. Data obtained at the light microscopical level indicated that WGA was specific to the dense vesicles and the outer portion of the envelope, while LCA exhibited specificity for the ring vesicles and the inner portion of the envelope. At the ultrastructural level, gold‐LCA labelling was seen associated with the cisternal elements (containing ring‐shaped structures), ring vesicles, and the inner layer of the fully formed envelope. These data demonstrated that (1) the ring vesicles are formed by fusion of cisternal elements containing ring‐shaped structures; (2) the two species of cortical vesicles are chemically heterogeneous; and (3) the components of each type of vesicle contribute to different integral parts (the outer and inner layers) of the hatching envelope.
Rat RIβ isoform of type I regulatory subunit of cyclic adenosine monophosphate‐dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis
Authors: Massa John S. , Fellows Robert E. , Maurer Richard A. 
Published: February 2005

AbstractA rat complementary DNA (cDNA) for the RIβ isoform of type I cyclic adenosine monophosphate (cAMP)‐dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3′‐untranslated regions, with a single (ATTAAA) poly‐adenylation site. The largest open reading frame was preceded by a short outof‐phase open reading frame, which is not seen in the corresponding mouse RIβ cDNA due to a single base substitution. The rat RIβ cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RIβ mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RIβ cDNA also detected RIβ mRNA in mouse brain, but not mouse testis, from 10‐week‐old BALB/c or 10‐ and 6‐week‐old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RIβ in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RIβ mRNA.
Carbohydrates and fertilization in animals
Authors: Miller David J. , Ax Roy L. 
Published: February 2005

AbstractA frequently used mechanism for sperm–egg recognition in many species involves complementary protein–carbohydrate interaction. The usual paradigm includes complex glycoconjugates in reproductive tract fluids or on the eggs which are recognized by carbohydrate‐binding proteins on the sperm surface. Various glycocojugates are utilized in the steps of sperm capacitation, sperm binding to the egg extracellular matrix and vitelline membrane and induction of the acrosome reaction. Several types of complex glycoconjugates are involved in these processes, including proteoglycans, lactosaminoglycans, sulfated fucose‐containing glycoconjugates, and glycoproteins. There appear to be some structural similarities between active glycoconjugates; they are large in molecular weight and complex, and they are often sulfated, fucosylated, and attached to a protein through serine or threonine residues. In some species, the protein core of the glycoconjugates also participates in the interaction by limiting the binding of carbohydrates to sperm only of the relevant species, likely by providing the proper steric arrangement for the interaction. In other cases the protein core seems to serve more as a crosslinker of the carbohydrate moieties. This review discusses the types of glycoconjugates implicated in fertilization and the complementary lectin‐like proteins found on sperm.
The effect of cytochalasin D on protein synthesis in Xenopus laevis oocytes
Authors: De Sousa Paul A. , Masui Yoshio 
Published: February 2005

AbstractDefolliculated fully grown oocytes of Xenopus laevis were treated with cytochalasin D (10 μg/ml) and their protein synthesis was studied by labelling with S‐35 methionine. This treatment brought about an alteration in pigment pattern as well as a reduction in amino acid uptake by the oocytes. However, the radioactive amino acid taken by cytochalasin‐treated oocytes was incorporated into protein in the same proportion as in untreated oocytes. These results suggested that subcortical pigment distribution and amino acid uptake in fully grown oocytes were microfilament‐dependent processes, whereas protein synthesis in the oocyte was not.
Ultrastructural localization of silver‐staining nuclear proteins at the onset of transcription in early bovine embryos
Authors: Antalíková Libuše , Fulka J. 
Published: February 2005

AbstractArgyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four‐, eight‐, and 16‐cell bovine embryo blastomere nuclei using two different silver‐staining procedures. Within the eight‐cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full‐capacity ribosome‐producing machinery is established. In the four‐cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus‐precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear‐cut localization of argyrophilic proteins was detected during the eight‐cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus‐associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16‐cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver‐staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component. These observations confirm and expand existing evidence of the localization of the onset of rDNA transcription to the eight‐cell stage in the early cow embryo.
Mouse zygotes express endogenous lectins
Authors: Legge Michael , Sellens Martin H. 
Published: February 2005

AbstractCell interactions during mouse development have been shown to involve carbohydrate‐containing macromolecules (glycoconjugates). We have therefore used a series of fluorescein‐labelled synthetic glycoproteins to determine if mouse oocytes and zygotes also express sugar binding molecules (endogenous lectins) which might participate in such interactions. Unfertilised secondary oocytes did not express endogenous lectins at 4°C but a low level of expression of fucose, mannose, and galactose‐binding activity could be detected at 37°C. In contrast, the zygote clearly expressed three classes of endogenous lectins, with preferential binding for i) fucose or mannose, ii) glucose or galactose, and iii) lactose. The expression of these lectins was much reduced at 4°C and maximal binding at 37°C was achieved only after 2 h incubation. We therefore conclude that a low level of endogenous lectin expression in the mouse oocyte is greatly enhanced after fertilisation and that, at both stages, expression, or the detection of expression, is markedly temperature dependent.
Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro
Authors: Nagai T. , Moor R. M. 
Published: February 2005

AbstractThe consequences of interactions between porcine sperm, eggs, and oviduct cells before and during fertilization in vitro (IVF) has been examined with particular reference to the block to polyspermy. The pattern of polypeptides secreted by porcine oviduct epithelial cells has been determined and its effects on sperm both during pre‐fertilization co‐culture and during fertilization have been examined. In standard IVF procedures with no oviduct cell involvement, high rates of penetration (91%) were accompanied by equally high rates of multiple sperm penetration (91% of penetrated eggs). Fertilization on oviduct cell monolayers or a combination of 1 h co‐culture of sperm and oviduct cells before the addition of in vitro matured oocytes did not reduce polyspermy. However, a sperm‐oviduct cell co‐culture period of 2.5 h followed by IVF on oviduct cells selectively reduced the rate of polyspermy by 40% and 50% in two separate series of trials (United Kingdom and Japan, respectively): Overall fertilization rates after this treatment were high (95% or 84%, respectively). A 3.5 h period of pre‐fertilization co‐culture further reduced polyspermy to only 14% of penetrated eggs, but this treatment was accompanied by a sharp drop in the fertilization rate from an overall mean of 88% for all other groups to 19% after 3.5 h co‐culture.
Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture
Authors: Sato E. , Miyamoto H. , Koide S. S. 
Published: February 2005

AbstractThe viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs‐Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose‐dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1‐x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE‐2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.