bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cleavage and 3H‐uridine incorporation in bovine embryos of high in vitro developmental potential
Authors: Plante Louise , L. Shepherd Dena , Allan King W. , Plante Claire 
Published: February 2005

AbstractThe timing of genome activation in bovine embryos is still not well defined. The objective of this study was therefore to investigate transcription in bovine embryos with a high potential to develop in culture after in vitro fertilization, by examining, autoradiographically, their incorporation of 3H‐uridine. Initial experiments determined that developmental potential in vitro could be related to the time of first division of the zygote. Embryos that completed their first cleavage within 30 hours of exposure to sperm were more likely to develop into blastocysts (65.7%) and to hatch (50.9%). Using such embryos, it was found that 10 of 12 8‐cell and all 11 4‐cell stage embryos were labeled after a 2–4‐hr exposure to 3H‐Uridine. Among 2‐cell stage embryos, 0 of 23, 3 of 17, 8 of 15, and 3 of 4 were labeled after exposure to 3H‐uridine of 2, 4, 7, and 10 hr, respectively. Treatment with α‐amanatin (10–100 m̈g/ml) blocked 3H‐uridine incorporation but did not inhibit cleavage during the first 4 cell cycles. It was concluded that transcription occurs as early as the 2‐cell stage in bovine embryos in vitro but is not critical to the first four cell cycles. © 1994 Wiley‐Liss, Inc.
Implication that potassium flux and increase in intracellular calcium are necessary for the initiation of sperm motility in salmonid fishes
Authors: Tanimoto Satomi , Nakazawa Tohru , Kudo Yoshihisa , Morisawa Masaaki 
Published: February 2005

AbstractFlux of K+ and changes in intracellular Ca2+ in the sperm of salmonid fishes were measured with spectrophotometry, ion electrode, microscopic fluorometry, and radioisotope accumulation. Release of K+ occurred at the initiation of sperm motility which is induced by decrease in external K+ and the K+ efflux and sperm motility were inhibited by K+ channel blockers. Intracellular Ca2+ increased within a short period in K+‐ free condition, and the accumulation of 45Ca in sperm cells was higher in motile sperm than that in immotile sperm. The efflux of K+ and the increase in intracellular Ca2+ were suppressed when external K+ concentration increased, i.e., sperm remained immotile. These results suggest that efflux of K+ through K+ channel and subseqent increase in intracellular Ca2+ are prerequisite for the initiation of sperm motility. © 1994 Wiley‐Liss, Inc.
Differential localization of two isoforms of the regulatory subunit RIIα of cAMP‐dependent protein kinase in human sperm: Biochemical and cytochemical study
Authors: Pariset Claude , Weinman Serge 
Published: February 2005

AbstractIn the present study, immunogold labeling of ultrathin sections of ejaculated sperm was used to obtain insight into the ultrastructural localization and presumable function of type II cAMP‐dependent protein kinase in sperm motion. In the flagellum, a human‐specific isoform of the RIIα subunit was located on the axonemal microtubule wall, whereas a different isoform of broader specificity was present in the cytoplasm at the periphery of the coarse fibers and fibrous sheath. This isoform was also found in the mitochondria. The human‐specific RIIα subunit is likely linked to microtubules by a unique binding protein of Mr 72kD. These findings are in agreement with the concept of a concerted mechanism involving phosphorylation of both the axonemal microtubules and the fibrous structures for the regulation of mammalian sperm motion. © 1994 Wiley‐Liss, Inc.
Comparison of the ability of progesterone and heat solubilized porcine zona pellucida to initiate the porcine sperm acrosome reaction in vitro
Authors: S. Melendrez Carman , Meizel Stanley , Berger Trish 
Published: February 2005

AbstractIt has been previously shown that progesterone can initiate the acrosome reaction (AR) of capacitated human and hamster sperm in vivo. We report here that progesterone can initiate a morphologically normal AR in porcine sperm that have undergone capacitation in a Hepes‐buffered medium in vitro. In addition, we have compared the abilities of progesterone and heat‐solubilized porcine zona pellucida (zona) to initiate the porcine sperm AR. Capacitated porcine sperm were treated with 1 m̈g/ml progesterone, 150 m̈g/ml porcine zona, or solvent control for 10 min. After treatment, sperm were incubated with the supravital dye Hoechst 33258, fixed and the acrosomal status determined in the previously viable sperm by fluorescence microscopy with fluorescein isothiocyanate‐labeled Pisum sativum agglutini (FITC‐PSA). There was no significant difference between the percentage of AR initiated by zona compared to that initiated by progesterone. In order to determine whether there was a synergistic interaction between the two AR initiators, both were added simultaneously to capacitated porcine sperm at optimal (1 m̈g/ml progesterone, 150 m̈g/ml zona) and suboptimal (75 ng/ml progesterone and 75 m̈g/ml zona) concentrations. Simultaneous addition of the two AR‐initiators at the two concentrations stimulated an additive AR‐initating response, rather than a synergistic one. Several possible explanations for the additive results are discussed. © 1994 Wiley‐Liss, Inc.
Translational regulation of the gradual increase in histone H1 kinase activity in maturing mouse oocytes
Authors: Hampl Aleš , Eppig John J. 
Published: February 2005

AbstractIn maturing mouse oocytes, p34cdc2‐associated histone H1 kinase activity gradually increases until it reaches its maximum at metaphase I (Choi et al., 1991: Development 113:789–795). In this study, treatment of oocytes with cycloheximide resulted in a failure to increase the level of histone H1 activity above that detected at approximately the time of germinal vesicle breakdown (GVB), which is ∼20‐30% of the level normally achieved at metaphase I. Cyclin B was detected in GV‐stage oocytes, but there was a 2–2.5‐fold increase in the amount of cyclin B in maturing oocytes from GV‐stage to metaphase I and a burst of cyclin B synthesis during the first 3 hr of maturation. Okadaic acid‐treatment of mouse oocytes did not accelerate activation of histone H1 kinase but rather arrested its activity at the same level observed in cycloheximide‐treated oocytes. Thus the components of the p34cdc2 kinase activating system in mouse oocytes are apparently not present in GV‐stage oocytes in an amount or configuration that would allow maximum kinase activation when meiosis is reinitiated by okadaic acid. Importantly, okadaic acid‐treatment dramatically inhibited protein synthesis. Therefore, the inhibition of protein synthesis by okadaic acid probably abrogates the possibility of de novo synthesis of the regulators of p34cdc2 kinase required to drive its activity to the maximum level normally achieved by metaphase I. It is concluded that there is a critical point in driving the continued activation of histone H1 kinase that occurs at approximately the time of GVB. Progression beyond this point requires de novo protein synthesis. Since newly synthesized cyclin B is immediately complexed with the p34cdc2 kinase in maturing mouse oocytes, cyclin B is a candidate for one of the proteins whose synthesis is required to drive the continued increase in histone H1 kinase activity after the time of GVB. © 1995 Wiley‐Liss, Inc.
Mitochondrial biogenesis in early mouse embryos: Expression of the mRNAs for subunits IV, Vb, and VIIc of cytochrome c oxidase and subunit 9 (P1) of H+‐ATP synthase
Authors: Taylor Kent D. , Pikó Lajos 
Published: February 2005

AbstractThe mouse egg contains about 90,000 mitochondria which undergo a buildup of mitochondrial cristae and increase in respiratory activity during cleavage. The mitochondrial DNA does not replicate during preimplantation development but is transcribed actively from the two‐cell stage onward (Pikó and Taylor, 1987: Dev Biol 123:364–374). To gain further insight into mitochondrial biogenesis, we have now determined the steady state amounts of the mRNAs for the cytochrome c oxidase (COX) subunits IV, Vb and VIIc and the H+‐ATPase subunit 9(P1) (all encoded by nuclear genes) in slot hybridization experiments of total RNA from oocytes and early embryos. All four mRNAs showed a similar developmental pattern of prevalence, characterized by a steady decline in mRNA copy numbers from the late growth‐phase oocyte through the two‐cell embryo, and an about 30‐fold rise during cleavage through the blastocyst stage. However, the ATPase subunit 9 (P1) mRNA was about three times more prevalent in cleavage‐stage embryos than the COX mRNAs. A similar pattern was obtained previously for the mitochondrial‐encoded COX I and II mRNAs, but the latter accumulate at a 30–50‐fold excess over the nuclear‐encoded COX subunit mRNAs during the cleavage stages. The results suggest a coordinated activation and transcription of the mitochondrial and nuclear genes for the components of the respiratory apparatus beginning with the two‐cell stage. It is estimated that new respiratory chains are produced at a rate of 50–100 chains hr‐1/mitochondrion in the early blastocyst, accounting for 3.5‐7% of the total protein synthetic activity at this stage. This rapid buildup of the mitochondrial oxidative phosphorylation system appears to be mostly preparatory for postimplantation development. © 1995 Wiley‐Liss, Inc.
Expression of the rabbit sperm protein Sp17 in cos cells and interaction of recombinant Sp17 with the rabbit zona pellucida
Authors: O'rand Michael G. , Richardson Richard T. , Yamasaki Noriyuki 
Published: February 2005

AbstractThis study extends our analysis of rabbit recombinant Sp17 (rSp17) by examining whether rSp17 synthesized in transfected COS cells will show a particular localization within the cell and whether the COS cell will bind with zona pellucida. We show, using the cross‐linking, reagent DSS that rSp17 can bind to rabbit zona glycoprotein R45 or R55. © 1995 Wiley‐Liss, Inc.
Effects of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development, and chromosomal abnormalities
Authors: Bouquet M. , Selva J. , Auroux M. 
Published: February 2005

AbstractIn a previous study, we have shown that the cryopreservation of mouse oocytes caused increases in the rates of degeneration and of digynic polyploid embryos, while the fertility of frozen‐thawed oocytes was decreased. In this study, we have attempted to determine the different stages in the complete freezing‐thawing process which are deleterious for the oocytes and the subsequent zygotes. IVF assays showed that DMSO decreased the fertility of oocytes, whereas cooling to 0°C had no effect. DMSO, used at 0°C, was less deleterious for oocytes. Thus, the prefreezing manipulations seem to be important for the quality and fertility of oocytes. However, neither DMSO nor cooling increased the incidence of chromosomal abnormalities in embryos obtained from inseminated exposed oocytes. Therefore, the increased frequency of polyploidy observed in embryos after the cryopreservation of mouse oocytes must correspond to disruption occurring during the freezing‐thawing process.
Regulation of the transforming growth factor‐β2 gene promoter in embryonal carcinoma cells and their differentiated cells: Differential utilization of transcription factors
Authors: Kelly David , Scholtz Beáta , Orten Dana Jo , Hinrichs Steven H. , Rizzino Angie 
Published: February 2005

AbstractPrevious studies demonstrated that differentiation of embryonal carcinoma (EC) cells increases the expression of the TGF‐β2 gene and identified a CRE/ATF‐like motif in the TGF‐β2 promoter that is necessary for its activity. This suggested that differentiation may increase the transcription of this gene by differential binding of transcription factors to the CRE/ATF‐like motif. To test this possibility, we performed gel mobility shift analysis using double‐stranded oligodeoxynucleotides containing the TGF‐β2 CRE/ATF‐like motif and nuclear extracts prepared from F9 EC cells and F9‐differentiated cells. We determined that the DNA/protein complexes formed by the EC nuclear extracts, but not the complexes formed by differentiated cell nuclear extracts, are recognized and supershifted by an ATF‐1 specific antibody. This observation is consistent with our Western immunoblot analysis that detects ATF‐1 in the EC cells, but not in their differentiated counterparts. In addition, we provide evidence that protein phosphorylation influences the formation of complexes between F9 nuclear proteins and the CRE/ATF‐like motif. Together, our studies identify a likely role for the CRE/ATF‐like motif in the regulation of TGF‐β2 and suggest that this site binds one set of nuclear proteins in EC cells, where the gene is not expressed, and a different set of nuclear proteins in the differentiated cells, where the gene is expressed. © 1995 Wiley‐Liss, Inc.
Identification and localization of integrin subunits in oocytes and eggs of the mouse
Authors: Evans Janice P. , Schultz Richard M. , Kopf Gregory S. 
Published: February 2005

AbstractResults of a recent study have implicated egg integrins in sperm binding to the egg plasma membrane (Blobel et al., 1991: Nature 356:248–252). In this report, immunoprecipitation was used to identify, and confocal immunofluorescence microscopy was used to localize, several different integrin subunits in mouse eggs. Antibodies to α2, α5, αv, and β1 subunits, as well as antibodies to the fibronectin receptor (FNR; α5β1 and/or α3β1) and vitronectin receptor (VNR; αvβ3 and/or αvβ5), detect polypeptides of the appropriate molecular weights following immunoprecipitation. β1 is localized preferentially to either the microvillar or amicrovillar membrane/cortical regions of eggs, and these asymmetric localizations depend on the antibody used. Proteins recognized by anti‐FNR antibodies are localized preferentially to the amicrovillar membrane/cortical region. Germinal vesicle‐intact oocytes display a symmetric plasma membrane distribution using β and FNR antibodies, and the asymmetric distribution develops as a consequence of oocyte maturation and is clearly observed by metaphase I. In contrast to the membrane localization of these integrin subunits, α2, α5, and VNR are predominantly localized in the cytoplasm of both oocytes and eggs. In the oocyte, each of these integrin subunits is uniformly distributed throughout the cytoplasm. Oocyte maturation is associated with a redistribution of α5 and VNR, leading to an asymmetric cytoplasmic distribution with an increased localization towards the spindle. αv, which is localized in the plasma membrane/cortex of both oocytes and eggs, does not show such a change during oocyte maturation. Results of these experiments are discussed in the context of a role for integrins in mediating sperm plasma membrane‐egg plasma membrane interactions leading to egg activation. © 1995 Wiley‐Liss, Inc.
Aneuploidy in late‐step spermatids of mice detected by two‐chromosome fluorescence in situ hybridization
Authors: Wyrobek A. , Lowe X. , Pinkel D. , Bishop J. 
Published: February 2005

AbstractA multicolor procedure employing fluorescence in situ hybridization is described for detecting chromosomal domains and germinal aneuploidy in late‐step spermatids in mice using DNA probes specific for repetitive sequences near the centromeres of chromosomes 8 and X. These probes were nick‐translated with biotin‐ or digoxigenin‐labeled nucleotides, and were detected with FITC or rhodamine. Probe and hybridization specificities were confirmed using metaphase chromosomes from spleen and bone marrow cells as well as from primary and secondary spermatocytes. Late‐step spermatids, identified in testicular preparations by their hooked shape, yielded compact fluorescence domains in ∼ 50% and > 99% of cells when hybridized with probes for chromosomes X and 8, respectively. In a survey of > 80,000 late‐step spermatids from 8 healthy young adult C57BL/6 or B6C3F1 mice, ∼ 3/10,000 spermatids had fluorescence phenotypes indicative of X‐X or 8–8 hyperhaploidy. These frequencies are consistent with published frequencies of aneuploidy in meiotic metaphase II and first cleavage metaphases of the mouse, providing preliminary validation of sperm hybridization for the detection of aneuploidy. No significant animal or strain differences were observed. In addition, the hyperhaploidy frequencies for murine spermatids were indistinguishable for those for sperm from healthy men obtained by a similar hybridization procedure. These procedures for detecting aneuploid male gametes are examples of “bridging biomarkers” between human and animal studies. They have promising applications for investigations of the genetic, reproductive, and toxicological factors leading to abnormal reproductive outcomes of paternal origin. © 1995 Wiley‐Liss, Inc.
Factors involved in nuclear reprogramming during early development in the rabbit
Authors: Pinto‐Correia Clara , Long Charles R. , Chang Thomas , Robl James M. 
Published: February 2005

AbstractMechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM‐2 antibody. Nuclear transfer embryos were derived from G1/early S‐phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rerelication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16‐32‐cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM‐2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32‐cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32‐cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM‐2 label. In contrast, the appearance of MPM‐2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus. © 1995 Wiley‐Liss, Inc.
Localization of fibrillarin and nucleolin in nucleoli of mouse preimplantation embryos
Authors: Baran V. , Veselá J. , Rehák P. , Koppel J. , Fléchon J. E. 
Published: February 2005

AbstractThe localization of fibrillarin and nucleolin in the nuclei of mouse two‐cell, four‐cell, and eight‐cell embryos has been studied using immunofluorescent staining with specific antibodies. In all of these cleavage stages, both antigens were associated exclusively with the peripheral region of the nucleolus precursor bodies (NPBs). The original speckled fluorescent staining pattern in the early two‐cell stage was progressively changed into a continuous fluorescent‐positive layer localized in the cortex of the NPBs in the four‐cell embryos. The compact central area of NPBs was never stained. Both proteins were colocalized in the same substructures of developing nucleoli. In order to analyze the interaction of chromatin, with NPBs, DNA structures were specifically immunolbelled. At the time of resumption of nucleolar transcription (in the two‐cell mouse embryo), DNA was detected at the periphery of, but not penetrating into, NPBs. Our results confirm the view that the cortical region of NPBs could represent a nucleolonemal area involved in the resumption of nucleolar transcription in the early mouse embryo. © 1995 Wiley‐Liss, Inc.
Effects of stimulators of protein kinases A and C and modulators of phosphorylation on plasminogen activator activity in porcine oocyte‐cumulus cell complexes during in vitro maturation
Authors: Kim Nam Hyung , Menino Alfred R. 
Published: February 2005

AbstractEffects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6‐dimethylami‐nopurine (6‐DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte‐cumulus cell complexes (POCC) in vitro were determined. Cumulus cell‐enclosed oocytes were collected from 1–4 mm antral follicles and cultured in TCM‐199 with 0.3% polyvinyl‐pyrrolidone for 48 hr PA activities in POCC were quantified using SDS‐PAGE, casein‐agar zymography, and densitometry. Two plasminogen‐dependent lytic zones (93–96 kD and 71–79 kD) were observed in POCC. Addition of amilorde to the zymograph, a competitive inhibitor of urokinase‐type PA, failed to reduce activities in either zone, suggesting that the 71–79 kD band is a tissue‐type PA (tPA) and the 93–96 kD band is possibly a tPA‐inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P <0.05) activity in dose‐dependent fashion, whereas 6‐DMAP and 10 and 100ng/ml PMA inhibited (P <0.05) PA activity. PA activity increased (P <0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P <0.05) at concentrations >75 nM. Treatment with 25 nM OA also induced the expression of an amiloride‐sensitive PA (49–52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P <0.05) by 2.5 mM dbcAMP and 2 mM 6‐DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P <0.05) only progression to metaphase II. These data suggest that PA production by POCC is influenced by protein kinases A and C and kinase inhibitors during oocyte maturation. Inhibition of intracellular phosphatases also induced novel PA production. © 1995 Wiley‐Liss, Inc.
Efficient production of transgenic cattle by retroviral infection of early embryos
Authors: Haskell R. E. , Bowen R. A. 
Published: February 2005

AbstractProduction of transgenic cattle by microinjection of DNA has been difficult and costly. To explore an alternative method, one‐ to four‐cell bovine embryos were exposed to a replication‐defective retrovirus by microinjection of retrovirus producer cells into the perivitelline space. Embryos were cultured in vitro for 3–4 days, then transferred to recipient cows for further development. Thirteen of 22 embryos recovered at 15 days gestation and each of four fetuses recovered at 90 days gestation were transgenic. Fetuses harbored between 2 and 12 pro‐viruses, and within each fetus, identical patterns of integration were observed in seven tissues tested. Estimates of the number of proviruses per cell suggested that in three of the four fetuses, most, and possibly all, cells were transgenic. This technique should facilitate application of transgenic technology to cattle and other agriculturally important species. © 1995 Wiley‐Liss, Inc.