bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pig membrana granulosa cells prevent resumption of meiosis in cattle oocytes
Authors: Kalous Jaroslav , Sutovsky Peter , Rimkevicova Zora , Shioya Yasuo , Lie Byong‐Lyul , Motlik Jan 
Published: February 2005

AbstractMembrana granulosa was isolated from healthy large antral follicles of prepubertal or cyclic gilts stimulated with PMSG or PMSG and hCG. Ultrastructural observations revealed that pieces of pig membrana granulosa were associated with the basement membrane. The cattle cumulus‐enclosed oocytes (COC) were placed in the rolled pieces of the pig membrana granulosa (PMG). After 8 and 24 hr of coculture with PMG from prepubertal gilts, only 16% and 21% of oocytes underwent GVBD, respectively. PMG from PMSG‐stimulated cyclic gilts blocked the resumption of meiosis in all COC. The inhibitory effect of heterologous granulosa cells was fully reversible. When COC were initially incubated for 2 and 4 hr, subsequent culture in PMG prevented GVBD in 100% and 36% of oocytes, respectively. This suggests that functional contact between COC and PMG was established during the first 2 hr of coculture. To follow metabolic cooperation between PMG and COC, PMG was prelabeled with 3H‐uridine and cocultured with COC. Autoradiography on semithin sections revealed the intensive passage of 3H‐uridine from PMG into the cumulus layer and an oocyte. COC placed in PMG after GVBD (8 and 12 hr of an initial incubation) did not extrude the first polar body. PMG isolated from cyclic gilts after PMSG and hCG stimulation also inhibited GVBD of COC. Since nearly all COC placed in PMG isolated 10 and 12 hr after hCG remained in the GV stage after 24 hr of coculture, the hCG stimulation did not substantially diminish the meiosis inhibiting activity of PMG. During coculture, cattle cumulus cells were closely associated with the basement membrane, but no gap junctions were formed among heterologous granulosa cells. These results suggest that an inhibitory factor secreted by pig granulosa cells is not species specific and it can act in vitro without the mediation of gap junctions. © 1993 Wiley‐Liss, Inc.
Functional significance of cumulus expansion in the mouse: Roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass
Authors: Chen L. , Russell P. T. , Larsen W. J. 
Published: February 2005

AbstractGonadotropin‐stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6‐diazo‐5‐oxo‐1‐norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2‐cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation. © 1993 Wiley‐Liss, Inc.
Bovine oocyte development following different oocyte maturation and sperm capacitation procedures
Authors: Yang Xiangzhong , Jiang Shie , Foote R. H. 
Published: February 2005

AbstractVarious procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2–7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 μg/ml, oLH 5.0 μg/ml, and E2 1.0 μg/ml) at 39°C in 5% CO2 and 95% air. For IVF, frozen‐thawed sperm were treated with either 0.1 μM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 μg/ml heparin (H10 or H100) for 15 min. Our results demonstrated the following: (1) both M199 + FCS and M199 + FCS + H supported maturation development to the metaphase II stage (90–95%, P > 0.05); (2) when oocytes were matured in M199 + FCS without added hormones, A23187 sperm treatment was superior to H10 or H100 treatment for fertilization and blastocyst development of the inseminated oocytes (P < 0.05); (3) when oocytes were matured in M199 + FCS + H, A23187 treated sperm again produced a higher fertilization rate than the H10 group (P < 0.05), but the development to the blastocyst stage was similar among all 3 sperm treatment groups (P > 0.05); (4) direct comparison of the 2 maturation systems with A23187 treated sperm resulted in no difference in all criteria measured; however, (5) when compared retrospectively, beneficial effects of added hormones are evident for blastocyst development (but not for fertilization) when sperm were treated with heparin procedures. © 1993 Wiley‐Liss, Inc.
Quantitative estimation of chimerism in mice using microsatellite markers
Authors: Delhaise Françoise , Zhao Xuyun , Bralion Valérie , Dessy Franz , Georges Michel 
Published: February 2005

AbstractAn embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ‐line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring.The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample.Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice. © 1993 Wiley‐Liss, Inc.
Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells
Authors: Francolini Maura , Lavitrano Marialuisa , Lamia Carla Lora , French Deborah , Frati Luigi , Cotelli Franco , Spadafora Corrado 
Published: February 2005

AbstractMature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area.Part of the sperm‐bound foreign DNA is further internalized into nuclei. Using end‐labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei.Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end‐labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end‐labelled DNA. © 1993 Wiley‐Liss, Inc.
Transient expression of a human β‐actin promoter/lacZ gene introduced into mouse embryos correlates with a low degree of methylation
Authors: Nilsson Erik , Lendahl Urban 
Published: February 2005

AbstractWe have analysed the correlation between expression and methylation for the human β‐actin promoter introduced into mouse embryos. The β‐actin promoter was fused to the reporter gene lacZ, and expression was analysed after pronuclear injection into fertilized mouse eggs. We analysed transient expression in in vitro cultured preimplantation embryos and expression after chromosomal integration in 5 independent lines of transgenic mice. The in vitro cultured preimplantation embryos expressed lacZ from the 2‐cell to the blastocyst stages, and most abundantly at the morula stage. By increasing the amount of injected DNA, a larger proportion of embryos expressed lacZ. Embryos expressing lacZ in only a subset of the blastomeres were detected at all preimplantation stages. In contrast to the transient expression after injection, we have not detected lacZ expression in any of the 5 analysed lines of transgenic mice carrying the same construct. The lack of expression in transgenic mice correlates with hypermethylation of C residues in the vast majority of CG sequences in the integrated β‐actin/lacZ construct, whereas the injected construct was completely nonmethylated. We discuss methylation and other possible reasons for the observed differences in expression between injected and integrated copies of the β‐actin/lacZ construct and for lacZ expression in only a subset of blastomeres in preimplantation embryos. © 1993 Wiley‐Liss, Inc.
Lipid globules at the midpieces of Glyptocidaris crenularis spermatozoa and their relation to energy metabolism
Authors: Mita Masatoshi , Nakamura Masaru 
Published: February 2005

AbstractSpermatozoa of the sea urchin, Glyptocidaris crenularis, use endogenous triglycerides (TG), not phosphatidylcholine, to produce energy for swimming. This study was undertaken to examine ultrastructurally the location of TG available for utilization in energy metabolism in these spermatozoa. Each spermatozoon contained several lipid globules at the bottom of the midpiece. Following incubation with sea water, both the number and size of the lipid globules decreased significantly. The total volume of lipid globules in each spermatozoon was roughly halved after 1 h of incubation. Similarly, about half of the TG was metabolized during the same incubation period. Oxygen consumption by spermatozoa during the incubation indicated the oxidation of fatty acid derived from TG. Thus it appears that G. crenularis spermatozoa obtain energy through oxidation of fatty acid from TG stored in the lipid globules within their midpieces. © 1993 Wiley‐Liss, Inc.
The differential expression of lamin epitopes during mouse spermatogenesis
Authors: Moss Stuart B. , Burnham Brenda L. , Bellvé Anthony R. 
Published: February 2005

AbstractThe presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti‐lamin AC and an anti‐lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti‐lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti‐lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti‐lamin B reacts with a ∼ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post‐meiotic nuclear matrices. Anti‐lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti‐lamin B, anti‐lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti‐lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley‐Liss, Inc.
Improved preservation of rat epididymal sperm for high‐resolution low‐voltage scanning electron microscopy (HR‐LVSEM)
Authors: Stoffel Michael H. , Frethem Chris , Hamilton David W. , Friess Armin E. 
Published: February 2005

AbstractVarious fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high‐resolution low‐voltage scanning electron microscopy (HR‐LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR‐LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OsO4 and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing. © 1993 Wiley‐Liss, Inc.
The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level
Authors: Fiser P. S. , Fairfull R. W. , Hansen C. , Panich P. L. , Shrestha J. N. B. , Underhill L. 
Published: February 2005

AbstractThe effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley‐Liss, Inc.
Fine structure of the spermatozoa of Crassostrea gigas (Mollusca, Bivalvia)
Authors: Bozzo Maria Gràcia , Ribes Enric , Sagrista Elena , Poquet Montserrat , Durfort Mercedes 
Published: February 2005

AbstractWe describe sperm ultrastructure and acrosome differentiation during spermiogenesis in Crassostrea gigas (Mollusca Bivalvia). The sperm cell is a uniflagellated cell of the primitive type. The head region contains a rounded or conical nucleus surmounted by small acrosome. This organelle consists of a membrane‐bound acrosomal granule, the contents of which have a homogeneous density, except in the anterior region, which is positive for PTA. The acrosome also surrounds the perforatorium, which includes oriented fibrillar elements: this is the axial body. The middle piece contains four mitochondria encircling two perpendicular centrioles. The distal centriole is provided with a system of mechanical fixation to the plasma membrane, consisting of nine fibers in radial arrangement. The tail flagellum, about 50 m̈m long, contains the usual microtubular axoneme. © 1993 Wiley‐Liss, Inc.
CDX2 co‐localizes with liver–intestine cadherin in intestinal metaplasia and adenocarcinoma of the stomach
Authors: Ko Samuel , Chu Kent‐Man , Luk John Moon‐Ching , Wong Bonnie Wing‐Yan , Yuen Siu‐Tsan , Leung Suet‐Yi , Wong John 
Published: February 2005

AbstractCDX2 and liver–intestine (LI)‐cadherin are intestine‐specific markers and both are physiologically expressed in the small intestine and colon. Recent studies have demonstrated that CDX2 regulates LI‐cadherin gene (CDH17) expression in colorectal cancer. The present study investigated the relationship of CDX2 and LI‐cadherin expression in gastric cancer. One hundred and nine pairs of tumour and non‐cancerous gastric mucosa were collected from gastrectomy specimens. Protein expression levels of CDX2 and LI‐cadherin were determined by immunohistochemical staining. Semi‐quantitative RT‐PCR showed that the mRNAs of both CDX2 and CDH17 were highly expressed in tumour compared with non‐cancerous mucosa. Overexpression of CDX2 was significantly associated with CDH17 in gastric adenocarcinoma. Furthermore, the expression of CDX2 and LI‐cadherin proteins was strongly coupled in intestinal metaplasia. In conclusion, overexpression of CDH17 is significantly associated with CDX2. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin‐embedded breast cancer
Authors: Lottner Christian , Schwarz Stephan , Diermeier Simone , Hartmann Arndt , Knuechel Ruth , Hofstaedter Ferdinand , Brockhoff Gero 
Published: February 2005

AbstractThe determination of HER2/neu status in breast carcinomas has become essential for the selection of breast cancer patients for Herceptin therapy. Herceptin treatment is used in patients with metastatic breast carcinoma with HER2/neu protein overexpression detected by immunohistochemistry (IHC) or gene amplification analysed by fluorescence in situ hybridization (FISH). A multiparametric fluorescent approach based on the simultaneous detection of HER2/neu gene amplification and protein expression was established to increase the accuracy, and to improve the reproducibility, of HER2/neu diagnostics. Based on four paraffin‐embedded breast cancer cell lines, a combined fluorescent immunostaining (FIHC) and FISH method was developed by using the PathVysion HER2 DNA Probe Kit (VYSIS) and the polyclonal antibody from the HercepTest (DAKO). Diagnostic applicability was documented on 215 formalin‐fixed primary breast carcinomas. Criteria for immunofluorescence quantification were chosen by analogy with the FDA‐approved HercepTest scoring, ranging from 0 to 3+. There was 97.7% concordance between conventional IHC and fluorescence IHC. The FISH data resulting from the multiparametric approach did not differ from conventional FISH. Breast carcinomas with HER2/neu protein overexpression and simultaneous gene amplification were detected with 100% sensitivity. In addition, five of the 215 cases (2.3%) had HER2/neu gene amplification without protein overexpression. The main advantage of this novel approach is that polysomy, aneuploidy, gene amplification, and protein content can be analysed simultaneously in the same cell. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Defect‐selective etching of SiC
Authors: Weyher J. L. , Lazar S. , Borysiuk J. , Pernot J. 
Published: February 2005

AbstractTwo methods of defect‐selective etching of SiC are described: (i) orthodox etching in molten KOH–NaOH eutectic with 10% of MgO powder (E + M etch) and (ii) electroless photo‐etching in aqueous KOH solutions (PEC method). The first etch shows similar effectiveness in revealing different type of dislocations, micropipes and stacking faults, as the commonly used molten KOH. The second approach is new for studying defects in SiC and is most suitable for revealing stacking faults both on the disoriented (0001) basal planes and on the ($1 \bar1 00$) cleavage surfaces. Cross‐calibration of both techniques and examples of calibration of PEC etch features by TEM are shown. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Forward‐bias degradation in 4H–SiC p+nn+ diodes: Influence of the mesa etching
Authors: Camara N. , Thuaire A. , Bano E. , Zekentes K. 
Published: February 2005

AbstractWe have investigated the appearance of defects in silicon carbide p+nn+ 4H–SiC diodes by observing the forward‐bias‐induced light emission through the substrate. The spatial intensity distribution, the temporal evolution and the spectral content of the electroluminescence (EL) signal have been measured in order to detect, identify and understand the defect formation during forward‐bias application. It was found that mesa etching is a main cause for the formation of extended defects. Reduction of mesa‐etching‐induced defects is the subject of our investigations. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)