bluuebunny/crossref_metadata_2025_split huggingface dataset:
PCR-generated cDNA libraries from reduced numbers of mouse oocytes
Authors: Revel Frédérique , Renard Jean-Paul , Duranthon Véronique 
Published: September 2008

SummaryWe describe a rapid and reproducible method for cloning cDNA amplified from 10 mouse oocytes. The procedure consists in priming cDNA synthesis from a crude cellular extract using an oligo d(T) containing primer and submitting the size-limited cDNA first strand to poly(dG) tailing. The whole cDNA population is then polymerase chain reaction (PCR) amplified using two primers complementary to oligo d(A) and oligo d(G) ends of the cDNA. In this procedure no purification steps are required. We obtained about 5 ×106clones from 10 oocytes. Screening of the library showed that the relative abundance of the transcripts was preserved during amplification and cloning and that the procedure allows cloning of low-abundance sequences at least as rare as 0.008% of the mRNA. The repeatable generation of representative cDNA libraries from reduced numbers of oocytes or embryos should open new opportunities for obtaining genetic information from mammalian preimplantation embryos.
Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes
Authors: Inoue Maki , Naito Kunihiko , Aoki Fugaku , Toyoda Yutaka , Sato Eimei 
Published: September 2008

SummaryTo investigate the involvement of mitogen-activated protein kinase(MAP kinase) in meiotic maturation of porcine oocytes, we assayed MAP kinase activity using basic protein(MBP) as a substrate. MAP kinase activity was low during the germinal vesicle stage, 0–20 h of culture. An abrupt increase was observed at metaphase I(30 h of culture), and activity remained significantly higher than that at 0 h until 50 h of culture, with a transient slight decrease at the time of first polar body extrusion (40 h). Detection of the kinase activity by an in-gel phosphorylation assay confirmed that the 42 and 44 kDa MAP kinases were significantly activated in 45 h cultured oocytes but not in 0 h oocytes, and just slightly in 20 h oocytes. In immunoblotting, however, the 42 and 44 kDa bands were detected in 0, 20 and 45 h cultured oocytes. Furthermore, the signal strength of the two bands did not change during the period of culture, but shifted up to 45 h, indicating that the activation of MAP kinase depended not on the synthesis but on the phosphorylation of this enzyme. These results suggest that the activation of MAP kinase is involved in the regulation of meiotic maturation of porcine oocytes, and especially in the regulation after germinal vesicle breakdown.
Response of avian nuclei to mammalian maturation promoting factor (MPF)
Authors: Trefil P. , Horská M. , Kaňka J. , Fulka J. , Jirmanová J. , Moor R.M. , Fulka J 
Published: September 2008

SummaryChicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.
Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis
Authors: Cuadros-Fernández Jorge M. , Esponda Pedro 
Published: September 2008

SummaryWe have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA Polymerase. I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar pricursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.
Microfilament assembly and cortical granule distribution during maturation, parthenogenetic activation and fertilisation in the porcine oocyte
Authors: Kim Nam-Hyung , Day Billy N. , Lee Hoon Taek , Chung Kil-Saeng 
Published: September 2008

SummaryIn this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation andin vitrofertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.
Comparison of method for activating mouse oocytes for spermatid nucleus tranfer
Authors: Sasagawa Isoji , Yanagimachi R. 
Published: September 2008

SummaryIn the mouse, mature oocytes injected with prespermatozoal cell nuclei remain unactivated. Additional stimulation is needed to trigger oocyte activation leading to embryo development. We compared various electrical stimulations, treatment with cycloheximide alone or in combination with electrical stimulation, and injection of sperm-borne oocyte-activating factor (oscillogen) in terms of their oocyte activation and embryo development rates. Of all the treatments tested, a single electrical pulse (1.0 kV / cm, 128 μs) was the simplest, yet very effective, in allowing the development of the oocytes injected with spermatid nuclei.
Cyclopiazonic acid induces accelerated progress of meiosis in pig oocytes
Authors: Petr Jaroslav , Rozinek Jirří , Jílek František 
Published: September 2008

SummaryIn mammalian oocytes, calcium plays an important role in the regulation of meiotic maturation. In our study, we used the mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, to mobilise intracellular calcium deposits duringin vitromaturation of pig oocytes. The CPA treatment of maturing oocytes significantly accelerated the progress of their maturation. Oocytes entered the CPA-sensitive period after 21 h ofin vitroculture. A very short (5 min) exposure to CPA (100 mM) is sufficient to accelerate maturation and it seems that accelerated maturation can be triggered by a transient elevation of intracellular calcium levels. The effect of CPA is not mediated through the cumulus cells, because maturation is accelerated by CPA treatment even in oocytes devoid of cumulus cells. Culture of oocytes with the calcium channel blocker verapamil (concentrations ranging from 0.01 to 0.04 mM) blocked the progress of oocyte maturation beyond the stage of metaphase I. This block can be overcome by the mobilisation of intracellular calcium deposits after CPA treatment (100 nM). The microinjection of heparin (20 pl, 50.1 mg/;ml), the inhibitor of inositol triphosphate receptors, before CPA treatment prevented the acceleration of oocyte maturation. This indicates that CPA mobilises the release of calcium deposits through inositol trisphosphate receptors. On the other hand, the microinjection of procaine (20 pl, 200 nM) or the microinjection of ruthenium red (20 pl, 50 mM), both inhibitors of ryanodine receptors, did not prevent accelerated maturation in CPA-treated oocytes. If present in pig oocytes, ryanodine receptors evidently play no part in the liberation of calcium from intracellular stores after CPA treatment.
Polyspermy in Bufo arenarum oocytes matured in vitro
Authors: Oterino J. , Toranzo G. Sánchez , Zelarayán L. , Bühler M.I. 
Published: September 2008

SummaryFull-grown ovarian oocytes of the amphibian Bufo arenarum were induced to mature in vitro by removing the follicular layers (spontaneous maturation) or by treatment with progesterone (hormone-induced maturation). These oocytes were then treated with trypsin and inseminated with homologous spermatozoa. Oocytes matured in vivo that had not undergone any influence of the oviducts (coelomic oocytes), inseminated under the same experimental conditions, were used as controls. The results show that oocytes induced to mature in vitro and exhibiting apparently normal signs of activation were polyspermic. In fact, 2 h after insemination numerous functioning pronuclei could be observed in the animal hemisphere. These results suggest that even though the oocytes which matured in vitro were able to undergo activation after insemination, they were unable to establish an effective block to polyspermy.
Strain difference in the timing of meiosis resumption in mouse oocytes: involvement of a cytoplasmic factor(s) acting presumably upstream of the dephosphorylation of p34cdc2kinase
Authors: Polanński Zbigniew 
Published: September 2008

SummaryOocytes from eight inbred strains of mice were screened for the timing of germinal vesicle breakdown (GVB)in vitro. This characteristic varied between strains, reaching most extreme values in oocytes from AKR and BALB/c mice (3.1 and 1.6h after release from dibutyryl cAMP block, respectively;p<0.0001). The difference between AKR and BALB/c mice was confirmed in experiments in which GVB was inducedin vivoby stimulation with exogenous gonadotrophins. Analysis of the rate of GVB in hybrids obtained after fusion of nuclear and cytoplamic fragments of oocytes from both strains suggests that the factor responsible for the difference between AKR and BALB/c mice is located in the cytoplasm of the proghase oocytes. Finally, in oocytes from both strains stimulated to resume meiotic maturation with okadaic acid, an inhibitor of protein phosphatases types 1 and 2A the rate of GVB was the same (2.2h and 2.3h for AKR and BALB/c, respectively;p= 0.48). This suggests that the difference between strains is not related to the amount or quality of the pre-MPF (Maturation Promoting Factor) stored in the prophase oocyte, but to the factor(s) acting upstream of the dephosphorylation ofp34cdc2. kinase in the pathway leading to pre-MPF activation.
Chlortetracycline fluorescence patterns andin vitrofertilisation of frozen-thawed boar spermatozoa incubated under various bicarbonate concentrations
Authors: Abeydeera Lalantha R. , Funahashi Hiroaki , Kim Nam-Hyung , Day Billy N. 
Published: September 2008

SummaryPorcine oocyte-cumulus complexes were cultured in bovine serum albumin(BSA)-free North Carolina State University (NCSU)23 medium containing porcine follicular fluid (10%), cysteine (0.1 mg/ml)and hormonal supplements (eCG and hCG: 10IU/ml each) for 22h. They were then cultured in the same medium but without hormonal supplements for an additional 22h. After culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed ejaculated boar spermatozoa in tissue culture medium (TCM)199 containing caffeine (5mM), fetal calf serum (FCS; 10%) and varying concentrations (26–56 mM)of NaHCO3for 9h(experiment 1). In experiment 2, chlortetracycline (CTC) was used to assess the functional state of spermatozoa incubated under different NaHCO3concentrations. Experiment 3 examined the effect of FCS (1% and 10%)and NaHCO3(26 and 46 mM) on fertilisation parameters. Compared with 26 mM, penetration rate was significantly higher (p<0.05)at 36–56mM NaHCO3. Polyspermy showed a similar pattern although no difference was observed between 26 and 36 mM. At 46mM NaHCO3, the mean number of spermatozoa (MNS) penetrated per oocyte increased significantly (p< 0.05). A significantly higher proportion of spermatozoa were capacitated and acrosome reacted at 46 and 56mM NaHCO3, respectively. The fertilisation medium containing 46mM NaHCO3and 1% FCS showed a higher penetration rate (84%)with a relatively low incidence of polyspermy (39%). The results indicate that NaHCO3stimulates capacitation and/or the acrosome reaction of boar spermatozoa in a dose-dependent manner and thus affects fertilisation parameters.
Caffeine overrides the S-phase cell cycle block in sea urchin embryos
Authors: Patel Rajnikant , Wright Elizabeth M. , Whitaker Michael 
Published: September 2008

SummaryDuring the early mitotic cell cycles of the sea urchin embryo, the cell oscillates between S-phase and M-phase. In the presence of aphidicolin, a DNA synthesis inhibitor, a checkpoint control blocks the activation of the p34cdc2protein kinase, by keeping it in the inactive, tyrosine phosphorylated form, and the embryos do not enter mitosis. Caffeine has been shown to bypass the G2/M-phase checkpoint in mammalian cells and in cyclingXenopusextracts and to induce mitosis despite the presence of damaged or unreplicated DNA. In this study we show that caffeine also induces mitosis and cell division in sea urchin embryos, in the presence of unreplicated DNA, by stimulating the tyrosine dephosphorylation of p34cdc2and switching on its protein kinase activity. We also show that the caffeine-induced activation of the p34cdc2protein kinase is not mediated by either of the two second messengers, calcium and cAMP, or by inhibition of the p34cdc2tyrosine kinase. Thus, none of the mechanisms proposed for caffeine's action can explain how it overrides the S-phase checkpoint in the early cell cycles of the sea urchin embryo.
Primordial germ cells and oocytes ofBranchiostoma virginiae(Cephalochordata, Acrania) are flagellated epithelial cells: relationship between epithelial and primary egg polarity
Authors: Frick Jennifer E. , Ruppert Edward E. 
Published: September 2008

SummaryPrimordial germ cells (PGCs) are described from the gonad ofc. 2 cm juvenileBranchiostoma virginiae; early oocytes (c. 10 μm) and enlarging, previtellogenic oocytes (c. 35 μm) are described from the ovary ofc. 5 cm adults. The germinal epithelium of the juvenile gonad and adult ovary is composed of both germinal and somatic cells. In the juvenile, somatic cells retain contact with the basal lamina of the germinal epithelium though their perikarya may be displaced towards the lumen; the germinal epithelium is, therefore, a simple but pseudostratified epithelium. In the adult ovary, somatic cells may lose contact with the basal lamina and the epithelium appears to become stratified. PGCs and oocytes are identified as germ cells by the presence of nuage. PGCs and oocytes are polarised epithelial cells. They rest on a basal lamina, extend apically towards a lumen, form adhering junctions with neighbouring cells, and exhibit apical-basal polarity. PGCs and early oocytes have an apical flagellum with an associated basal body, accessory centriole, and one or more striated rootlet fibres. The flagellum is surrounded by a collar of microvilli. Once oocytes begin to enlarge and bulge basally into the connective tissue layer, the flagellum is lost, but the basal bodies and ciliary rootlets are present at the apex of 35 μm oocytes. Similarities of the oogenic pattern in cephalochordates and echinoderms indicate that the establishment of egg polarity in deuterostomes is influenced by the polarity of the germinal epithelium.
Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos
Authors: Plusa Berenika , Ciemerych Maria A. , Borsuk Ewa , Tarkowski Andrzej K. 
Published: September 2008

SummaryThe aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.
Chromatin dynamics inTriturus cristatusoogenesis: an epigenetic approach
Authors: Burlibaşa Liliana , Zărnescu Otilia , Cucu Natalia , Gavrilă Lucian 
Published: September 2008

SummaryOogenesis is a critical event in the formation of female gametes, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the chromatin architecture in newt oocytes. Using fluorescence microscopy, as well as transmission electron microscopy (TEM), immunohistochemical method and RE-ChIP assay, some peculiar aspects of chromatin and chromosome organization and evolution in crested newt oogenesis were investigated. We focussed our investigations on detection of certain epigenetic modifications (H4 hyperacetylation, H2A ubiquitinylation and cytosine methylation) at the rRNA gene (18S–5.8S–28S) promoter region. Our findings suggest that there is an involvement of some epigenetic modifications as well as of linker histone variants in chromatin architecture dynamics during crested newt oogenesis.
The current strategy of repair of tetralogy of Fallot in children and adults
Authors: He Guo-Wei , Liu Xiao-Cheng , Kong Xiang-Rong , Liu Li-Xin , Yan Ying-Qun , Chen Bao-Jun , Li Zong-Xiao , Jing Wen-Bin , Wang Zheng-Qing , Wang Kai , Zhang Wei , Chen Tie-Nan , Wang Ping-Shan , Lu Wan-li , Zhang Jian-Liang , Guo Zhi-Peng , Xue Lan-Gang , Zhu Yu-Xiang , Wang Xiu-Li , Xi Lei 
Published: September 2008

AbstractObjectivesThe strategies of repair of tetralogy of Fallot change with the age of patients. In children older than 4 years and adults, the optimal strategy may be to use different method of reconstruction of the right ventricular outflow tract from those followed in younger children, so as to avoid, or reduce, the pulmonary insufficiency that is increasingly known to compromise right ventricular function.MethodsFrom April, 2001, through May, 2008, we undertook complete repair in 312 patients, 180 male and 132 female, with a mean age of 11.3 years ±0.4 years, and a range from 4 to 48 years, with typical clinical and morphological features of tetralogy of Fallot, including 42 patients with the ventriculo-arterial connection of double outlet right ventricle. The operation was performed under moderate hypothermia using blood cardioplegia. The ventricular septal defect was closed with a Dacron patch. When it was considered necessary to resect the musculature within the right ventricular outflow tract, or perform pulmonary valvotomy, we sought to preserve the function of the pulmonary valve by protecting as far as possible the native leaflets, or creating a folded monocusp of autologous pericardium.ResultsThe repair was achieved completely through right atrium in 192, through the right ventricular outflow tract in 83, and through the right atrium, the outflow tract, and the pulmonary trunk in 36 patients. A transjunctional patch was inserted in 169 patients, non-valved in all but 9. There were no differences regarding the periods of aortic cross-clamping or cardiopulmonary bypass. Of the patients, 5 died (1.6%), with no influence noted for the transjunctional patch. Of those having a non-valved patch inserted, three-tenths had pulmonary regurgitation of various degree, while those having a valved patch had minimal pulmonary insufficiency and good right ventricular function postoperatively, this being maintained after follow-up of 8 to 24-months.ConclusionsBased on our experience, we suggest that the current strategy of repair of tetralogy of Fallot in older children and adults should be based on minimizing the insertion of transjunctional patches, this being indicated only in those with very small ventriculo-pulmonary junctions. If such a patch is necessary, then steps should be taken to preserve the function of the pulmonary valve.
Mindfulness Approaches in Cognitive Behavior Therapy
Authors: Singh Nirbhay N. , Lancioni Giulio E. , Wahler Robert G. , Winton Alan S. W. , Singh Judy 
Published: September 2008

AbstractMindfulness is the latest addition to the armamentarium of cognitive behavioral therapists. Mindfulness methods from the wisdom traditions, as well as from current psychological theories, are beginning to be used as cognitive behavioral strategies for alleviating psychological distress and for personal transformation. The use of mindfulness as a clinical tool is in its infancy, with attendant growing pains in theory, research and practice. We briefly discuss the historical context of the use of mindfulness, recent developments in theory, research and practice, and future developments. We conclude that mindfulness shows a lot of promise as a clinical treatment modality, but there are inherent pitfalls in the developing approaches.