bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nuclear transplantation in bovine embryo: Fine structural and autoradiographic studies
Authors: Kaňka Jiřia , Fulka Josef , Fulka Josef , Petr Jaroslav 
Published: February 2005

AbstractNucleolar fine structure, “blebbing” activity of nuclear evelope, and activation of heterogeneous nuclear RNA (hnRNA) synthesis were studied in bovine reconstructed embryos obtained by electrofusion of a single eight‐cell blastomere with an enucleated oocyte. Developmental progress of nucleolar fine structure and hnRNA synthesis are arrested during three cell cycles following fusion. The activation of both appears during the eight‐cell stage of the reconstructed embryo, after the same number of cell cycles after fusion as in nonmanipulated bovine embryos after fertilization. “Blebbing” activity of nuclear envelope, which is already absent in original blastomeres, reappears after fusion and continues for the next two cell cycles. From the present results, it can be concluded that the donor nuclei are arrested after fusion in morphology and function. Their reactivation corresponds to the developmental pattern typical for normal bovine embryos.
Situs inversus in the developing mouse: Proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy)
Authors: Van Keuren Margaret L. , Layton William M. , Iacob Ruxandra A. , Kurnit David M. 
Published: February 2005

AbstractTo decipher genes that are important in the determination of laterality, we compared two‐dimensional protein gels from wild‐type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer‐analyzed two‐dimensional protein gels from wild‐type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post‐coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild‐type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two‐dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild‐type, C57BL/6J iv/iv, or C57BL/6J wild‐type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.
Cross‐linking a maturation‐dependent ram sperm plasma membrane antigen induces the acrosome reaction
Authors: McKinnon Christine A. , Weaver Frances E. , Yoder Jeffrey A. , Fairbanks Grant , Wolf David E. 
Published: February 2005

AbstractESA152 is a highly hydrophobic 18 kDa sialoglycoprotein, which becomes expressed on ram sperm in the proximal cauda epididymis. ESA 152 is expressed on all regions of the sperm surface, most strongly on the posterior region of the head, most weakly on the anterior region of the head. In this paper, we show that induction of the acrosome reaction with Ca2+ ionophore causes ESA152 to be redistributed from the posterior to the anterior region of the head plasma membrane. Cross‐linking ESA152 with bivalent antibody causes similar redistribution and induces the acrosome reaction, Induction of the acrosome reaction with ESA152 antibody requires Ca2+ but is insensitive to (10 ng/ml) pertussis toxin.
Induction of c‐fos transcripts in early postimplantation mouse embryos by tgf‐α, EGF, PDGF, and FGF
Authors: Nielsen Loretta L. , Werb Zena , Pedersen Roger A. 
Published: February 2005

AbstractThe activity of growth factor receptors in the early postimplantation mouse embryo was studied by analyzing changes in expression of mRNA transcripts of an early response gene, c‐fos, after binding of specific ligands. Reverse transcription of mRNA coupled with the polymerase chain reaction was used to detect gene transcription in single embryos after exposure to growth factors. Postimplantation embryos (at 7.5 days of gestation) had physiologically active receptors for transforming growth factor‐α (TGF‐α), epidermal growth factor (EGF), human platelet‐derived growth factor (PDGF), recombinant PDGF‐AA homodimer, and basic fibroblast growth factor (FGF), as indicated by induced expression of c‐fos mRNA. c‐fos expression was not induced in untreated embryos or in embryos incubated with active recombinant PDGF‐BB homodimer. These results show that growth factor receptors are functional during early mammalian embryogenesis.
In vitro maturation of the potential for movement of carp spermatozoa
Authors: Redondo‐Müller C. , Cosson M.‐P. , Cosson J. , Billard R. 
Published: February 2005

AbstractCarp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3,000‐fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145–1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation‐reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially “poor” quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2°C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation‐reactivation occurred in ATP‐Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year‐round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.
Thiol status in human sperm
Authors: Rufas Onit , Fisch Benjamin , Seligman Judith , Tadir Yona , Ovadia Jardena , Shalgi Ruth 
Published: February 2005

AbstractThe passage of spermatozoa along the epididymis is characterized by a gradual stabilization of intracellular organelles mainly through the oxidation of thiol groups. In this study, we examined the relationship between the thiol–disulfide status of human spermatozoa (using a specific fluorescent probe, monobromobimane) and routine semen analysis parameters. Fluorescence intensity was measured by spectrofluorimeter and its frequency distribution within samples, using a fluorescence‐activated cell sorter. The mean proportion of reactive thiols SH/(SS + SH) in 29 semen samples was 29.8% ± 2.5%. When comparing thiol labeling patterns, oligozoospermic samples differed from normozoospermic ones (P < 0.05). However, within the normozoospermic group, no correlation was found between thiol‐labeling patterns and routine sperm parameters or fertilizing capacity in vitro. No difference in thiol labeling patterns was found between “swim‐up” and “whole semen” preparations.
Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa
Authors: Phillips David M. , Jones Roy , Shalgi Ruth 
Published: February 2005

AbstractThe surface memrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of ∼200 kDa in immature spermatozoa, but later in mature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of ∼160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.
Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes
Authors: Shalgi R. , Maymon R. , Bar‐Shira (Maymon) B. , Amihai D. , Skutelsky E. 
Published: February 2005

AbstractCarbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm‐egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus‐oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR‐Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin‐tagged lectins as probes, and avidin‐biotin‐peroxidase complex as visualant. For EM examination, thin LR‐Gold sections were labeled with RCA‐I colloidal gold complex (RCA/G) and stained with uranyl acetate.LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S‐WGA), Griffonia simplisifolia agglutinin‐I (GS‐I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA‐I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA‐I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S‐WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP. The results clearly reflect a change in both the content and distribution of sugar residues of the ZP following ovulation, which may have significance in fertilization.
Effect of 6‐dimethylaminopurine on germinal vesicle breakdown of bovine oocytes
Authors: Fulka J. , Leibfried‐Rutledge M. L. , First N. L. 
Published: February 2005

AbstractThe effect of 6‐dimethylaminopurine (6‐DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%; 120/124) in control medium, presence of 6‐DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6‐DMAP were used (100–500 μM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis‐metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6‐DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6‐DMAP, respectively. Whereas in cycloheximide‐supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6‐DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6‐DMAP supplemented medium, the level of the maturation‐promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.
In situ histochemical analysis of region‐specific gene expression in the adult rat epididymis
Authors: Garrett Susanne H. , Garrett James E. , Douglass James 
Published: February 2005

AbstractThe epididymal tubule is a dynamic structure, in which spermatozoa undergo distinct physiological and morphological changes. The epithelial cells lining the ductuli vary dramatically in their histochemical and cytological properties according to the region of the tubule in which they are located. Additionally, regional variation is observed regarding the biosynthetic, secretory, and absorptive properties of the epithelial cells. Using in situ histochemical analysis, we document here the region‐specific expression of a variety of genes that are transcriptionally active in the adult rat epididymis. Radiolabeled antisense riboprobes were used to localize, within the efferent duct/caput epididymis, transcripts encoding protein B/C, protein D/E (acidic epididymal glycoprotein), sulphated glycoprotein 1, sulphated glycoprotein 2, cellular retinol‐binding protein, and the neuroendocrine peptide precursor proenkephalin. Each species of mRNA exhibits a unique pattern of hybridization, revealing that gene transcription within the efferent duct/caput epididymis is also highly region specific. This observation may partially elucidate the molecular basis underlying the phenomenon of regional alternations in the composition of protein factors within the tubule lumen.
Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development
Authors: Valdimarsson Gunnar , De Sousa Paul A. , Beyer Eric C. , Paul David L. , Kidder Gerald M. 
Published: February 2005

AbstractDe novo assembly of gap junctions begins during compaction in the eight‐cell stage of mouse development, and intercellular coupling mediated by gap junctions appears to be required for maintenance of the compacted state. We have begun to explore the expression of the family of genes encoding the connexins, the proteins that form the gap junction channels. We recently reported that a protein with antigenic and size similarity with connexin32, the rat liver gap junction protein, is inherited as an oogenetic product by the mouse zygote, but its gene appears not to be transcribed prior to implantation (Barron et al., Dev Genet 10:318–323, 1989). Here we report that another member of this gene family, connexin43, is transcribed by the embryonic genome from shortly after the time of genomic activation. As revealed by Northern blotting, connexin43 mRNA is absent from ovulated oocytes, becomes detectable in the 4‐cell stage, and accumulates steadily thereafter to reach a maximum in blastocysts. In contrast, no transcripts of connexin26 could be detected in any preimplantation stage. A protein with antigenic and size similarity with connexin43 from rat heart was found by Western blotting to accumulate from the four‐cell stage onward. Immunofluorescence analysis with embryo whole mounts was used to demonstrate that this protein is incorporated into punctate interblastomeric foci during compaction, consistent with its assembly into gap junction plaques. We conclude that connexin43 is one member of the connexin gene family whose zygotic expression is critical for preimplantation morphogenesis.
Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells
Authors: Czlonkowska Maria , Eysymont Urszula , Guszkiewicz Andrzej , Kossakowski Maciej , Dziak Jacek 
Published: February 2005

AbstractControl ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty‐one (38%) reached morula/blastocyst stage. Twenty‐one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty‐one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.
Comparison of sperm binding potential of uninseminated, inseminated‐unfertilized, and fertilized‐noncleaved human oocytes under hemizona assay conditions
Authors: Franken Daniel R. , Windt M. L. , Kruger Thinus F. , Oehninger Sergio , Hodgen Gary D. 
Published: February 2005

AbstractIn this study, human oocytes obtained after ovarian hyperstimulation for in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were utilized to evaluate sperm/zona pellucida binding potential. Three groups of oocytes were evaluated: (1) uninseminated; (2) inseminated‐unfertilized; and (3) fertilized‐uncleaved. All oocytes had undergone germinal vesicle breakdown at the time of retrieval and were salt‐stored (pH 7.2) for not more than 30 days. Sperm binding was recorded under hemizona assay (HZA) conditions using spermatozoa from eight fertile men (HZA control) and from (1) four teratozoospermic (HZA test) and (2) four normozoospermic (HZA test) infertile men. First, the mean numbers (±SD) of sperm tightly bound for fertile controls and teratospermic men to hemizonae from uniseminated oocytes were 69.7 ± 16 and 14.5 ± 7, respectively (P = 0.02). Likewise, hemizonae from uninseminated oocytes bound 102.0 ± 19 and 114.0 ± 28, respectively, for fertile controls and normospermic men (P = 0.5). Second, hemizonae obtained from inseminated‐unfertilized IVF oocytes bound 44.2 ± 12 and 19.7 ± 6 for fertile controls and teratospermic men, respectively (P = 0.02). This category of oocytes bound 100.5 ± 7 and 108.5 ± 11 sperm, respectively, for fertile controls and normospermic semen (P = 0.3). Third, HZA results of fertilized but uncleaved oocytes showed a mean number of tightly bound sperm of 6.0 ± 4 compared with 65.0 ± 1 in control, uniseminated oocytes using fertile sperm. These results demonstrate that uninseminated and inseminated‐unfertilized human oocytes, salt‐stored under controlled pH conditions, give reliable information regarding sperm binding potential under HZA conditions.
In vivo secretion and association of clusterin (SGP‐2) in luminal fluid with spermatozoa in the rat testis and epididymis
Authors: Mattmueller Danette R. , Hinton Barry T. 
Published: February 2005

AbstractClusterin (sulfated glycoprotein‐2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two‐dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two‐dimensional gel electrophoresis of proteins secreted directly into the epididymal liminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.
Spermatogenesis in XY, XYSxra and XOSxra mice: A quantitative analysis of spermatogenesis throughout puberty
Authors: Sutcliffe Maxine J. , Darling Susan M. , Burgoyne Paul S. 
Published: February 2005

AbstractAdult XYSxra mice exhibit varying degrees of spermatogenic deficiency but are usually fertile, while XOSxra mice have severe spermatogenic failure and are always sterile. The present quantitative spermatogenic analysis documents when these anomalies first appear during puberty. The results demonstrate that in XYSxra mice there was increased degeneration of pachytene spermatocytes and, to a lesser extent, meiotic metaphase stages. On average, there were only one‐half the number of spermatids compared with the XY controls. The defect in XOSxra mice appeared a little later, with an almost complete arrest and degeneration during the meiotic metaphases, so that the number of spermatids produced was only 3% of the control value. These results are discussed in relation to an hypothesis that links sex chromosome univalence during meiotic prophase with spermatogenic failure.
Expression of SV40‐lacZ gene in mouse preimplantation embryos after pronuclear microinjection
Authors: Takeda Satoshi , Toyoda Yutaka 
Published: February 2005

AbstractIn order to study the expression of an exogenous gene in developing mouse embryos during the preimplantation period, DNA carrying the SV40 early promoter fused with the Escherichia coli β‐galactosidase gene (lacZ) was microinjected into the pronucleus of fertilized mouse eggs. Expression of lacZ gene was detected by staining embryos with 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal) as a substrate at pH 7.2. The embryos expressing the lacZ gene showed various intensities of blue staining, all showing a mosaic pattern. The exogenous gene was expressed from the 4‐cell stage until the blastocyst stage. The proportion of embryos expressing the lacZ gene was maximal (38%) at the morula stage, and the expression was dependent on the presence of the SV40 promoter.