bluuebunny/crossref_metadata_2025_split huggingface dataset:
Requirement for protein synthesis during the onset of meiosis in bovine oocytes and its involvement in the autocatalytic amplification of maturation‐promoting factor
Authors: Tatemoto Hideki , Horiuchi Toshitaka 
Published: February 2005

AbstractThe present study was carried out using the method of electrofusion, or treatment with okadaic acid (OA), to determine whether protein synthesis at the onset of culture was required for the meiotic resumption of bovine follicular oocytes. Germinal vesicle breakdown (GVBD) occurred in bovine oocytes at 6 hr after separation from their follicles in vitro. Following this, immature germinal vesicle (GV) oocytes, preincubated for 0,2,4, and 6 hr, were fused to mature oocytes. When immature oocytes, preincubated for 0 hr, were fused to mature oocytes and then cultured for 3 hr in basic medium, GVBD was observed in all fused cells, whereas in the case of cultivation in medium supplemented with the protein synthesis inhibitor (25 μg/ml cycloheximide; CX), 39% of the fused cells possessed an intact GV within their cytoplasm. In immature oocytes preincubated for 4 or 6 hr, however, this proportion was significantly reduced to 7% and 4%, respectively, without protein synthesis after fusion. In addition, the CX‐dependent block of GVBD could be overcome in only 13% of bovine follicular oocytes by the addition of 2 μM OA, although 51% of oocytes which synthesized the protein during the first 6 hr of culture induced GVBD in subsequent culture with CX plus OA. Thus, we conclude that the initiation of GVBD in bovine oocytes requires protein synthesized at the onset of meiosis, which is related to the autocatalytic amplification of the maturation‐promoting factor. © 1995 Wiley‐Liss, Inc.
Calcium oscillations and protein synthesis inhibition synergistically activate mouse oocytes
Authors: Bos‐Mikich A. , Swann K. , Whittingham D. G. 
Published: February 2005

AbstractWe have examined the ability of the two parthenogenetic agents, strontium (Sr2+) and cycloheximide, to activate mouse oocytes. We demonstrate that Sr2+ and cycloheximide act synergistically to promote parthenogenetic activation up to the pronuclear stage in oocytes collected immediately after ovulation. These two agents appeared to act independently, since incubation in Sr2+ media triggered a series of intracellular Ca2+ rises without affecting protein synthesis and cycloheximide inhibited protein synthesis without causing any intracellular Ca2+ changes. In addition, cycloheximide did not alter the pattern of Ca2+ oscillations induced by Sr2+. In contrast, we show that another commonly used parthenogenetic activation treatment, the Ca2+ ionophore A23187, has dual effects. Exposure of oocytes to the Ca2+ ionophore, A 23187, in Ca2+‐ and Mg2+‐free medium leads to the activation of young oocytes. However, as well as generating a Ca2+ increase, the treatment of mouse oocytes with A23187 and Ca2+‐ and Mg2+‐free media led to a marked inhibition of protein synthesis. Our data show that parthenogenetic agents may have two important loci for activating mammalian oocytes and that the combined effect on Ca2+ release and protein synthesis is most effective. © 1995 Wiley‐Liss, Inc.
Cis‐regulatory elements and transcription factors involved in the regulation of the transforming growth factor‐β2 gene
Authors: Scholtz Beáta , Kelly David , Rizzino Angie 
Published: February 2005

AbstractEmbryonal carcinoma (EC) cells and embryonic stem (ES) cells provide useful model systems for studying differentiation during early mammalian development. Previous studies have demonstrated that differentiation of two restricted mouse EC cell lines is accompanied by activation of the TGF‐β2 gene. Moreover, one negative and two positive regulatory regions upstream of the transcription start site were identified, which appear to play key roles in the transcriptional regulation of the human TGF‐β2 gene. In this report, we demonstrate that the same three regulatory regions strongly influence the activity of the TGF‐β2 promoter in differentiated cells derived from the multipotent human EC cell line, NT2/D1, and from the murine totipotent ES cell line, CCE. We also determined that the same three regions are active in the regulation of the TGF‐β2 gene in the murine parietal endoderm‐like cell line, PYS‐2. However, an additional negative regulatory region appears to contribute to the regulation of the TGF‐β2 gene in PYS‐2 cells. Last, mutation of a CRE/ATF element located just upstream of the transcription start site of the TGF‐β2 gene reduces significantly the activity of the TGF‐β2 promoter in the differentiated cells. However, in contrast to our previous findings, our gel mobility shift analyses demonstrate that this CRE/ATF element is bound by similar proteins in nuclear extracts prepared from undifferentiated and differentiated mouse EC cells as well as from undifferentiated human EC cells. Taken together, our findings suggest strongly that four regulatory regions, including a critical CRE/ATF element, help control the expression of the TGF‐β2 gene during early mammalian development. © 1995 Wiley‐Liss, Inc.
Effect of microinjection time during postfertilization S‐phase on bovine embryonic development
Authors: Gagné Marc , Pothier François , Sirard Marc‐André 
Published: February 2005

AbstractMicroinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken β‐actin promoter‐lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H‐thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21–22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9–18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin‐treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P<0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (>16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin‐ and cycloheximide‐treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P<0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage. © 1995 Wiley‐Liss, Inc.
Endocytosis in mouse blastocysts: Characterization and quantification of the fluid phase component
Authors: Dunglison Greta F. , Kaye Peter L. 
Published: February 2005

AbstractFluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H‐dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy‐requiring process, with only 20% accountable by diffusion as indicated by analysis at 4°C. Insulin stimulated uptake of 3H‐dextran by 30% (P<0.05) over the first hr. The rate of uptake then decreased in both control and insulin‐treated blastocysts. However, by 2 hr, insulin‐treated blastocysts contained 38% more 3H‐dextran (38%; P<0.01) than control blastocysts. Incubation of blastocysts in protein‐free medium increased 3H‐dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 ± 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/I and 10 g BSA/I, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid‐phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient‐containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells. An analysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant contributions of both phases of endocytosis to blastocyst metabolism in vivo. © 1995 Wiley‐Liss, Inc.
An isoform of ferritin as a component of protein yolk platelets in Schistosoma mansoni
Authors: Schüßler Peter , Pötters Elke , Winnen Ralf , Bottke Werner , Kunz Werner 
Published: February 2005

AbstractSchistosoma mansoni possesses two isoforms of the iron storage protein ferritin, Fer1 and Fer2. At the mRNA level as well as at the protein level, Fer1 is much more abundant than Fer2; females contain an about 15‐fold excess of Fer1 compared with males. In contrast, nearly equal amounts of Fer2 occur in both sexes. By electron microscopy we identified ferritin as a component of electron dense membrane‐bound bodies in cells of the vitellarium. The mode of formation of these inclusions (as inferred from electron microscopy) and the abundance of phospholipid multilayered membranes suggest that these bodies are of a lysosomal nature. Here we interpret these ferritin‐containing inclusions as protein yolk platelets. To date, most of the literature does not contain any hints of the existence of protein yolk in trematodes. The possible function of ferritin in embryonic development is discussed. © 1995 Wiley‐Liss, Inc.
Perivitelline space of marsupial oocytes: Extracellular matrix of the unfertilized oocyte and formation of a cortical granule envelope following the cortical reaction
Authors: Dandekar Pramila , Mate Karen E. , Talbot P. 
Published: February 2005

AbstractThe purpose of this study was to characterize the structure of the vestments surrounding unfertilized and cortical granule‐reacted oocytes from a marsupial, the grey short‐tailed opossum Monodelphis domestica and to determine if a cortical granule envelope (CGE) forms in the perivitelline space (PVS) following the cortical reaction. Unfertilized oocytes collected from mature ovarian follicles and oviducal oocytes that had undergone a cortical reaction were fixed for electron microscopy in the presence of ruthenium red which stabilizes extracellular matrices (ECM) and facilitates demonstration of a CGE. Unfertilized oocytes were surrounded by a zona pellucida and had a PVS which contained a thick ECM comprised of granules and filaments. This matrix appeared to attach to the oolemma and was structurally similar to matrices reported previously in the PVS of unfertilized oocytes from eutherian mammals and two other marsupials, the Virginia opossum and the fat‐tailed dunnart. The cortex of unfertilized oocytes contained cortical granules which were absent in oocytes recovered from the oviducts of mated females. Oviducal oocytes which lacked cortical granules exhibited a new coat within the PVS between the zona pellucida and the tips of the oocyte microvilli. This coat, the CGE, appeared structurally similar to CGEs described previously around fertilized eutherian oocytes. The CGE of the grey short‐tailed opossum is approximately 1 μm thick and is made up of numerous small dense granules. The coats of the opossum oocyte are compared to those present around other marsupial and eutherian oocytes. © 1995 Wiley‐Liss, Inc.
Chromatin condensation activity and cortical activity during the first three cell cycles of a mouse embryo
Authors: Ciemerych Maria A. 
Published: February 2005

AbstractOne‐cell parthenogenetic haploid embryos and blastomeres of the 2‐ and 4‐cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one‐cell embryos and of blastomeres.The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2‐ and 4‐cell embryos did not exhibit any cortical activity.In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2‐cell and 4‐cell stage blastomeres did not reveal condensation activity. © 1995 Wiley‐Liss, Inc.
Different transmission rates of herpesvirus thymidine kinase reporter transgenes from founder male parents and male parents of subsequent generations
Authors: Ellison Aaron R. , Wallace Helen , Al‐Shawi Raya , Bishop John O. 
Published: February 2005

AbstractPreviously we demonstrated that lines of transgenic mice carrying the herpes simplex type 1 virus thymidine kinase (HSV1‐tk) reporter gene are malesterile. Ectopic transcription of the HSV1‐tk reporter in the testis was initiated downstream of the normal translation initiation codon and truncated proteins consistent with translational initiation at the second and third ATG codons were synthesized. Here we describe the effects on fertility (1) of converting the second and third ATG codons of the HSV1‐tk reporter to CTG codons and (2) of utilizing the HSV type 2 thymidine kinase (HSV2‐tk) reporter gene, in which the second ATG codon is located downstream of the ATP‐binding pocket of the enzyme. Both reporters were coupled to the bovine thyroglobulin promoter (bTG‐tk1α and bTG‐tk2 transgenes). The level of ectopic expression of these transgenes in the testis, relative to expression in the thyroid, was one to two orders of magnitude less than that of bTG‐tk1.Sixty percent of male founders carrying the bTG‐tk1α and bTG‐tk2 transgenes were fertile but did not transmit the transgene. In contrast, most males from subsequent generations were fertile and transmitted the transgenes at the expected frequency. This difference between founder males and male descendants is also observed with certain constructs in which the HSV1‐tk reporter is coupled to other promoters. We attribute the effect to mosaicism among male founders, leading to competition between transgenic and nontransgenic spermatozoa and/or spermatogenic precursor cells and resulting in a lack of fertilization by transgenic sperm that would successfully fertilize eggs in the absence of competition. © 1995 Wiley‐Liss, Inc.
Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth
Authors: Yelian Frank D. , Yang Yan , Hirata Janie D. , Schultz Jeffery F. , Armant D. Randall 
Published: February 2005

AbstractTo investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum‐free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin‐binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell‐binding domain (FN‐120) and the Arg‐Gly‐Asp (RGD) recognition sequence. A 40 kD COOH‐terminal chymotryptic fragment of fibronectin containing both a heparin‐binding region and an alternate (non‐RGD) cell‐binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin‐binding domain, including the CS1 alternate cell‐binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val‐Thr‐Gly‐Arg‐Gly‐Asp‐Ser‐Pro‐Ala‐Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin‐mediated outgrowth. These findings demonstrate the key role of the central cell‐binding domain of fibronectin in trophoblast adhesion, and suggest four RGD‐binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley‐Liss, Inc.
Localization of intracellular calcium during the acrosome reaction in ram spermatozoa
Authors: Watson P. F. , Plummer J. M. , Jones P. S. , Bredl J. C. S. 
Published: February 2005

AbstractCalcium was identified by a pyroantimonate‐osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39°C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time.Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a “sawtooth” arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. © 1995 Wiley‐Liss, Inc.
Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria
Authors: Martin Renée H. , Rademaker Alfred 
Published: February 2005

AbstractAneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one‐half‐domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one‐domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one‐half‐domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley‐Liss, Inc.
Localization of epitopes for monoclonal antibodies at the N‐terminus of the porcine zona pellucida glycoprotein pZPC
Authors: Gupta S. K. , Yurewicz Edward C. , Afzalpurkar Abhijit , Rao K. V. S. , Gage Douglas A. , Wu H. , Sacco A. G. 
Published: February 2005

AbstractZona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse‐phase HPLC of the tryptic digest yielded twenty two peaks (T1–T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb‐455 and mAb‐467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb‐455 and mAb‐467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB‐MS identified T11 as the N‐blocked decapeptide pyro‐Gln‐Pro‐Val‐Trp‐Gln‐Asp‐Glu‐Gly‐Gln‐Arg derived from the N‐terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenie activity for mAb‐455 and mAb‐467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb‐455 and W,Q,D,E as critical for binding mAb‐467. Both mAbs inhibited binding of boar sperm to zona‐encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species. © 1995 wiley‐Liss, Inc.