bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epidermal growth factor and its receptor gene expression and peptide localization in porcine ovarian follicles
Authors: Singh Balwant , Rutledge Jean M. , Armstrong David T. 
Published: February 2005

AbstractThe present study was undertaken to determine the expression of genes for epidermal growth factor (EGF) and its receptor (EGF‐R) in various components of medium‐sized porcine ovarian follicles by reverse transcription‐polymerase chain reaction (RT‐PCR), and to localize their peptides during folliculogenesis by immunocytochemistry. A strong band for EGF mRNA transcript was detected in the oocyte, whereas the signal in cumulus, granulosa, and theca cells was very weak but detectable. In contrast, a very strong EGF‐R mRNA signal was observed in cumulus, granulosa, and theca cells, whereas the signal in the oocyte was very weak. EGF peptide was localized in the oocyte, cumulus, and granulosa cells of all stages of follicle. In the oocyte, the intensity of immunostaining was more pronounced in primordial and primary follicles, compared to antral follicles. In large antral follicles, immuno‐staining was pronounced in granulosa cells, whereas theca cells showed little or no detectable staining for EGF. EGF staining was also observed in the cumulus and granulosa cells of follicles undergoing atresia. EGF‐R immunostaining was observed in the oocytes of primordial and primary follicles, and in cumulus, granulosa, and theca cells of all stages of follicle, including atretic follicles. In large antral follicles, the intensity of immunostaining was more pronounced in theca cells than in granulosa cells, and the oocyte showed little or no detectable staining. No immunostaining was observed when the primary antibody was replaced with preimmune serum (EGF), or preabsorbed with the control peptide (EGF‐R), confirming the specificity of the staining procedures. These results suggest a local follicular production of EGF and its receptor in the porcine ovary, and thus a role for EGF of follicular origin in the regulation of follicular development in autocrine/paracrine fashion. © 1995 Wiley‐Liss, Inc.
Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts
Authors: Van Stekelenburg‐Hamers Annelies E. P. , Van Achterberg Tanja A. E. , Rebel Heggert G. , Fléchon Jacques E. , Campbell Keith H. S. , Weima Sjerp M. , Mummery Christine L. 
Published: February 2005

AbstractInner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal‐stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight‐cell block. © 1995 Wiley‐Liss, Inc.
Mitogen‐activated protein (MAP) kinase during the acquisition of meiotic competence by growing oocytes of the mouse
Authors: Harrouk Wafa , Clarke Hugh J. 
Published: February 2005

AbstractDuring the growth phase of oogenesis, oocytes acquire the ability to undergo meiotic maturation. Although the molecular basis of this meiotic competence is unknown, specific differences in microtubular organization exist between incompetent and competent mammalian oocytes. Mitogen‐activated protein (MAP) kinase has been implicated in microtubular regulation and is present in fully grown competent oocytes of mice, suggesting a possible role for this protein in the acquisition of meiotic competence. We report that the MAP kinase species, p42ERK2 and p44ERK1, were detectable by immunoblotting in incompetent oocytes at the early stages of oocyte growth and throughout subsequent growth and acquisition of competence. In partially competent oocytes, which can enter metaphase but cannot complete the first meiotic division, both p42ERK2 and p44ERK1 became phosphorylated, as judged by retarded electrophoretic mobility, and a morphologically normal spindle was assembled. In incompetent oocytes, which cannot enter metaphase, p42ERK2 and p44ERK1 remained nonphosphorylated. When these oocytes were treated with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, a portion of them entered metaphase and the slow‐migrating phosphorylated forms of p42ERK2 and p44ERK1 were observed. These phosphorylated forms appeared more rapidly, relative to the time of entry into metaphase, than during maturation of fully competent oocytes. The remaining incompetent oocytes, which did not enter metaphase during okadaic acid treatment, also did not generate slow‐migrating p42ERK2 and p44ERK1. These results suggest that the acquisition of meiotic competence during oocyte growth is not linked to the de novo appearance of p42ERK2 or p44ERK1, that the failure of partially competent oocytes to complete meiosis I reflects a defect acting downstream or independently of MAP kinase phosphorylation, and that the ability of meiotically incompetent oocytes to generate phosphorylated forms of p42ERK2 and p44ERK1 in response to okadaic acid is linked to the ability to enter metaphase. © 1995 Wiley‐Liss, Inc.
Differential effects of protein synthesis inhibitors on porcine oocyte activation
Authors: Nussbaum Diana J. , Prather Randall S. 
Published: February 2005

AbstractThis study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L‐[35S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D‐SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25‐kDa polypeptide to a 22‐kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley‐Liss, Inc.
Possible role of variant RNA transcripts in the regulation of epidermal growth factor receptor expression in human placenta
Authors: Ilekis John V. , Stark Benjamin C. , Scoccia Bert 
Published: February 2005

AbstractEpidermal growth factor receptor (EGFR) plays an important role in growth and differentiation. The human placenta expresses high levels of the receptor. In the placenta, as in many other human tissues, EGFR is encoded by two RNA transcripts of 5.8 kb and 10.5 kb. The placenta also expresses a putative truncated EGFR transcript of 1.8 kb, which encodes only the ligand binding domain of the receptor. The etiology and role of these variant EGFR transcripts is unknown. Using the human placenta as a model to study this area, we report (1) the relationships among these transcripts suggest that the induction of alternate pathways of EGFR RNA processing is involved in their etiologies; (2) the 10.5 kb transcript may be the principal transcript involved in determining the level of the protein receptor; and (3) the isolation of a soluble protein with characteristics consistent with a translational product corresponding to the 1.8 kb transcript, which may act in regulating the activity of EGFR. Together these results suggest that alternate processing of EGFR RNA into variant transcripts may represent a novel mechanism involved in the regulation of the receptor protein. © 1995 Wiley‐Liss, Inc.
Nonoverlapping expression of Cx43 and Cx26 in the mouse placenta and decidua: A pattern of gap junction gene expression differing from that in the rat
Authors: Pauken Christine M. , Lo Cecilia W. 
Published: February 2005

AbstractWe characterized the expression of two gap junction genes (Cx26 and Cx43) in the mouse decidua and placenta. In the decidua, in situ hybridization analysis and immunostaining studies revealed a high level of Cx43 expression. In contrast, Cx26 expression was not detected. Analysis of the placenta revealed that both Cx43 and Cx26 transcripts are expressed, but in nonoverlapping cell populations. Cx26 transcripts were observed only in the labyrinthine trophoblast layer of the placenta, a tissue of ectoplacental cone derivation. In contrast, no Cx43 transcripts were found in the placenta proper, but only in the maternally derived decidual cap covering the placenta. These results, in conjunction with previous observations in the mouse and rat, indicate that there may be species‐specific differences in the pattern of Cx43/Cx26 expression in the placenta and decidua. © 1995 Wiley‐Liss, Inc.
Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility
Authors: Young R. J. , Bodt B. A. , Heitkamp D. H. 
Published: February 2005

AbstractAn earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Trisbuffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer‐directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb2+ and Cd2+, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb2+ was time‐ and concentration‐dependent; the average percentage of hyperactivated sperm decreased from ∼ 30% to<5% (n = 5) in 1 hr at a Pb2+ concentration of 25 μM. Cd2+ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 μM, the decrease in the percent of hyperactivated sperm was ∼ 50% (n = 3). Hg2+, Zn2+, and Cr6+ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb2+ and Cd2+, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis. © 1995 Wiley‐Liss, Inc.
Sperm‐induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery
Authors: Tesarik Jan , Sousa Mario , Mendoza Carmen 
Published: February 2005

AbstractTemporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady‐state period of sperm‐induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal‐induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm‐induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca2+‐induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator. © 1995 Wiley‐Liss, Inc.
Sry‐negative XX sex reversal in the American cocker spaniel dog
Authors: Meyers‐Wallen V. N. , Palmer V. L. , Acland G. M. , Hershfield B. 
Published: February 2005

AbstractThe Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY‐negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y‐specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. © 1995 Wiley‐Liss, Inc.
Amino acid composition and endocrine control of vitelline envelope proteins in European sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata)
Authors: Hyllner Sven Johan , Barber Hipolito Fernández‐Palacios , Larsson D. G. Joakim , Haux Carl 
Published: February 2005

AbstractThe vitelline envelopes of European sea bass and gilthead sea bream are both composed of mainly four proteins with the molecular masses of 90, 52, 48, 45 kDa and 75, 50, 48, 44 kDa, respectively. Each protein has an amino acid composition that is characterized by a high content of proline and glutamic acid and a low content of cysteine, similar to the whole vitelline envelope of both species. The amino acid composition suggests that each protein is distinct but related to the other vitelline envelope proteins. The use of homologous antisera shows that both species have vitelline envelope proteins that are induced by estradiol‐17β. As males of both species synthesize these proteins after treatment with estradiol‐17β, the origin is not restricted to the ovaries. Vitellogenin of both Eurpoean sea bass and gilthead sea bream has the apparent molecular mass of 170 kDa. © 1995 Wiley‐Liss, Inc.
Distribution of the intermediate filament proteins vimentin, keratin, and desmin in the bovine ovary
Authors: van den Hurk R. , Dijkstra G. , van Mil F. N. , Hulshof S. C. J. , van den Ingh Th. S. G. A. M. 
Published: February 2005

AbstractThe distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co‐localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3–7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunore‐active, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small (<3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin‐positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport. © 1995 Wiley‐Liss, Inc.
Isolation, culture, and characterization of equine oviduct epithelial cells in vitro
Authors: Thomas Philip G. A. , Ignotz George G. , Ball Barry A. , Miller Paula G. , Brinsko Steven P. , Currie Bruce 
Published: February 2005

AbstractOviduct epithelial cells (OEC) increasingly are used to support embryonic development and to study gamete interactions with the female reproductive tract in vitro. This series of experiments was designed to characterize monolayers derived from oviduct epithelium. Epithelial cells harvested from the isthmus and ampulla of the oviducts of five estrous mares were cultured with or without the basal lamina extract, Matrigel. Within each group OEC were cultured in the presence of either estradiol‐17β or a carrier control. All groups were subcultured three times. Epithelial cell morphology and function were examined by microscopy, analysis of secreted proteins, and immunocytochemistry. Epithelial cells attached more rapidly and reached confluence sooner when cultured on Matrigel than in uncoated wells. Cells showed variable evidence of ciliary activity up to 12 days in primary culture. Cells grown on Matrigel had a more polarized appearance in primary culture than those in uncoated wells, although no morphologic difference between anatomic site of origin or between steroid treated groups was noted. Anatomic site of origin had no effect, and steroid treatment had minimal effects, on patterns of secreted proteins. However, some differences were noted in protein secretion between cells grown with or without Matrigel. These data suggest that culture substrate may affect structure and function of OEC monolayers. © 1995 Wiley‐Liss, Inc.
Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization
Authors: Mozingo Nancy M. , Vacquier Victor D. , Chandler Douglas E. 
Published: February 2005

AbstractAbalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley‐Liss, Inc.
UV irradiation of chromosomal DNA and its effect upon MPF and meiosis in mammalian oocytes
Authors: Bradshaw J. , Jung T. , Fulka J. , Moor R. M. 
Published: February 2005

AbstractBovine oocytes were irradiated at germinal vesicle (GV) or metaphase II (MI) stage, after Hoechst staining, with chromosomally focused UV‐C (254 nm) or UV‐A (> 330 nm). UV‐C irradiation at GV stage did not inhibit germinal vesicle breakdown (GVBD) or chromosomal condensation; spindle formation was abolished and maturation promoting factor (MPF) levels failed to increase. UV‐A irradiation at GV stage caused meiotic arrest at anaphase I; MPF levels were lower than control. UV‐C irradiation at MII stage led to subsequent abnormal parthenogenetic activation when MPF levels failed to decrease. A normal male but no female pronucleus was formed at fertilization. UV‐A irradiation at MII stage also caused abnormal activation; MPF levels declined normally. A normal male and abnormal female pronucleus formed at fertilization. UV‐A irradiation results have implications for oocyte evaluation during development using Hoechst staining. UV‐C irradiation is a potential means for oocyte enucleation in nuclear transfer. © 1995 Wiley‐Liss, Inc.