bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of pH on the reactivation of human spermatozoa demembranated with triton X‐100
Authors: Giroux‐Widemann V. , Jouannet P. , Pignot‐Paintrand I. , Feneux D. 
Published: February 2005

AbstractThe aim of this work was to study the role of different parameters involved in the motility of human spermatozoa. Human spermatozoa were totally demembranated with 0.05% Triton X‐100, and the demembranation was checked using electron microscopy. We have shown that, with a concentration of ATP‐Mg lower than 2 mM, a pH effect was observed with a dose‐dependent motility reactivation at pH 7.1, with 14% ± 2.0% motile cells at 1 mM ATP‐Mg and a straight line velocity (VSL) of 12.0 ± 1.4 μm/sec. However, at pH 7.8, more than 65% of the spermatozoa were reactivated with as low as 0.02 mM ATP‐Mg and 77.8% ± 2.5% of them were motile at 1 mM ATP‐Mg and had a VSL of 23.4 ± 3.9 μm/sec. The depletion of free calcium by the addition of 0.5 mM EGTA in the reactivation medium (RM) improved the percentage of motile cells and the VSL most markedly at low ATP‐Mg and low pH. If no MgSO4 was added in RM, cells were not motile at pH 7.8, but 30–40% reactivated at pH 7.1. If 5 mM Ca2+ was added to the RM, up to 88% of the cells became reactivated at both pHs, but the beat frequencies were very low, suggesting different mechanisms of reactivation when Mg2+ or when Ca2+ is present in the RM.
Protein synthesis and phosphorylation patterns of bovine oocytes maturing in vivo
Authors: Kastrop P. M. M. , Bevers M. M. , Destrée O. H. J. , Kruip T H. A. M. 
Published: February 2005

AbstractTo investigate protein synthesis and phosphorylation during bovine oocyte maturation in vivo, oocytes were collected at consecutive times after the preovulatory luteinizing hormone (LH) peak. Therefore, heifers treated for superovulation were ovariectomized between 3 and 20 h after the maximum of the LH peak. Subsequently, cumulus‐enclosed oocytes, selected from nonatretic follicles >10 mm, were radiolabeled with 35S‐methionine or 32P‐orthophosphate for 3 h and individually prepared for gel electrophoresis. Changes in the protein synthesis patterns were observed coinciding with germinal vesicle breakdown (GVBD). No changes were detected during the ensuing maturation period or coinciding with the extrusion of the first polar body. In addition, the protein phosphorylation patterns exhibited striking differences around GVBD. In particular, a phosphoprotein band of 19 kDa and the two heavily phosphorylated proteins with apparent molecular weights between 50 and 60 kDa were present in patterns of oocytes in the germinal vesicle stage. The results are discussed in relation to previous data obtained during maturation in vitro.
A monoclonal antibody, MN13, that recognizes specifically a novel substance between the postacrosomal sheath and the overlying plasma membrane in the mammalian sperm head
Authors: Toshimori K. , Tanii I. , Ōura C. , Eddy E. M. 
Published: February 2005

AbstractMonoclonal antibody MN13 raised against mouse spermatozoa specifically recognizes the postacrosomal region of the sperm head in several mammalian species. Colloidal gold‐immunoelectron microscopy of demembranated mouse spermatozoa indicated that the antigen is associated with the outer layer of the periodic substructure apparently linking the postacrosomal sheath to the overlying plasma membrane. The antigen recognized by MN13 may cotribute to the intimate association of the postacrosomal sheath with the overlying plasma membrane.
Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos
Authors: Carriero Filomena , Campioni Nadia , Cardinali Beatrice , Pierandrei‐Amaldi Paola 
Published: February 2005

AbstractA large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.
Rabbit blastocysts accumulate [3H]quinuclidinyl benzilate in vitro
Authors: Jones M. A. , Harper M. J. K. 
Published: February 2005

AbstractDay‐6 rabbit blastocysts were able to accumulate [3H]quinuclidinyl benzilate (QNB) from their environment. This accumulation was reduced approximately 50% in the presence of 1.5 × 10−4 M atropine (an accepted antagonist for ligands which bind to muscarinic cholinergic receptors). The accumulation of QNB was sensitive to temperature and was apparently saturable. In the presence of 2 nM QNB, Day‐6 blastocysts accumulated 30.3 ± 2.0 fmoles per blastocyst. When the cellular elements alone were examined, lesser amounts of specific binding were detected. Owing to the complexity of this multicompartmental system, Scatchard analysis did not provide meaningful results. This accumulation appears higher than that reported for other tissues such as rabbit heart homogenates or rabbit uterine endometrial cells. This muscarinic cholinergic accumulation may have some role in blastocyst‐maternal recognition.
Effect of RU486 on development and implantation of rat embryos
Authors: Roblero Luis S. , Croxatto Horacio B. 
Published: February 2005

AbstractThis study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1+2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose‐related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486‐treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486‐treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486‐treated pseudopregnant recipients failed to implant completely when the dose was ≥ 1.0 mg.The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.
Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis
Authors: Srivastava Archana , Olson Gary E. 
Published: February 2005

AbstractTo investigate surface glycoprotein changes during post‐testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS‐PAGE fractionated samples using biotinylated lectins and Vectastain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation‐dependent sperm surface glycoproteins co‐migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation‐related surface changes occurring within the epididymis than are noted with surface labeling procedures.
Stored mRNA in early embryos of a fern Marsilea vestita: A paternal and maternal origin
Authors: Kuligowski Janine , Ferrand Michèle , Chenou Eliane 
Published: February 2005

AbstractIn early embryos of the fern Marsilea vestita, we have shown that transcription did not begin until the 8‐cell stage, even though protein synthesis was taking place. As in animal species, the pre‐existence of stored maternal mRNA has been suspected in embryonic cells. To investigate the presence of such long‐lived mRNA we have used in situ hybridization method using [3H]polyuridylic acid, [3H]poly(U), as a probe.Temporal and spatial changes in the distribution of poly(A)+ RNA were followed throughout all the different phases of embryogenesis. Poly(A)+ RNA was present in the cytoplasm of both unfertilized and fertilized eggs and early embryos, while the nucleus exhibited no, or a moderate, [3H]‐poly(U) binding activity. New poly(A)+ RNA was detected in the nucleus only at the 16‐cell stage, when cytological differentiation of the root apical cell became morphologically detectable.After in situ hydridization with the labeled probes, the numerous silver grains detected autoradiographically over the mature sperm, indicate the presence of poly(A)+ RNA molecules associated with the male genome.We discuss the possible role of poly(A)+ RNA of maternal origin in supporting early translation prior to synthesis of new mRNA. Additional studies are needed to elucidate the role of stored paternal mRNA.
Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo
Authors: Cohen Jacques , Feldberg Dov 
Published: February 2005

AbstractThe present studies were performed to establish the effects of the size and number of artificial holes produced in the zona pellucida (ZP) on hatching and trophoblast outgrowth in vitro. Limited partial zona dissection (PZD) produced small, narrow incisions, and zona drilling with acidic Tyrode's (AT) across a larger area in the ZP was used to produce bigger round holes. Some embryos were micromanipulated once; others were micromanipulated several times. Blastocysts hatched preferentially through the artificial gaps, but completion of hatching was dependent on the size of the hole. Only 16% (26/167) of PZD embryos migrating through narrow holes hatched completely; the remainder were trapped in a typical figure‐eight shape. Seventy‐two percent (43/60) of those migrating through larger PZD holes hatched, but trophoblast outgrowth was not observed. Significantly (P < 0.001) more AT‐blastocysts hatched (248/270; 92%) and showed trophoblast outgrowth (176/248; 70%). Simultaneous hatching through several openings was rarely observed in AT‐embryos (14/167; 8%), but this did occur in 36% (73/201) of the PZD embryos. Trapping of PZD‐embryos could be almost entirely avoided by drilling with AT elsewhere on the ZP. Embryos with multiple holes in their zonae preferentially hatched through the largest opening. The results suggest that the ability of microsurgically treated human embryos to fully hatch in vitro, should be carefully (re)assessed prior to application of clinical micromanipulation systems. Micromanipulated embryos with small holes in their zonae may be rescued by performing an additional more aggressive opening procedure elsewhere on the ZP.
Quantitative light and scanning electron microscopy of ferret sperm
Authors: Van Der Horst Gerhard , Curry Patrick T. , Kitchin Robert M. , Burgess Warren , Thorne E. Tom , Kwiatkowski Don , Parker Michael , Atherton Robert W. 
Published: February 2005

AbstractSperm were obtained via electro‐ejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black‐footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black‐footed ferret.
Qualitative and quantitative changes in protein sythesis of bovine follicular cells during the preovulatory period
Authors: Rabahi Flora , Monniaux Danielle , Pisselet Claudine , Chupin Daniel , Durand Philippe 
Published: February 2005

AbstractTo understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H‐methionine or with 35S‐methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the prevoulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH‐stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.
Improvement of fertilizing performance by normal and abnormal mouse semen after zona opening of mature oocytes
Authors: Barlow P. , Van Der Zwalmen P. , Schoysman R. , Leroy F. 
Published: February 2005

AbstractThe usefulness of opening the zona pellucida by partial zona dissection (PZD) to enhance fertilization of mature mouse oocytes was studied after insemination with three types of semen: normal and diluted semen and semen from long‐term‐vasectomized males. Zona opening did not by itself induce parthenogenetic cleavage of mature oocytes and did not significantly increase mono‐ and polyspermic fertilization of oocytes inseminated with normal semen. While a fertilization rate of 62% was obtained among intact oocytes, of which 4.5% were polyspermic, a 66.8% fertilization rate was observed among PZD oocytes, 6.3% of which were polyspermic. However, after using diluted semen, only 54 of 193 intact oocytes were fertilized (28%), and PZD improved the fertilization rate to 65.4%. Cleavage rate of nonmanipulated oocytes inseminated with abnormal semen from vasectomized males was dramatically decreased in comparison with those inseminated with normal semen (7.6% vs. 65%). PZD induced a moderate but significant improvement of fertilization performance when using this abnormal semen (19.6%).
Time course and pattern of cortical granule breakdown in hamster eggs after sperm fusion
Authors: Stewart‐Savage J. , Bavister Barry D. 
Published: February 2005

AbstractWe have determined the temporal relationship between sperm fusion and cortical granule breakdown in the hamster egg. Sperm fusion was determined by the Hoechst‐transfer method (Stewart‐Savage and Bavister: Dev Biol 128:150–157, 1988), and cortical granules were visualized with fluorescein isothiocynate‐conjugated Lens culinaris agglutinin (Cherr et al. J Exp Zool 246:81–93, 1988). By 55 min after insemination, there was an 85% reduction in the density of cortical granules (fewer than four granules/100 m̈m2). Taking this value as the completion of the cortical reaction, analysis of the data indicate that the cortical reaction was completed 9 min after sperm fusion and 3 min after the formation of the zona and cell surface blocks to polyspermy. There was no obvious spatial pattern of granule loss in eggs that had a Hoechst‐positive sperm but had not completed the cortical reaction.
Ontogeny of Ha‐ras and c‐myc mRNA levels in rabbit embryo and extraembryonic tissues by quantitative in situ hybridization
Authors: Martinoli M. G. , Lambert R. D. , Pothier F. , Pelletier Georges 
Published: February 2005

AbstractA large variety of proto‐oncogenes are known to be of key importance in cellular growth and differentiation during embryonic development. Using quantitative during in situ hybridization, we studied in detail the levels of the proto‐oncogenes Ha‐ras and c‐myc mRNA in embryos and extraembryonic tissues (maternal and embryonic placentas, trophoblast, and endometrial epithelium) during prental life of rabbit. cDNA probes encoding for Ha‐ras (fragment Kpn 1‐BstE II of 883 bp) and c‐myc (fragment Pst 1‐Pst 1 of 490 bp) were used to detect specific transcripts in fixed cryostat sections. High levels of Ha‐ras and c‐myc mRNA were detected in the rabbit embryo as well as in the decidua and in the trophoblast as early as day 9 of gestation. At 12 and 15 days of gestation, Ha‐ras and c‐myc mRNA levels decreased in both embryonic and maternal placenta while in the embryo a significant increase of Ha‐ras and c‐myc expression was detected with particular evidence in the central nervous system. Finally, at 25 days of gestation the expression of the two proto‐oncogenes, Ha‐ras and c‐myc, was greatly decreased in both the embryo and extraembryonic tissues, and was undetectable by 30 days of gestation. These results show that in rabbit the expression of the two proto‐oncogenes Ha‐ras and c‐myc is localized in the same tissues with similar intensity and follows an unparallel temporal modulation in the embryo and in the extraembryonic tissues during prental development.
Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation
Authors: Rochwerger Leonora , Cuasnicu Patricia S. 
Published: February 2005

AbstractRat epididymal glycoprotein DE (37kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in virto and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibitied the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus‐associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.
Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae
Authors: Vasiliev Aleksej O. , Kapkov Denis V. , Kandror Konstantin V. , Stepanov Aleksandr S. 
Published: February 2005

AbstractA homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme‐linked immunosorbent assay (ELISA), and immunochemical isolation on protein A‐Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two‐step model of casein kinase 2 regulation during heat shock.
The main adenosine triphosphate‐binding component of amphibian oocytes is ferritin
Authors: Kandror Konstantin V. , Stepanov Aleksandr S. , Tsuprun Vladimir L. 
Published: February 2005

AbstractAfter fractionation of mitochondrion‐free extracts of Xenopus laevis and Rana temporaria oocytes in sucrose gradients, a distinct peak of adenosine triphosphate (ATP)/guanosine triphosphate (GTP)‐binding activity in the 50–70 S range has been detected. This substance has a boyant density in Cs2SO4 of 1.45 g/cm3. The nucleotide‐binding substance has been purified to apparent homogenety. By means of electron microscopy, sodium dodecyl sulfate‐electrophoresis and other methods it has been identified as ferritin.