bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stage‐specific appearance of the mouse antigen TEC‐3 in normal and nuclear transfer bovine embryos: Re‐expression after nuclear transfer
Authors: van Stekelenburg‐Hamers Annelies E. P. , Rebel Heggert G. , van Inzen Wouter G. , de Loos Frans A. M. , Drost Maarten , Mummery Christine L. , Weima Sjerp M. , Trounson Alan O. 
Published: February 2005

AbstractBovine embryos, recovered from the uterus in vivo or derived from in vitro matured and in vitro fertilized oocytes, were investigated for the presence of the developmentally regulated mouse antigen TEC‐3 by indirect immunofluorescence. During preimplantation embryo development TEC‐3 is expressed on bovine morulae and blastocysts. It is absent from unfertilized and fertilized oocytes, and from all stages before the 32‐cell stage. The finding that TEC‐3 is not expressed before the onset of embryonic transcription, which occurs at the eight‐cell stage in the bovine, but only when the embryonic genome is active, makes it a potential marker for studying nuclear reprogramming after nuclear transfer. Nuclear transfer embryos were made by electrical fusion of blastomeres from morulae derived in vivo with enucleated metaphase II oocytes. Indirect immunofluorescence with the TEC‐03 antibody showed that the TEC‐3 antigen, present on blastomeres of the morula stage embryo, disappeared after fusion and was expressed again when the nuclear transfer embryos developed to the morula and blastocyst stage. These data suggest that the bovine embryonic nucleus may be able to revert to the equivalent of an earlier developmental stage when transferred to ooplasm, and is then capable of following the normal developmental program. © 1994 Wiley‐Liss, Inc.
Protein databases for compacted eight‐cell and blastocyst‐stage mouse embryos
Authors: Shi C. Z. , Collins H. W. , Garside W. T. , Buettger C. W. , Matschinsky F. M. , Heyner S. 
Published: February 2005

AbstractHigh‐resolution two‐dimensional sodium dodecyl sulfate‐polyacrylamide (2D‐SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight‐cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30–50 embryos were labelled with L‐[35S]methionine for 2 hr. The embryos were then lysed in 30 μl hot SDS sample buffer, and the lysates were stored at −80°C until the gels were run. Five replicates were run for eight‐cell embryos, and four for blastocyst‐stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight‐cell embryo standards, >79% of spots had a percentage error (S.E.M./average) <50%, and >45% had a percentage error <30%. Similarly, of 1,653 total spots in blastocyst‐stage embryo standards, 74% of spots had a percentage error <50%, and approximately 47% of spots had a percentage error <30%. Forty‐three spots (approximately 3% of the total spots) were found to be detected only in the eight‐cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty‐nine proteins showed a greater than threefold increase in isotope incorporation from the eight‐cell to the blastocyst stage, with a percentage error <50% in both the eight‐cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight‐cell stage to the blastocyst stage of mouse preimplantation development. These databases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development. © 1994 Wiley‐Liss, Inc.
Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes
Authors: Cheong H. T. , Takahashi Y. , Kanagawa H. 
Published: February 2005

AbstractThe present study was conducted to examine the relationship between nuclear remodeling and subsequent embryonic development in nuclear transplant mouse embryos. Metaphase II oocytes were enucleated without staining and fused with transferred donor nuclei from two‐, four‐, or eight‐cell embryos. Fusion and oocyte activation were performed by means of electric fields. High rates of enucleation (89.1%), fusion (88.0–91.6%), and activation (95.2–96.9%) were obtained using this system. Nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by various pronuclear‐like formations upon oocyte activation. Development to blastocysts was obtained from both PCC (17.9%) and non‐PCC (NPCC; 52.9%) embryos fused with the two‐cell nuclei. However, development to term was obtained only in PCC embryos with a single pronucleus‐like structure and a polar body (12.5%). In vitro development of nuclear transplant embryos with four‐ and eight‐cell nuclei was limited. All the NPCC embryos examined had tetraploid chromosome constitutions, but chromosome constitutions of PCC embryos varied. Only 37.5% of the PCC embryos had diploid chromosome constitutions. The results indicated that the development of nuclear transplant embryos is affected by the types of nuclear remodeling and that oocyte activation in relation to their chromosome constitutions. The results also indicated that the PCC of the donor nucleus in nonactivated cytoplasm is important for the development of the nuclear transplant embryos. © 1994 Wiley‐Liss, Inc.
Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone
Authors: McLaughlin E. A. , Ford W. C. L. 
Published: February 2005

AbstractThe intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura‐2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P < 0.0001 paired t‐test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca2+]i to a peak value > 1 μM after 10–20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30–40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley‐Liss, Inc.
Spatially restricted imprinting of mouse chromosome 7
Authors: Villar Angela J. , Pedersen Roger A. 
Published: February 2005

AbstractThree of the four known imprinted genes (Igf‐2, H19, and Snrpn) map to mouse chromosome 7. We used mRNA phenotyping to examine the tissuespecific transcription of Igf‐1r, H‐ras‐1, and Gabrb3, which map to chromosome 7 between Snrpn and the Igf‐2/H19 domain, and Myod‐1, which maps proximal to Snrpn. We found that all of these genes were expressed by both parental alleles in tissues from day 1 neonates. The fact that imprinted genes can flank or map closely to genes that escape such epigenetic modification suggests that autosomal imprinting is not manifested globally along imprinted chromosomes but rather is spatially restricted, perhaps even defined by specific DNA consensus sequences or an “imprint box” associated with imprintable genes. © 1994 Wiley‐Liss, Inc.
Synthesis and processing of mammalian protamines and transition proteins
Authors: Green G. R. , Balhorn R. , Poccia D. L. , Hecht N. B. 
Published: February 2005

AbstractMouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]‐arginine, [3H]‐histidine, [35S]‐cysteine, or [32P]‐PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication‐resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre‐mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R‐250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre‐mP2 increased to match those of their predominant forms. These changes are consistent with initial charge‐neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady‐state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre‐mP2‐3, a major processing intermediate derived by proteolysis of pre‐mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley‐Liss, Inc.
Effects of freezing of bovine preimplantation embryos derived from oocytes fertilized in vitro on survival of their inner cell mass cells
Authors: Iwasaki S. , Yoshikane Y. , Li X. , Watanabe S. , Nakahara T. 
Published: February 2005

AbstractThe morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen‐thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three‐step and one‐step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co‐cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three‐step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one‐step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three‐step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method. © 1994 Wiley‐Liss, Inc.
Inhibition of germinal vesicle breakdown in bovine oocytes by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)
Authors: Farin Charlotte E. , Yang Li 
Published: February 2005

Abstract5,6‐Dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) is an analog of the nucleoside adenosine that has been used to inhibit transcription in a variety of cell types. The present studies were designed to evaluate the ability of DRB to block germinal vesicle breakdown (GVBD) in bovine oocytes matured in vitro and to characterize culture conditions required for DRB‐mediated arrest of meiosis. Administration of DRB (60–90 μM) at 2–3 h intervals during culture of COC blocked GVBD in approximately 70 percent of oocytes. The inhibitory effect of DRB was reversible and required the presence of cumulus cells. Treatment with DRB was associated with a 57% decrease in 3H‐uridine incorporation into total COC RNA and a 90.8% decrease into mRNA but did not affect the incorporation of 3H‐leucine into COC proteins. The ability of DRB to arrest meiosis was significantly compromised if supplemental gonadotropin preparations were absent from the maturation media. Gonadotropin‐induced GVBD as well as cumulus cell expansion was blocked by treatment with DRB but no with adenosine. GVBD in cultured bovine COC was initially inhibited and then stimulated when supplemental gonadotropin preparations were included in the culture media. DRB treatment in the presence of gonadotropin supplementation blocked the stimulatory effect of gonadotropins on GVBD. In conclusion, DRB can be used to arrest GVBD in bovine COC in a specific and reversible manner. The data support the hypothesis that gene transcription is required for the stimulatory phase of gonadotropin‐mediated GVBD in cultured bovine COC. © 1994 Wiley‐Liss, Inc.
Flow cytometric analysis of mouse sperm using monoclonal anti‐sperm antibody A‐1
Authors: Kawai Yuichi , Ohnishi Shigeru , Miyake Masaharu , Hama Takao , Mayumi Tadanori 
Published: February 2005

AbstractWe have previously prepared an anti‐mouse sperm monoclonal antibody (A‐1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A‐1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol‐fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A‐1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence‐positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A‐1 antibody was reduced to 55% by incubation in zona‐containing media from the 80% obtained in zona‐free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A‐1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A‐1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Production of motile acrosome‐reacted mouse sperm with nanomolar concentration of calcium ionophore A23187
Authors: Tanphaichitr N. , Hansen C. 
Published: February 2005

AbstractA method to generate a population of motile, acrosome‐reacted mouse sperm is described. Sperm retrieved from the cauda epididymis and vas deferens were first capacited in a 3% bovine serum albumin (BSA) containing medium. Sperm were then resuspended in medium with low BSA content (0.01%) and treated with 30 nM of the calcium ionophore, A23187, which was added as a singel dose of 30 nM for 15 min at 37°C; or three sequential 10 nM doses over three 5 min intervals. Approximately 55–60% of the treated sperm population became acrosome reacted. The motility of the treated sperm sample was 40–65%, slightly lower than that of the control sperm, following addition of medium containing 3% BSA, This is in contrast to the <10% motility observed for capacitated mouse sperm treated with 10 μM A23187, a concentration that had been used by other investigators to induce the acrosome reaction. The ultrastructure of the 30 nM A23187‐induced acrosome‐reacted sperm ws similar to that of the acrosome‐reacted sperm induced by solubilized zonae pellucidae. These motile, acrosome‐reacted sperm were able to penetrate zone‐free mouse eggs at a higher rate than the control sperm. Thus this method of treatment will be useful for further physiological experimentation with acrosome‐reacted sperm. © 1994 Wiley‐Liss, Inc.
Expression of insulin‐like growth factors I and II in conceptuses from normal and diabetic mice
Authors: Chernicky C. L. , Redline R. W. , Tan H. Q. , Gwatkin R. B. L. , Johnson T. R. , Ilan Joseph , Ilan Judith 
Published: February 2005

AbstractInsulin‐like growth factors (IGF‐I and IGF‐II) play an important regulatory role in fetal growth and development. Alterations in expression of these growth factors may result in developmental abnormalities, macrosomia, and intrauterine growth retardation, which occur with a higher incidence in diabetic pregnancies. In situ hybridization histochemistry was employed to investigate the distribution and abundance of IGF‐I and IGF‐II in peri‐implantation and postimplantation conceptuses from normal and streptozotocin‐treated diabetic mice. Animals were sacrificed on gestational days 5, 6, 7, 8, and 9. The entire uterine horn was prepared for hybridization with antisense and sense α‐35S‐dATP labeled oligonucleotide probes for IGF‐I, IGF‐II, and mouse β‐actin. IGF‐I transcript was apparent only in myometrium at 6 days of gestation in normal and diabetic mice. IGF‐II transcripts were restricted to trophoectoderm cells within the implantation chamber on day 5. Following implantation, IGF‐II transcripts were found in trophoectodermal derivatives, primitive endoderm, mesoderm, heart, walls of the foregut, and mesenchyme in normal and diabetic postimplantation conceptuses. There were no apparent differences between normal and diabetic samples in the distribution and abundance of the IGF‐II transcript from gestational days 7, 8, and 9. The embryos from the diabetic mother at day 6 were growth retarded and had a significant decrease in the expression of IGF‐II. These results suggest that maternal hyperglycemia may retard development of the early implanting conceptus in a narrow window around day 6 through a mechanism involving decreased IGF‐II expression. Fetuses from diabetic pregnancies that escape this critical period appear to develop and express IGF‐II in an equivalent manner to those of the control group. © 1994 Wiley‐Liss, Inc.
Characterization of the major proteins of bovine seminal fluid by two‐dimensional polyacrylamide gel electrophoresis
Authors: Desnoyers L. , Théarien I. , Manjunath P. 
Published: February 2005

AbstractRecently, we demonstrated that the major proteins from bovine seminal plasma BSP‐A1, ‐A2, ‐A3 and ‐30‐kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D‐PAGE. BSP‐A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP‐A2 at 16 kDa and at a pl of 4.9–5.2; BSP‐A3 at 15 kDa and at a pl of 4.8–5.2, and BSP‐30‐kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP‐30‐kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP‐A1, ‐A2 and ‐A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D‐PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP‐30‐kDa is mainly due to its sialic acid content. © 1994 Wiley‐Liss, Inc.
Spermophagy in semen in the red wolf, Canis rufus
Authors: Koehler James K. , Platz Carrol C. , Waddell Will , Jones Michael H. , Smith Roland , Behrns Susan 
Published: February 2005

AbstractThe red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley‐Liss, Inc.