bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gelation in Multiple Link System
Authors: Suematsu Kazumi 
Published: August 2003

AbstractThe critical point theory is generalized to include gelation in multilink system with f functional units and J junction points. The equations derived include, as special cases, the cyclotrimerization model of J = 3, and the R–Af model of J = 2. The theory is applied to the recent observation of the cyclotrimerization of bisphenol‐A dicyanate. The theoretical prediction agrees exactly with the Stutz‐Simak observation, Dc = 0.504, and accords with the Georjon‐Galy‐Pascault observation, giving a confirmation of the physical soundness of the theory. Under the smoothness assumption, we derive post‐gelation relationships with loop formation, the result suggesting the formation of permanent sol molecules that resist being absorbed into gel phase throughout an entire reaction process.Representation of binary branching.imageRepresentation of binary branching.
Involvement of the Akt/mTOR pathway on EGF‐induced cell transformation
Authors: Nomura Masaaki , He Zhiwei , Koyama Ichiko , Ma Wei‐Ya , Miyamoto Ken‐ichi , Dong Zigang 
Published: August 2003

AbstractOur previous study demonstrated that phosphatidylinositol 3‐kinase (PI3K) is necessary for epidermal growth factor (EGF)–induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as PI3K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF‐induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM‐Akt1). We found that the treatment of cells with rapamycin inhibited EGF‐induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a PI3K inhibitor, attenuated EGF‐induced activator protein 1 (AP‐1) activation, treatment with rapamycin did not affect AP‐1 activity. Treatment with rapamycin inhibited EGF‐induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF‐induced phosphorylation of extracellular signal–regulated protein kinases (ERKs). We showed that introduction of DNM‐Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF‐induced cell transformation without blocking cell proliferation. The expression of DNM‐Akt1 also suppressed EGF‐induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF‐induced cell transformation in JB6 cells. © 2003 Wiley‐Liss, Inc.
A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm
Authors: Hill Francesca S. , Marchetti Francesco , Liechty Melissa , Bishop Jack , Hozier John , Wyrobek Andrew J. 
Published: August 2003

AbstractDe novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three‐color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of ∼80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, ∼0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology. Mol. Reprod. Dev. 66: 172–180, 2003. © 2003 Wiley‐Liss, Inc.
Effects of donor oocytes and culture conditions on development of cloned mice embryos
Authors: Gao Shaorong , McGarry Michelle , Priddle Helen , Ferrier Tricia , Gasparrini Bianca , Fletcher Judy , Harkness Linda , De Sousa Paul , McWhir Jim , Wilmut Ian 
Published: August 2003

AbstractMice have been successfully cloned from somatic and embryonic stem (ES) cells using the “Honolulu method.” In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM‐1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 × DBA/2) and B6CBAF1 (C57BL/6 × CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre‐implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos. Mol. Reprod. Dev. 66: 126–133, 2003. © 2003 Wiley‐Liss, Inc.
Role of adrenergic receptor subtypes in the control of human placental blood vessels
Authors: Resch BÉla Endre , Ducza Eszter , GÁspÁr RÓbert , Falkay George 
Published: August 2003

AbstractStatements of the Problem: The use of β2‐Adrenergic Receptor (AR) agonists and the potential use of α1‐AR blockers as tocolytics raise the question of how they influence placental circulation. The receptor profile was characterized via the amounts of the mRNA of α1‐AR subtypes and β2‐ARs. The mRNAs were detected by reverse transcription‐polymerase chain reaction. Electric field stimulation (EFS) was applied to test the pharmacological reactivity of the placental vessels. Expressions of β2‐ and all subtypes of α1‐AR mRNA were demonstrated in human placental vessels, and were significantly higher in the arteries. A significant difference was not found between the veins and the arteries as concerns the amount of α1D‐AR mRNA. There was a preponderance of α1A‐ and α1B‐AR mRNA as compared to α1D‐AR mRNA both in the arteries and in the veins. β2‐AR agonists and α1‐AR antagonists antagonized the EFS‐induced contractions of the placental arteries in a dose‐dependent manner. These effects were significantly less marked on veins at all applied doses. Urapidil antagonized the EFS‐induced contractions of both the placental arterial and vein rings in a dose‐dependent manner. The β2‐, α1A‐, and α1B‐AR are the important subtypes involved in the regulation of the contractility of the human term placental vessels. The possible increase in placental blood flow mediated by these ARs can even be beneficial during pregnancy. Accordingly, the use of β2‐AR agonists and the potential use of α1‐blockers as tocolytics seem safe on the basis of in vitro examinations as concerns the placental circulation. Mol. Reprod. Dev. 66: 166‐171, 2003. © 2003 Wiley‐Liss, Inc.
Identification and quantification of differentially expressed transcripts in in vitro‐produced bovine preimplantation stage embryos
Authors: Tesfaye Dawit , Ponsuksili Siriluck , Wimmers Klaus , Gilles Markus , Schellander Karl 
Published: August 2003

AbstractIn this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT‐PCR) to analyze the mRNA expression patterns in in vitro‐produced bovine 8‐cell, 16‐cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8‐cell (n = 188), 16‐cell (n = 94), morula (n = 35), and blastocyst (n = 15) were reverse transcribed and subjected to DDRT‐PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8‐cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty‐two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8‐cell stage and the rest derived from the 16‐cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT‐PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT‐PCR. In conclusion, we have successfully applied mRNA DDRT‐PCR to identify and isolate stage‐specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT‐PCR, quantitative real time PCR provides quantitative data on the expression of target genes. Mol. Reprod. Dev. 66: 105–114, 2003. © 2003 Wiley‐Liss, Inc.
A study of the nature of embryonic lethality in LIS1−/− Mice
Authors: Cahana A. , Jin X.L. , Reiner O. , Wynshaw‐Boris A. , O'Neill C. 
Published: August 2003

AbstractHomozygous deletion of the Lis1 gene (Lis1−/−) in mouse resulted in early embryonic lethality immediately after embryo implantation by an undefined mechanism. We seek to define the nature of this demise. LIS1 (pafah1b1) is a 46 kDa protein with seven tryptophan‐aspartate (WD) repeats. It docks with many proteins and has been implicated in microtubular function, cell division, intercellular transport, and nuclear and cellular motility. Combined Western and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) analyses showed that LIS1 expression from the blastocyst stage required new transcription from the embryonic genome. Consequently, the death of post‐implantation embryos may not reflect the first time during development that LIS1 was required, rather, it may reflect the first time following depletion of gametic stores that its actions were essential. Following culture of blastocysts in vitro for 96 hr the inner cell mass (ICM) of null embryos were significantly smaller than ICM of wild‐type siblings. Normal blastocyst outgrowths after 96‐hr culture had high levels of LIS1 expression in the outer cells of developing ICM and extensive expression in trophoblast cells. Lis1−/− embryos had significantly smaller trophoblast nuclei than wild‐type embryos. The results show that LIS1 expression is required for the continued normal development of the ICM and optimal trophoblast giant cell formation. Mol. Reprod. Dev. 66: 134–142, 2003. © 2003 Wiley‐Liss, Inc.
Evaluation of gene targeting by homologous recombination in ovine somatic cells
Authors: Williams Sarah H. , Sahota Virender , Palmai‐Pallag Timea , Tebbutt Scott J. , Walker Jean , Harris Ann 
Published: August 2003

AbstractMouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post‐natal fibroblasts generated G418‐resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post‐natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability. Mol. Reprod. Dev. 66: 115–125, 2003. © 2003 Wiley‐Liss, Inc.
Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization
Authors: Takahashi Toshifumi , Takahashi Eiji , Igarashi Hideki , Tezuka Naohiro , Kurachi Hirohisa 
Published: August 2003

AbstractIn vivo post‐ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca2+ oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca2+ homeostasis mainly through direct effects on the machinery involved in intracellular Ca2+ storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca2+‐oscillation‐dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 μM hydrogen peroxide (H2O2) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H2O2‐treated fresh oocytes. The frequency of Ca2+ oscillations was significantly increased whereas the amplitude of individual Ca2+ transients was lowered in the aged and H2O2‐treated fresh oocytes. The rates of rise and decline in individual Ca2+ transients were decreased in these oocytes, indicating impaired Ca2+ handling. When lipid peroxidation was assessed using 4,4‐difluoro‐5‐(4‐phenyl‐1,3‐buttadienyl)‐4‐bora‐3a, 4a‐diaza‐s‐indacene‐3‐undecanoic acid (C11‐BODIPY) in unfertilized oocytes placed in a 5% CO2 in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca2+ oscillations play a key role in the development of fertilized aged oocytes. Mol. Reprod. Dev. 66: 143–152, 2003. © 2003 Wiley‐Liss, Inc.
Differential expression of lysosomal associated membrane protein (LAMP‐1) during mammalian spermiogenesis
Authors: Moreno Ricardo D. 
Published: August 2003

AbstractThe mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent‐mannose‐6‐phosphate receptor (CI‐MPR) is transiently expressed in the cytoplasm of mid‐stage spermatids (steps 5–11). On the other hand, γ‐adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap‐phase spermatids (steps 1–5). Our major finding is that the lysosomal protein LAMP‐1 is differentially expressed during spermiogenesis. LAMP‐1 appears late in spermatogenesis (Acrosome‐phase) contrasting with LAMP‐2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI‐MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis. Mol. Reprod. Dev. 66: 202–209, 2003. © 2003 Wiley‐Liss, Inc.
β‐Alanine but not taurine can function as an organic osmolyte in preimplantation mouse embryos cultured from fertilized eggs
Authors: Hammer Mary‐Anne , Baltz Jay M. 
Published: August 2003

AbstractEarly preimplantation mouse embryos are susceptible to the detrimental effects of increased osmolarity and, paradoxically, their in vitro development is significantly compromised by osmolarities near that of oviductal fluid. In vitro development can be restored, however, by several compounds that are accumulated by 1‐cell embryos to act as organic osmolytes, providing intracellular osmotic support and cell volume regulation. Taurine, a substrate of the β‐amino acid transporter that functions as an organic osmolyte transporter in other cells, had been proposed to function as an organic osmolyte in mouse embryos. Here, however, we found that taurine is neither able to provide protection for in vitro embryo development against increased osmolarity nor is it accumulated to higher intracellular levels as osmolarity is increased, indicating that it cannot function as an organic osmolyte in early preimplantation embryos. In contrast, β‐alanine, the other major substrate of the β‐amino acid transporter, both protects against increased osmolarity and is accumulated to somewhat higher levels as osmolarity is increased, indicating that it is able to function as an organic osmolyte in embryos. However, we also found that β‐alanine is displaced from embryos by glycine—the most effective organic osmolyte in embryos previously identified—and β‐alanine does not increase protection above that afforded by glycine at concentrations near those in vivo. Thus, the β‐amino acid transporter is likely present in early preimplantation embryos to supply β‐amino acids such as taurine for purposes other than to serve as organic osmolytes. Mol. Reprod. Dev. 66: 153–161, 2003. © 2003 Wiley‐Liss, Inc.
Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa
Authors: Baxendale Rhona W. , Fraser Lynn R. 
Published: August 2003

AbstractIn addition to a bicarbonate‐regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein‐regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane‐associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2′,5′‐dideoxyadenosine significantly blocked these responses. Furthermore, GTPγS and NaF stimulated cAMP, but GDPβS and mastoparan had no apparent effect, consistent with recent evidence that Gs, but not Gi, contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (∼50–60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP. Mol. Reprod. Dev. 66: 181–189, 2003. © 2003 Wiley‐Liss, Inc.
Experimental demonstration that pre‐ and post‐conceptional mechanisms influence sex ratio in mouse embryos
Authors: JimÉnez Adela , FernÁndez RaÚl , Madrid‐Bury Ninoska , Moreira Pedro Nuno , Borque Concepción , Pintado BelÉn , GutiÉrrez‐AdÁn Alfonso 
Published: August 2003

AbstractPreviously we have demonstrated in two monotocous species (bovine and sheep), a relationship between time of insemination, moment of ovulation, and embryo sex ratio. Here, we have analyzed in a polytocous specie (mice) if in addition to pre‐conceptional mechanisms, also post‐conceptional ones affect the offspring sex ratio. To verify this hypothesis we carried out two experiments. In the first experiment, we analyzed the effect of mating dynamics on the sex ratio of mice with synchronic male and female embryo development. Females were mated before and after ovulation and sacrificed 13 days later for sex determination of embryos and reabsorptions. A decreased litter size, and an increased offspring sex ratio in matings occurring later in oestrus, supported the view that a biased sex ratio may occur as the result of behavioral differences between the populations of X‐ or Y‐bearing spermatozoa. In the second experiment, embryos developmentally synchronic and asynchronic with the recipient female endometrium were transferred, and again, 13 days later, females were sacrificed for sex determination of embryos and reabsorptions. The male proportion per litter found, indicated that our developmentally asynchronic transfers favored a sex ratio disbalance at birth. When combined, these results become the first experimental evidence supporting the view that both pre‐ and post‐conceptional mechanisms of sex ratio distortion in polytocous species are not mutually exclusive and both may explain, under different conditions, sex ratio deviations at birth. Mol. Reprod. Dev. 66: 162–165, 2003. © 2003 Wiley‐Liss, Inc.