bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo
Authors: Brazolot C. L. , Petitte J. N. , Etches R. J. , Gibbins A. M. Verrinder 
Published: February 2005

AbstractChicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli β‐galactosidase (β‐gal). A reporter construct (phspPTlacZpA) containing a mouse heat‐shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 μg LipofectinTM (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55‐3.1 μg DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn2+‐inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken β‐actin (pmiwZ) or cytomegalovirus (pMaori3) promoters.Endogenous chicken β‐gal and transferred bacterial β‐gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6–12 h with 150 μM ZnCl2. Bacterial β‐gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have b̃‐gal activity 16–24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct. Plated PCFs were lipofected as well, with stable incorporation of the gene construct indicated in 10% of positive events.Lipofected CBCs were injected into the subgerminal cavity of stage X (Eyal‐Giladi and Kochav, 1976) chicken embryos in a manner that has been shown to produce somatic and germline chimeras for untreated CBCs (Petitte et al., 1990). After 65 h of incubation, cells expressing β‐gal activity were observed in the prosencephalon, head ectoderm, and ventricle of the heart of a stage 11 (Hamburger and Hamilton, 1951) embryo. In other cases, bacterial β‐gal activity was detected extraembryonically, both in individual cells and in foci of expressing cells, 24, 48, and 65 h after injection. Few, if any, single expressing cells and no foci were detected following injection of the LipofectinTM:DNA mixture directly into the embryo.Refinement of these procedures could contribute to the development of transgenic poultry, without reliance on retroviral vectors for DNA transmission or incorporation.
Effects of maturation and co‐culture treatments on the developmental capacity of early bovine embryos
Authors: Wiemer Klaus E. , Watson Andrew J. , Polanski Voyteck , McKenna Ann I. , Fick Gordon H. , Schultz Gilbert A. 
Published: February 2005

AbstractA total of 901 cumulus–oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n=451), were cultured in TCM‐199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n=450) were cultured in TCM‐199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P <0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co‐cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co‐cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co‐cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co‐culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P <0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16‐cell stage was also greater (P <0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P<0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co‐culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups. Results indicate that the combination of in vitro maturation in TCM‐99 + 10% FBS and hormones followed by co‐culture on oviductal monolayers is a superior system for the production of early bovine embryos.
Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: Involvement in the segregation of mitochondrial populations
Authors: Tourte Monique , Besse Colette , Mounolou Jean‐Claude 
Published: February 2005

AbstractAn organized microtubular cytoskeleton was discovered in the cytoplasm of Xenopus laevis oocytes. The microtubules were observed in 10‐ to 30‐m̈m cryostat sections by indirect immunoperoxidase labeling using an antibody to tubulin. A gradual extraction of cells with a nonionic detergent was essential for good penetration of the antibody into the cells. In the cytoplasm of all previtellogenic oocytes, a dense network of criss‐crossed long microtubules was associated in a basket‐like structure surrounding the mitochondrial mass. At the beginning of vitellogenesis, the network meshes enlarged, while clusters of mitochondria migrated, in close association with microtubule bundles. At the beginning of vitellogenesis, the reorganization of the microtubular network, mostly in the vegetal hemisphere, occurred during the segregation of the mitochondrial populations. Reorganization is characterized by (1) a temporary enlargement of the network and close association of mitochondrial clusters with microtubular bundles, and (2) a progressive organization of a ring‐shaped microtubular structure in the crown elaboration area. It is hypothesized that these modifications of the microtubular cytoskeleton contribute to the maintenance of cell shape and the polarized organization of the cell.
Equine oocyte in vitro maturation: Influences of sera, time, and hormones
Authors: Willis P. , Caudle A. B. , Fayrer‐Hosken A. 
Published: February 2005

AbstractObjectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 μg/ml), equine luteinizing hormone (eLH; 100 μg/ml), bovine follicle‐stimulating hormone (FSH; 5 μg/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P < 0.05) increased by MS, MSO, and FCS as compared with the control (BSA). In vitro culture for either 15 or 32 hr significantly (P < 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P < 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.
Detection of a soluble acrosome reaction‐inducing factor, different from serum albumin, associated with the ovulated egg‐cumulus complex
Authors: Boatman Dorothy E. , Robbins Robert S. 
Published: February 2005

AbstractSoluble extracts of the ovulated hamster egg‐cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona‐bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt‐stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona‐bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that (1) the ovulated ECC contains a soluble ARIF that augments zona‐induced ARs and sperm penetration and (2) the ARIF is not serum albumin.
Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro
Authors: Yoshida Mitsutoshi , Ishigaki Koji , Pursel Vernon G. 
Published: February 2005

AbstractThe present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM‐199, Waymouth MB 752/1, and mTLP‐PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P < 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/1 with or without pFF than in oocytes matured in the other two media (P < 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP‐PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P < 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/1, can remarkably promote this ability.
Electrofusion‐induced intracellular Ca2+ flux and its effect on murine oocyte activation
Authors: Rickords L. F. , White K. L. 
Published: February 2005

AbstractThese experiments were designed to monitor influx of extracellular Ca2+ into the murine ooplasm following a 1.56 kV · cm−1 direct current (DC) electrofusion pulse and subsequently to determine its effect on rate of activation. Pulse media consisted of nonelectrolyte (0.3 M mannitol) and electrolyte (phosphatebuffered saline; PBS) media each containing 0.0, 0.05, or 0.9 mM Ca2+ (groups T1–T3 and T4–T6, respectively). Cumulus‐free oocytes were incubated in 100 μl drops of PBS containing 2 μM of the calcium indicator fluo‐3/AM for 60 min at 37°C. Fluo‐3/AM‐loaded oocytes were equilibrated for 7 min in assigned treatment media (T1–T6) prior to application of DC pulse. Change in fluorescent intensity was monitored for 6.5 min after DC pulse by photon counting spectrofluorometry. Fluorometric measurements demonstrate a dramatic rise in intracellular free Ca2+ (Ca2+i) following DC pulse is associated with Ca2+ ion concentration in the pulse medium. Significantly (P < 0.01) higher Ca2+i levels were observed when 0.9 mM Ca2+ was added to the pulse medium (T3 and T6) compared with pulse medium containing lower Ca2+ ion concentrations (T1, T2, T4, and T5; P > 0.05). Differences (P < 0.01) were observed in peak Ca2+i levels 18 sec after pulse with mean percent change in fluorescence of 5.1%a, 33.9%b, 112.7%c, 1.2%a, 9.3%a, and 99.9%c for T1–T6, respectively (values with different superscripts are significantly different at P < 0.01). Increased oocyte membrane permeability to Ca2+ ion after DC pulse was observed for a minimum of 5 min after delivery of the 1.56kV · cm−1 pulse. Nonloaded oocytes receiving the same pulse treatments were cultured in 100 μl drops of Whitten's medium for 8 h prior to evaluation of oocyte activation. Activation was defined as the formation of one pronucleus and one polar body, two or more pronuclei, or two cells each containing a nuclear structure. Activation rates of 28.2%b, 31.1%b, 70.4%d, 35.6%b, 57.3%c, 71.8%d 8.7%a (P < 0.01) were observed for T1–T6 and control nonpulsed oocytes, respectively. These data demonstrate that supplementation of 0.9 mM of Ca2+ ion to pulse medium results in a dramatic rise in Ca2+i after DC pulse. This influx of Ca2+ ion has a positive effect on oocyte activation rate.
Sex‐related differences in developmental rates of bovine embryos produced and cultured in vitro
Authors: Xu K. P. , Yadav B. R. , King W. A. , Betteridge K. J. 
Published: February 2005

AbstractThe classical concept of sex determination in mammals is that a Y chromosomal gene controls the development of the indifferent gonad into a testis. Subsequent divergence of sexual phenotypes is secondary to this gonadal determination. The most likely candidate gene is SRY (sex‐determining region Y) in humans, and Sry in mouse. However, several lines of evidence indicate that sexual dimorphism occurs even before the indifferent gonad appears. Here we present evidence that bovine male embryos generally develop to more advanced stages than do females during the first 8 days after insemination in vitro. Corresponding relationships between both cell numbers and mitotic indices and sex were also seen. Although it is not clear whether this phenomenon involves factors originating before or after fertilization, these findings suggest that sex‐related gene expression affects the development of embryos soon after activation of the embryonic genome and well before gonadal differentiation.
Expression cloning of TGF‐β receptors
Authors: Lin Herbert Y. , Wang Xiao‐Fan 
Published: February 2005

AbstractUsing a powerful expression cloning method in COS cells, we have cloned the TGF‐β types II and III receptors. The type III TGF‐β receptor is a membrane‐bound proteoglycan with a core protein of about 110 kDa. Stable expression of the type III receptor in L6 myoblasts leads to an apparent increase in the ability of the type II receptor to bind iodinated TGF‐β1. The cloned type II receptor has a predicted protein core of about 60 kDa with a cysteine‐rich extracellular domain, a single transmembrane domain, and a functional serine/threonine kinase domain that is homologous to the activin receptor and to the C. elegans protein daf‐1. These results implicate serine/threonine phosphorylation as an important mechanism of TGF‐β action. © 1992 Wiley‐Liss, Inc.
Negative regulation of gene expression by TGF‐β
Authors: Matrisian Lynn M. , Ganser Gail L. , Kerr Lawrence D. , Pelton Ron W. , Wood Lauren D. 
Published: February 2005

AbstractStromelysin gene expression is transcriptionally activated by a number of growth factors (e.g., EGF and PDGF), tumor promoters (e.g., TPA), and oncogenes (e.g., ras, src) through an AP‐1‐dependent mechanism. TGF‐β repression of stromelysin induction is mediated at the level of transcription by an element located at position −709 in the rat stromelysin promoter referred to as the TGF‐β inhibitory element (TIE). A TIE‐binding protein complex is induced by treatment of rat fibroblasts with TGF‐β. This protein complex contains the protooncogene c‐fos, and induction of c‐fos by TGF‐β is required for the repressive effects of TGF‐β on stromelysin gene expression. Interestingly, c‐fos induction is also required for stimulation of stromelysin expression by EGF in rat fibroblasts. Preliminary studies suggest that differential regulation of members of the jun family of early‐response genes may explain this apparent paradox and determine whether stromelysin is induced or repressed by growth factors. TGF‐β stimulation therefore initiates a cascade of events that results in a specific pattern of gene expression: the direct stimulation of early‐response genes can lead to subsequent induction or repression of other genes.Growth factor regulation of matrix metalloproteinases appears to play a role in embryonic development in the morphogenesis of the murine lung. Treatment of embryonic lungs in organ culture with the growth factors EGF or TGF‐β results in stimulation of growth and inhibition of branching morphogenesis. A similar inhibition of branching was observed when these lung rudiments were treated with the matrix metalloproteinase collagenase. Most interestingly, the effects of EGF and TGF‐β can be completely reversed by the tissue inhibitor of metalloproteinases, TIMP. TGF‐β has the opposite effect on growth of murine lung rudiments—growth is inhibited in a dose‐dependent manner. This example illustrates a potential role for growth factor regulation of matrix‐degrading metalloproteinases in complex developmental processes. © 1992 Wiley‐Liss, Inc.
TGF‐β3‐Mediated tissue interaction during embryonic heart development
Authors: Runyan Raymond B. , Potts Jay D. , Weeks Daniel L. 
Published: February 2005

AbstractA critical process during early heart development is the formation of mesenchymal cells which will contribute to valves and septa of the mature heart. These cells arise by an epithelial‐mesenchymal transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract areas of the heart. Adjacent endothelial cells in the atrium and ventricle remain epithelial. A three‐dimensional collagen gel culture system has been exploited to examine the interactions that mediate this transformation. The AV canal myocardium produces a stimulus that is transmitted through an intervening extra‐cellular matrix to the AV canal endothelium. This interaction is regionally specific, such that ventricular myocardium does not provide an adequate stimulus and ventricular endothelium does not respond to the AV canal myocardial stimulus. Exogenous TGF‐β1 (or TGF‐β2) can complement ventricular myocardium to produce transformation by AV canal endothelium. A blocking antibody, effective against several TGF‐β, prevents cell transformation. To identify the specific member of the TGF‐β family that functions in situ, antisense oligonucleotides for each of the numbered TGF‐β were topically added to AV canal explant cultures. Only the oligonucleotide targeted to TGF‐β3 was an effective inhibitor of mesenchymal cell formation. Studies have been undertaken to localize specific mRNas by in situ hybridization and RNase protection assays. These assays have concentrated on the regional and temporal appearance of TGF‐β2 and 3. Surprisingly, RNase protection assays with a TGF‐β3 sense probe showed the presence of a transcript complementary to TGF‐β3. Further analysis of this tissue interaction included the testing of a variety of signal transduction mechanisms including kinases, G‐proteins, and intracellular calcium. Tissue interaction in the heart is a complex interaction in which regulation of the induction process occurs in both the inducing and target tissues. © 1992 Wiley‐Liss, Inc.
Marsupial fertilization: Some further ultrastructural observations on the dasyurid Sminthopsis Crassicaudata
Authors: Breed W. G. , Leigh C. M. 
Published: February 2005

AbstractVarious morphological aspects of in vivo egg maturation and sperm—egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron‐dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron‐dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron‐dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm—egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.
drop out: A third chromosome maternal‐effect locus required for formation of the Drosophila cellular blastoderm
Authors: Galewsky Samuel , Schulz Robert A. 
Published: February 2005

Abstractdrop out (dop) is a recessive maternal‐effect locus identified in a screen for female‐sterile mutations in Drosophila polytene region 71C‐F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularizaiton in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.