bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein and nuclear changes in pig eggs at fertilization
Authors: Ding Jianchi , Clarke N. , Nagai T. , Moor R. M. 
Published: February 2005

AbstractThe nuclear restructuring that occurs between insemination and full pronuclear formation in pig eggs is accompanied by posttranslational changes to specific egg proteins. Sperm penetration begins in vitro at 3 hr postinsemination (hpi). By 5 hr, decondensing sperm heads and anaphase II plates are observed in 50% of eggs, and, by 8 hpi, both male and female pronuclei have formed. Three consistent changes to the pattern of newly synthesised proteins are triggered in this period; they affect the 46K, 25K, and 22K polypeptides. Changes are also triggered in the 180–200K polypeptides and in the 14K polypeptides, but these are highly variable. The same changes in the prefertilization pattern were observed when prelabelled eggs were used and new protein synthesis was suppressed. The first and most abrupt change involves the apparent catabolic elimination of a group of 46K unphosphorylated polypeptides (pl 7.3–6.4), whose synthesis was greatest before germinal vesicle breakdown but declined slowly in the final phase of maturation, then declined precipitously after activation. Ageing (beyond maturation) also leads to the disappearance of these polypeptides. The progressive disappearance of a set of 25K polypeptides and the concomitant appearance of a dominant 22K polypeptide is the most characteristic fertilization‐induced modification to porcine egg proteins. These modifications begin within 1 hr of sperm penetration or activation, are specific to the pig, and involve heavily phosphorylated polypeptides (25K, pl 6.7–6.0) whose synthesis is begun in the early metaphase I stage. Dual ([35S] and [32P]) labelling, protein blocking experiments, and use of alkaline phosphatase suggest that dephosphorylation selectively affects these 25K polypeptides and is mainly or wholly responsible for converting them (completely within 6 hr) to a single, new (22K, pl 7.6) species that is positively charged. The 25K/22K polypeptide modification has a close temporal relationship with the formation of the male and female pronuclei.
Human zona pellucida during in vitro fertilization: An ultrastructural study using saponin, ruthenium red, and osmium‐thiocarbohydrazide
Authors: Familiari Giuseppe , Nottola Stefania A. , Macchiarelli Guido , Micara Giulietta , Aragona Cesare , Motta Pietro M. 
Published: February 2005

AbstractThe human zona pellucida (ZP) and its changes during in vitro fertilization in oocytes at different maturational stages and polypronuclear ova at one‐ to four‐cells stages were studied by transmission electron microscopy (TEM) and correlative scanning electron microscopy (SEM). To define the microstructure of the ZP, its amorphous masking material was removed using a detergent (saponin), and its structural glycoproteins were stabilized with a cationic dye, ruthenium red, followed by osmium‐thiocarbohydrazide treatment. These methods allowed in all samples the clear visualization of variously arranged networks of filaments composing the outer and inner surfaces of the ZP. These filaments were straight or curved, 0.1–0.4 μ in length and 10–14 nm thick as seen via TEM or 22–28 nm thick as seen via SEM (the difference in thickness was due to the presence of the metal coating for SEM). The filament arrangement was remarkably different between the inner and outer surfaces of the ZP and among the various stages studied. The filaments of the outer surface of the ZP were basically arranged in “large” and “tight” meshed networks. Mature oocytes and fertilized (polypronuclear) ova had a regular alternating pattern of wide and tight meshed networks of filaments. On the other hand, immature and atretic oocytes displayed almost exclusively a tight meshed network of filaments. The inner surface filaments of the ZP of unfertilized oocytes at any stage were arranged in repetitive structures characterized by numerous short and straight filaments anastomosing with each other and sometimes forming at the intersections small, rounded structures. After fertilization, the inner surface of the ZP displayed numerous areas where filaments fused together. Collectively, these data clearly reveal that oocyte maturation and fertilization in humans are accompanied by changes of ZP filaments arrangement, which may be relevant in the processes of binding, penetration, and selection of spermatozoa. © 1992 Wiley‐Liss, Inc.
Differential expression of the TGF‐β isoforms in embryogenesis suggests specific roles in developing and adult tissues
Authors: Roberts Anita B. , Sporn Michael B. 
Published: February 2005

AbstractThe TGF‐β's are multifunctional, pleiotropic molecules with major effects in control of cellular migration, cellular proliferation, and elaboration of extracellular matrix. Thus far, five distinct isoforms of TGF‐β have been described, each approximately 65–85% homologous and containing the characteristic 9 positionally conserved cysteine residues. Although the actions of the activated mature forms of the different isoforms on cells are qualitatively similar in most cases, there are a few examples of distinct activities. For example, TGF‐β's 1 and 3, but not TGF‐β2, inhibit the growth of large vessel endothelial cells, and TGF‐β's 2 and 3, but not TGF‐β1, inhibit the survival of cultured embryonic chick ciliary ganglionic neurons. In addition, selective targeting of the latent forms of the TGF‐β's is suggested by the observation that latent TGF‐β2 is the prominent isoform found in body fluids such as amniotic fluid, breast milk, and the aqueous and vitreous humor of the eye; it is noteworthy in this regard that TGF‐β2 is unique among various isoforms in that it lacks a RGD integrin‐binding sequence in its precursor. The most dramatic differences in the TGF‐β isoforms are seen at the level of expression, where there is now a wealth of data demonstrating both spatially and temporally distinct expression of both the mRNAs and proteins in developing tissues, regenerating tissues, and in pathologic responses. Moreover, the post‐transcriptional regulation of TGF‐β expression by members of the steroid/retinoid family of nuclear receptors is also isoform‐specific; thus, treatment of keratinocytes with retinoids induces secretion of TGF‐β2, whereas treatment of breast cancer cells with gestodene, a synthetic progestin, induces secretion of TGF‐β1. Recent characterization of the 5′ regulatory regions of the human TGF‐β 1, 2, and 3 genes suggests that distinct features of the promoters, including the presence of TATAA boxes and transcription factor binding sites, form the basis for the observed differential transcription. © 1992 Wiley‐Liss, Inc.
Regulation of mammary growth and function by TGF‐β
Authors: Daniel Charles W. , Robinson Stephen D. 
Published: February 2005

AbstractWe have previously shown that TGF‐β1 rapidly and reversibly inhibits ductal growth in vivo when administered by miniature slow‐release plastic implants. A possible role for endogenous TGF‐β1 was suggested by the observation that the normal gland displayed substantial, developmentally regulated levels of TGF‐β1 transcripts and protein. These studies have now been extended to include the other two mammalian TGF‐β isoforms. When tested with slow‐release plastic implants, TGF‐β2 and TGF‐β3 also caused disappearance of the proliferating mammary stem cell layer, with rapid involution of ductal end buds and cessation of glandular growth. None of the isoforms was active in inhibiting alveolar morphogenesis. We conclude that under the conditions of these tests, the three mammaliian isoforms are functionally equivalent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF‐β2 was expressed only at low levels, and mainly during pregancy. TGF‐β3 was expressed in ductal stroma and epithelium, and was the only isoform detected in myoepithelial cells. Developing alveolar tissue and its associated ducts displayed striking TGF‐β3 gene expression and immunostaining, which were greatly reduced during lactation. We are now investigating the possibility that the observed high levels of TGF‐β expression in pregnancy, particularly of TGF‐β3, and the absence of substantial expression of any isoform during lactation, may indicate a role for the TGF‐β in regulating functional differentiation or the onset of milk secretion. © 1992 Wiley‐Liss, Inc.
Localization of ras proto‐oncogene expression during development in Xenopus Laevis
Authors: Andéol Yannick , Méchali Marcel , Hourdry Jacques 
Published: February 2005

AbstractThe expression of the ras protooncogene was investigated in Xenopus laevis, throughout development, by in situ hybridization using a 35S‐labelled antisense RNA probe. During oogenesis, the ras RNA was strongly expressed in the cytoplasm of previtellogenic oocytes and further diluted between yolk platelets; no specific localization of transcripts was observed. The signal density was particularly weak over embryo sections until the tailbud stage. On the other hand, a high level of ras RNA expression was detected on sections through the young tadpoles. An intense labelling was observed in several areas, including the branchial apparatus, gut, somites, nervous system, and lens. It is noteworthy that the heterogeneity of labelling increases as tadpoles grow older. Together, these results are discussed in relation to cellular events appearing throughout the early development.
Transcription of the H19 gene in differentiating cytotrophoblasts from human placenta
Authors: Rachmilewitz J. , Gileadi O. , Eldar‐Geva T. , Schneider T. , De‐Groot N. , Hochberg A. 
Published: February 2005

AbstractPlacental differentiation is closely correlated with the appearance of specific proteins, yet factors regulating cytotrophoblast differentiation are unknown. One strategy employed to search for such factors makes use of differential screening of cDNA libraries. For this purpose, cytotrophoblasts were isolated from human term placentae and cultured for 24 and 120 hr. cDNA libraries were constructed from the cell's RNA, and differential screening resulted in the isolation of three identical clones highly expressed after 120 hr. A DNA sequencing of 139 bp at the 3′ end of these clones and a search of the data bank revealed that the sequence was identical to the parallel domain in the human H19 gene. This highly conserved gene is unusual in that it may not encode a protein. In the mouse, its RNA was shown to accumulate to high levels in embryonic tissues of endodermal and mesodermal origin. Our present findings imply that, in humans, the H19 gene is efficiently expressed in placental tissue and differentiated cytotrophoblasts, which are of ectodermal origin. RNA blot hybridization revealed a unique bimodal pattern of expression for the H19 gene in cultured cytotrophoblasts. The modulation in expression of H19 during cytotrophoblast growth was not due to the increase in the number of multinuclear cells. Size fractionation of cytotrophoblasts by centrifugal elutriation revealed that H19 expression is correlated with the stage of cell differentiation. We therefore propose that H19 transcripts might play a regulatory role in the process of cytotrophoblast differentiation.
Microtubule distribution during fertilization in the rabbit
Authors: Yllera‐Fernandez Maria Del Mar , Crozet Nicole , Ahmed‐Ali Michèle 
Published: February 2005

AbstractThe distribution of microtubles was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti‐α‐tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.
A rat sperm flagellar surface antigen that originates in the testis and is expressed on the flagellar surface during epididymal transit
Authors: Toshimori K. , Tanii I. , Araki S. , Oura C. 
Published: February 2005

AbstractWe identified a rat sperm flagellar surface antigen using an IgG1 monoclonal antibody (MC31) against rat epididymal sperm. Avidin‐biotin‐peroxidase immunohistochemistry demonstrated that the antigen was first expressed in the cytoplasm of early primary spermatocytes, then gradually became restricted to the principal piece of the sperm flagellum during spermatogenesis. However, when the sperm reached the corpus epididymidis, the antigen was expressed on the surface of both the principal piece and the midpiece of the flagellum. The epithelial cells of the epididymis were not stained with MC31. Immunogold electron microscopy showed that the antigen was present on the surface of the sperm flagellar plasma membrane. Immunoblotting of Triton X‐100 extracts of epididymal sperm after one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) under nonreducing conditions demonstrated that MC31 detected a major antigen of 26,000–28,000 daltons (26–28K). Two‐dimensional isoelectric focusing and SDS‐PAGE indicated that the 26–28K antigen had an isoelectric focusing point (pl) of 5.8‐5.3; minor antigens were also detected from 26K (pl 5.8) to 35K (pl 5.0). These results indicate that the antigen recognized by MC31 is an acidic 26–35K protein that originates in the testis, is integrated into the sperm flagellar plasma membrane of the principal piece during spermatogenesis, and then is expressed on the entire flagellar surface during epididymal transit. © 1992 Wiley‐Liss, Inc.
Extraplacental human fetal tissues express mRNA transcripts encoding the human chrorionic gonadotropin‐β subunit protein
Authors: Rothman Paula A. , Chao Victor A. , Taylor Mark R. , Kuhn Robert W. , Jaffe Robert B. , Taylor Robert N. 
Published: February 2005

AbstractThe glycoprotein hormone human chorionic gonadotropin (hCG) is synthesized in large quantities by the developing placenta, reaching peak concentrations in maternal blood during the late first trimester and early midtrimester of pregnancey. In general it is believed that the β‐subunit of this dimeric hormone is expressed in pituitary gonadotropes, thyrotropes, and trophoblasts, while the β‐subunit is expressed exclusively by trophoblasts. Studies from our laboratory and other laboratories have shown that some midtrimester human fetal tissues, in addition to the placenta, can synthesize proteins that appear to be very similar to the β‐subunit of hCG. To define precisely the nature of this putative hCG‐β‐subunit in extraplacental fetal tissues, we have examined the mRNA from a variety of human fetal and adult tissues using nucleic acid hybridization and reverse transcription‐polymerase chain reaction (PCR) methods. Our results demonstrate that midtrimester fetal kidney and adrenal tissues contain hCG‐β mRNA transcripts at concentrations comparable to that of placenta, while fetal lung, brain, muscle, and adult adrenal contain only trace to undetectable levels of hCG‐β mRNA. By restriction endonuclease mapping of PCR fragments from fetal tissue cDNAs, we show that the hCG‐β transcript expressed in midtrimester human fetal organs is a bone fide copy of hCG‐β gene No. 5 of the β‐subunit gene family located on chromosome 19. © 1992 Wiley‐Liss, Inc.
Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes
Authors: Ding Jianchi , Moor Robert M. , Foxcroft George R. 
Published: February 2005

AbstractIn vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis inhibitors (35 μM or 350 μM cycloheximide or a combination of inhibitors) (study 1); to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52% vs. 72%, P < 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the oocyte. © 1992 Wiley‐Liss, Inc.
F‐Actin in acrosome‐reacted boar spermatozoa
Authors: Castellani‐Ceresa L. , Brivio M. F. , Radaelli G. 
Published: February 2005

AbstractBiochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F‐actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein–phalloidin complex and an anti‐fluorescein antibody, followed by labeling with protein A‐gold complex. Gold particles, indicating the presence of F‐actin, were localized on the sperm surface of the acrosome‐reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP‐40 extraction, the SDS‐PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric. © 1992 Wiley‐Liss, Inc.
Genetic manipulation of mammalian dictyate oocytes: Factors affecting transient expression of microinjected DNA templates
Authors: Bevilacqua Arturo , Kinnunen Leann H. , Mangia Franco 
Published: February 2005

AbstractTranscription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: (1) the mouse hsp68 gene; (2) the human β‐actin gene; and (3) simian virus 40 (SV40) early genes. Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the β‐galactosidase activity was then cytochemically evaluated in single cells. In middle‐sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 103 plasmid copies/nucleus and was first observed 10–12 hr after injection. Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used. Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small‐ and middle‐sized growing oocytes, while the construct carrying the β‐actin promoter was expressed only in small‐sized cell. In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes. DNA linearization depressed construct activity regardless of the site of cleavage. These results show that: (1) lacZ is a valuable reporter gene in the analysis of eukaryotic promote activity in dictyate mouse oocytes; (2) transient construct expression requires the injection of DNA in circular form; and (3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed. © 1992 Wiley‐Liss, Inc.
A comparison of two autoradiographic methods for detecting radiolabeled nucleic acids in embryos
Authors: Plante Louise , Pollard John W. , King W. Allan 
Published: February 2005

AbstractTwo methods for preparing embryos for autoradiographic study of newly synthesized nucleic acids are described and compared. The first method consists of rapidly fixing radiolabeled embryos with acetic acid:methanol, spreading them on glass slides and exposing them for 8 days with a photographic emulsion. The second method consists of fixing, embedding in resin, and sectioning the embryos before their exposure with the emulsion for 3 weeks. Both techniques have many applications in studies of early embryonic activity, but the spread technique is very sensitive, simpler, and faster. © 1992 Wiley‐Liss, Inc.
Unexpected position‐dependent expression of H‐2 and β2‐microglobulin/lacz transgenes
Authors: Cohen‐Tannoudji M. , Morello D. , Babinet C. 
Published: February 2005

AbstractIn order to study sequences in volved in the developmentally regulated and tissue‐specific expression of the class I Major Histocompatibility Complex (MHC) genes, we have constructed several H‐2/lacZ transgenic lines in which the 5′ regulatory sequences of the H‐2Kbgene are linked to theEscherichia coliβ‐galactosidase (lacZ) gene. In five H‐2/lacZ lines, the pattern of lacZ expression, detected histochemically varied greatly from line to line. None of the H‐2/lacZ transgenes were transcribed in cells normally expressing a high level of endogenous H‐2 molecules, although these H‐2 regulatory sequences have been shown to be sufficient to drive tissuespecific expression of other reporter genes. Interstingly, when constructs containing 5′ β2‐microglobulin (β2m) regulatory sequences linked to lacZ were used to derive transgenic lines, similar results were obtained. A survey of lacZ labeling in H‐2/lacZ and β2m/lacZ transgenic mice strongly suggests that these transgenes are very sensitive to position effect, lacZ expression being controlled by endogenous chromosomal regulatory elements specific for each insertion site. Here we describe the complex pattern of lacZ expression in the different transgenic lines during development; we discuss the unusual properties of these transgenes and underline their potential use for developmental studies and characterization of genomic sequences involved in spatiotemporal gene expression. © 1992 Wiley‐Liss, Inc.