bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection and location of amphiregulin and Cripto‐1 expression in the developing postnatal mouse mammary gland
Authors: Kenney N. J. , Huang R.‐P. , Johnson G. R. , Wu J.‐X. , Okamura D. , Matheny W. , Kordon E. , Gullick W. J. , Plowman G. , Smith G. H. , Salomon D. S. , Adamson E. D. 
Published: February 2005

AbstractAmphiregulin (Ar) and Cripto‐1 (Cr‐1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr‐1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transciption‐polymerase chain reaction (RT‐PCR) was used to detect transcripts for Ar and Cr‐1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4‐week to mature 12‐week‐old mouse fourth inguinal mammary gland, Ar and Cr‐1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr‐1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4‐week and 6‐week‐old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr‐1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr‐1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGFα), Ar and Cr‐1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland. © 1995 Wiley‐Liss, Inc.
Analysis of G protein α subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT‐PCR approach
Authors: Rambhatla Lakshmi , Patel Bela , Dhanasekaran N. , Latham Keith E. 
Published: February 2005

AbstractWe have developed a novel reverse transcription‐polymerase chain reaction (RT‐PCR)‐based approach for systematically quantifying in a single experiment the abundances of many different mRNAs in preimplantation mouse embryos. With this approach, the entire mRNA population from a small number of embryos is amplified while preserving the relative abundance of each mRNA in the cDNA population. The cDNA is analyzed by quantitative hybridization to radiolabeled probes. The approach is very sensitive and provides reliable, quantitative data regarding changes in mRNA abundance. A major advantage of this method is that estimates of mRNA copy number can be obtained and compared between different mRNAs. With this approach, we analyzed the patterns of expression of nine G protein α subunit mRNAs (Gαs, Gαi, Gαq′, Gαo′, and Gα11–15) in oocytes, eggs, and preimplantation embryos from fertilization to the blastocyst stage. Six α subunit mRNAs were expressed at significant levels, all of which underwent significant temporal alterations in expression. The mRNAs encoding some α subunit types were expressed predominantly in the egg and 1‐cell embryo, underwent sharp reductions during the 2‐cell stage, and were re‐expressed between the 8‐cell and blastocyst stages. One α subunit mRNA increased in abundance at the early blastocyst stage. The possible significance of these alterations in G protein mRNA abundance to embryonic development is discussed. © 1995 Wiley‐Liss, Inc.
Separation of round spermatids from the rat using an immunoselection panning technique
Authors: Pelengaris S. A. , Moore H. D. M. 
Published: February 2005

AbstractA method was devised for the isolation of round spermatids from the rat using a positive immunoselection technique (panning). A testis suspension was prepared from adult rats by enzymatic digestion of seminiferous tubules with collagenase. Specific mouse monoclonal antibody (97.25) was indirectly attached to Petri dishes and used in a panning protocol to purify spermatids from the testis cell suspension. The quantity and purity of cells isolated were determined by cell counts and histochemical (periodic acid‐Schiff stain) or by immunostaining with acrosome‐specific antibodies. A mean yield of 1.38 ± 0.15 × 107 cells per dish was obtained with a purity of more than 90%. The viability of the cells was confirmed by epifluorescent microscopy with propidium iodide/carboxyfluorescein acetate probes. Northern blot analysis of RNA extracted directly from the dish indicated good integrity of a spermatid‐specific transcript of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). © 1995 Wiley‐Liss, Inc.
Composition of human zona pellucida as revealed by SDS‐PAGE after silver staining
Authors: Bercegeay S. , Jean M. , Lucas H. , Barriere P. 
Published: February 2005

AbstractThe macromolecular composition of zona pellucida (ZP) isolated from human oocytes and embryos was characterized by one‐ and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1‐D‐ and 2‐D‐SDS‐PAGE) under reducing conditions after silver staining. ZP specimens obtained after in vitro fertilization were removed from pools of heavily fragmented embryos and inseminated oocytes that failed to fertilize. For unfertilized oocytes, two major bands with an apparent molecular weight of, respectively, 96 and 76–54 kDa were observed after 1‐D‐SDS‐PAGE and silver staining. When ZP were isolated from fragmented embryos, the electrophoretic pattern showed a marked attenuation of the 96‐kDa band. Silver‐stained 2‐D‐SDS‐PAGE analysis of ZP components from unfertilized oocytes revealed the presence of four protein trains: ZP1 (Mr = 92–80 kDa, pl = 4.9–5.9), ZP2 (Mr = 66–58 kDa, pl = 5.0–6.0), ZP3H (high) (Mr = 72–58 kDa, pl = 3.5–5.1), and ZP3L (low) (Mr = 62–54 kDa, pl = 3.5–5.1). The human ZP3 family (ZP3H and ZP3L) showed marked heterogeneity. Fertilization‐associated changes were apparent in the electrophoretic pattern. ZP1 (Mr = 92–86 kDa, pl = 5.0–5.8) displayed a dramatic decrease in intensity, and a new component had migrated to a position similar to that of ZP2. This modification may have been responsible for one aspect of the zona reaction, and could have contributed to a zona block to polysperma. © 1995 Wiley‐Liss, Inc.
Effect of coculturing spermatozoa with oviductal cells on the incidence of polyspermy in pig in vitro fertilization
Authors: Dubuc A. , Sirard M.‐A. 
Published: February 2005

AbstractIt is known that oviductal cells play an important role in fertilization in vivo. We were interested in the effect of those cells on spermatozoa and their influence on the incidence of polyspermy in pig in vitro fertilization (IVF) when cocultured with spermatozoa. Oviductal cells are believed to select a highly fecund population of spermatozoa. By coculturing spermatozoa with oviductal cells it is possible to reduce the number of spermatozoa confronting the eggs at fertilization, reducing the incidence of polyspermy. In our study, spermatozoa were cocultured with oviductal cells for 30 min so that they could bind to the oviductal cells. Both bound and unbound spermatozoa were used for fertilization. The results show that the spermatozoa that were bound to the oviductal cells were capable of fertilizing the eggs, but the nonbound spermatozoa had a reduced penetration incidence. With the bound sperm, polyspermy incidence decreased and the two‐pronuclei proportion increased. We also found that with time the spermatozoa released from the cells had better motility than those that did not bind. Therefore, it is our belief that oviductal cells can be used for porcine IVF resulting in a lower polyspermy incidence and higher pronuclei incidence. © 1995 Wiley‐Liss, Inc.
Developmental control of transcription of the cat reporter gene by a truncated mouse alphafetoprotein gene regulatory region in transgenic mice
Authors: Ghebranious N. , Knoll B. J. , Yavorkovsky L. , Ilic Z. , Papaconstantinou J. , Lozano G. , Sell S. 
Published: February 2005

AbstractA truncated mouse alphafetoprotein (AFP) gene promoter/enhancer region was tested for its ability to regulate the expression of theEscherichia colichloramphenicol acetyltransferase (CAT) reporter gene in the livers of transgenic mice. The AFP regulatory region lacked any AFP gene structural DNA, included one enhancer sequence together with the proximal promoter sequence, and an element believed to be responsible for the postnatal repression of AFP gene transcription. The neonatal livers of AFP/CAT transgenic mice showed a high level of CAT enzyme expression, which was dramatically reduced between 7 and 14 days after birth. The staining of liver sections with anti‐CAT antibodies showed that this expression was limited to hepatocytes. In one lineage, reexpression of CAT in the adult liver could be achieved by restitutive proliferation of hepatocytes following partial hepatectomy or CCl4‐induced necrosis; reexpression in young animals (3–4 weeks of age) was even greater. These studies show that a truncated AFP promoter/enhancer region functions in a tissue‐specific and developmental stage‐specific fashion, and may be used to control the expression of other genes in the livers of transgenic mice. © 1995 Wiley‐Liss, Inc.
In vitro pluripotency of epiblasts derived from bovine blastocysts
Authors: Talbot Neil C. , Powell Anne M. , Rexroad Caird E. 
Published: February 2005

AbstractTwo experiments were conducted to compare the utility of in vitro‐ and in vivo‐derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo‐collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro‐produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo‐collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro‐produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte‐like cells, and macrophage‐like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo‐derived blastocysts, especially from early‐hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley‐Liss, Inc.This artilce is a US Government work and, as such, is in the public domain in the United States of America.
Effect of donor embryo cell number and cell size on the efficiency of bovine embryo cloning
Authors: Zakhartchenko V. , Wolf E. , Palma G. A. , Brem G. 
Published: February 2005

AbstractTo establish reliable criteria for the evaluation of nuclear donor embryos, we studied the effect of cell number and cell size of in vitro produced day 6 donor morulae on the rate of blastocyst formation following nuclear transfer to in vitro matured oocytes. In experiment 1, donor embryos were divided into three groups with low (25–34), intermediate (40–55), and high (60–81) blastomere numbers. Transfer of nuclei from day 6 morulae with intermediate and high cell numbers resulted in a significantly higher blastocyst rate (31% and 32%, respectively) than use of nuclei from day 6 morulae with low cell numbers (17%) or nuclei from day 7 morulae with 50–83 blastomeres (19%). This suggests that blastomeres from the developmentally advanced day 6 morulae are more viable than blastomeres from retarded embryos. In experiment 2, we evaluated the effect of blastomere size in day 6 donor morulae with intermediate (40–55) or high (60–81) cell numbers on the efficiency of nuclear transfer. In both classes of embryos, small blastomeres were better nuclear donors than large blastomeres. The rates of development to the blastocyst stage were 28% versus 15% (40–55 cells) and 41% versus 25% (60–81 cells), suggesting that small blastomeres include a higher proportion of totipotent cells than the polarized large blastomeres. Our results demonstrate that blastomere number and size markedly affect the efficiency of nuclear transfer and therefore are useful criteria for evaluating nuclear donor embryos. These parameters are easy to determine and may therefore be helpful to improve the efficiency of cattle cloning. © 1995 wiley‐Liss, Inc.
Cloning of complementary DNA encoding a 135‐kilodalton protein secreted from porcine corpus epididymis and its identification as an epididymis‐specific α‐mannosidase
Authors: Okamura Naomichi , Tamba Michiko , Liao Hong‐Jun , Onoe Sinji , Sugita Yoshiki , Dacheux Françloise , Dacheux Jean‐Louis 
Published: February 2005

AbstractIn the preceding study (Okamura et al., 1992; Biol Reprod 47:1040–1052) we suggested that a 135‐kDa protein secreted by porcine epididymis is involved in the sperm maturation. In this work, we have isolated the cDNA clone coding the 135‐kDa protein in an effort to investigate its structure and function.The 135‐kDa protein was purified from porcine cauda epididymal fluid. Three oligonucleotide probes were synthesized according to the amino acid sequences of N‐termini of the native protein and trypsin‐digested peptides. A cDNA clone hybridizing with these three probes was isolated from the cDNA library derived from the porcine proximal corpus epididymis. It encodes a novel protein with 1,006 amino acid residues in an open reading frame.Its overall amino acid sequence was significantly homologous (25.7%) to the α‐mannosidase precursor of Dictiostelium discoideum (P34098). The 135‐kDa protein could digest both p‐nitro‐phenyl‐α‐D‐mannoside and high mannose oligo saccharide (Man8‐GlcNAc2), strongly suggesting that it is an α‐mannosidase homologue.The expression of this protein was specific to porcine and was localized to the very narrow parts of epididymis: the border of the caput and corpus epididymis. This protein may serve as a good marker for the functional differentiation in porcine epididymis. A possible role of this protein in the species‐specific sperm‐egg interaction is discussed. © 1995 wiley‐Liss, Inc.
Analysis of stimulatory and inhibitory amino acids for development of hamster one‐cell embryos in vitro
Authors: McKiernan Susan H. , Clayton Murray K. , Bavister Barry D. 
Published: February 2005

AbstractHamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1‐cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium‐5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM‐6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM‐5 and −6 (chemically defined, protein‐free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley‐Liss, Inc.
Messenger RNA encoding an estrogen‐dependent oviduct secretory protein in the sheep is localized in the apical tips and basal compartments of fimbria and ampulla epithelial cells implying translation at unique cytoplasmic foci
Authors: Murray Mary K. , DeSouza Mary M. 
Published: February 2005

AbstractData from our laboratory have shown that an estrogen (E2)‐dependent Mr 90,000–92,000 protein and its mRNA are synthesized and expressed in abundant amounts at estrus from the fimbria and ampulla, not isthmus, oviduct of the sheep. Immunocytochemical studies have shown that the Mr 90,000–92,000 protein is contained in apical secretory granules of oviduct epithelial cells. The objective of this study was to determine whether the mRNA for the E2‐dependent oviduct protein was localized and compartmentalized in similar manner. Fimbria, ampulla, and isthmus oviducts obtained from estrous ewes were flash frozen in liquid nitrogen, cryosectioned, fixed in 4% paraformaldehyde, hybridized with digoxigenin (DIG)‐labeled oviduct‐specific riboprobes, incubated in anti‐DIG antibodies conjugated with alkaline phosphatase, and developed in color substrate. Oviduct protein‐specific transcripts were localized to basal perinuclear compartments and, surprisingly, at sites distant from the nucleus in the apical cytoplasm of epithelial cells in the fimbria and ampulla. No specific reaction product was observed in the underlying mucosa or smooth muscle layers. Oviduct protein mRNA was contained predominantly in the apical cytoplasm of epithelial cells at the free margins of mucosal folds and in the basal regions of cells located at the crypts of longitudinal folds. No reaction product was present when sections of the fimbria and ampulla oviduct of estrous ewes were incubated in sense riboprobe to the oviduct protein. In addition, when sections of the isthmus oviduct obtained from estrous ewes or fimbria and ampulla oviducts from long‐term ovariectomized ewes were hybridized with antisense riboprobes no specific reaction product was detected. Electron microscopy of oviduct protein mRNA containing areas revealed the presence of secretory granules, rough endoplasmic reticulum (RER) and Golgi in the apical cytoplasm, and RER in the basal regions of epithelial cells. These data show that the mRNA encoding an E2‐dependent oviduct‐specific protein is distributed in epithelial cells at perinuclear foci and at sites distant from the nucleus, which are also the sites of protein localization and protein synthesizing organelles, implying translation at unique cytoplasmic foci. © 1995 wiley‐Liss, Inc.
5‐aza deoxycytidine‐induced inhibition of differentiation of spermatogonia into spermatocytes in the mouse
Authors: Raman Rajiva , Narayan Gopeshwar 
Published: February 2005

AbstractIn order to explore the significance of DNA methylation in proliferation and differentiation of germ cells in testis, 5‐aza,2′‐deoxycytidine (5‐azaCdR), a hypomethylating agent, was administered in vivo to neonatal mice having only spermatogonial (premeiotic) cells. End‐labeling of the Mspl, Hpall, and Hhal digested DNA revealed considerable loss of methylation following the treatment. Cellular and histological preparations of the testis showed complete inhibition of differentiation into spermatocytic stage. Analysis of protein synthesis in the treated and control testis by growing the cells in 35S‐Methionine medium and resolving the lysate by SDS‐PAGE revealed that the programme of expression of at least 5 polypeptides (35.0, 31.5, 27.0, 22.5, and 18.0 KD) was altered as a result of 5‐azaCdR incorporation. It appears that DNA methylation plays a critical role in the differentiation of gonia into primary spermatocytes. © 1995 wiley‐Liss, Inc.
Initiation of transcription and nucleologenesis in equine embryos
Authors: Brinsko S. P. , Ball B. A. , Ignotz G. G. , Thomas P. G. A. , Currie W. B. , Ellington J. E. 
Published: February 2005

AbstractThe time of activation of the embryonic genome (maternal‐embryonic transition) in equine embryos was investigated by assessing incorporation of3H‐uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with3H‐uridine (560 μCi/ml) for 10 hr, while eight‐cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5–1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter‐stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight‐cell, and five‐cell stages incorporated3H‐uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four‐ to six‐cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five‐ to six‐cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight‐cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of3H‐uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four‐ to eight‐cell stage in equine embryos. © 1995 wiley‐Liss, Inc.
Mechanism of action of epidermal growth factor‐induced porcine oocyte maturation
Authors: Coskun Serdar , Lin Young C. 
Published: February 2005

AbstractEGF has been reported to promote oocyte maturation in several species, although the mechanism of action is not yet known. The present study is designed to determine the pathway used by EGF to enhance porcine oocyte maturation. Oocytes were aspirated from 2–5 mm follicles and cultured with various treatments in Medium‐199 at 37°C, 100% relative humidity, and 5% CO2 for 48 hr for the maturation study and 3 hr for intracellular cAMP measurement. Although treatment with 100 IU/ml hCG stimulated both intracellular cAMP formation and oocyte maturation, 10 ng/ml EGF stimulated oocyte maturation only. Dibutyryl cAMP (dbcAMP) inhibited oocyte maturation at 10−5, 10−4, and 10−3 M concentrations in the control medium. However, in the presence of 10 ng/ml EGF, dbcAMP inhibited oocyte maturation only at a concentration of 10−3 M. Increasing concentrations of EGF (i.e., 25 and 50 ng/ml) were ineffective in overcoming the inhibitory effect of dbcAMP at 10−3 M. In contrast, EGF reversed the decreased maturation rate caused by transforming growth factor‐β. Phorbol myristate acetate (PMA), a tumor‐promoting phorbol ester, enhanced the spontaneous maturation rate; 4α‐phorbol dideconoate, an inactive phorbol ester, did not show this effect. PMA‐ and EGF‐stimulated porcine oocyte maturation is reversed by calphostin‐C, a PKC inhibitor. In conclusion, EGF's promotional activity on porcine oocyte maturation is independent of the cAMP pathway and probably mediated by the PKC pathway. © 1995 wiley‐Liss, Inc.