bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric detection of bicarbonate‐induced changes in lectin binding in boar and ram sperm populations
Authors: Ashworth P. J. C. , Harrison R. A. P. , Miller N. G. A. , Plummer J. M. , Watson P. F. 
Published: February 2005

AbstractBoar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO2, a component believed essential for capacitation. At intervals, samples were stained with a range of FITC‐lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA‐E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate‐free medium. The bicarbonate‐induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin‐binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate‐induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA‐E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times. © 1995 Wiley‐Liss, Inc.
Characterization of a novel spermatogenic cell antigen specific for early stages of germ cells in mouse testis
Authors: Koshimizu Uichi , Nishioka Hiromi , Watanabe Daisuke , Dohmae Kayoko , Nishimune Yoshitake 
Published: February 2005

AbstractTo study the mechanism of spermatogenesis during the premeiotic phase, a hybridoma producing monoclonal antibody (mAb) specific for early stages of spermatogenic cells was obtained. In immunohistochemical staining of adult testis, this mAb, designated as EE2, was able to react with type A to B spermatogonia and early meiotic cells, but not with Sertoli cells, Leydig cells, and other somatic tissues. Precursor cells of type A spermatogonia (gonocytes) were also positive for EE2 in perinatal mouse testis. The antigenic molecule recognized by mAb EE2 was a novel glycoprotein with molecular weight of 114 kDa, which had affinity with Con A and WGA lectins, and was susceptible to N‐glycanase, suggesting the presence of asparagine‐linked sugar chains. Furthermore, EE2 antigen was found to localize on the germ cell surface. The specific expression of this antigenic molecule suggests that it may play an important role in early spermatogenesis, of which only a little information is available at present. © 1995 Wiley‐Liss, Inc.
Developmental and tissue‐specific expression of human CD4 in transgenic rabbits
Authors: Snyder Ben W. , Vitale James , Milos Patrice , Gosselin Joe , Gillespie Frances , Ebert Karl , Hague Bishop F. , Kindt Thomas J. , Wadsworth Sam , Leibowitz Paul 
Published: February 2005

AbstractA major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV‐1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hCD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952–2958). The hCD4 transgene was detected in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T‐cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4−rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV‐1 p24 core protein following HIV‐1 infection in vitro than HIV‐1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high‐level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV‐1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV‐1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV‐1 infection. In vivo HIV‐1 infection studies with these rabbits are under way. © 1995 Wiley‐Liss, Inc.
Galactosyl receptor in human testis and sperm is antigenically related to the minor C‐type (Ca2+‐dependent) lectin variant of human and rat liver
Authors: Goluboff Erik T. , Mertz James R. , Tres Laura L. , Kierszenbaum Abraham L. 
Published: February 2005

AbstractGalactosyl receptor, a cell surface Ca2+‐dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin‐2/3, RHL‐2/3), and its human liver‐equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL‐1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C‐type Ca2+‐dependent lectin with possible roles in cell‐cell interaction during spermatogenesis and sperm‐zona pellucida binding at fertilization. © 1995 Wiley‐Liss, Inc.
Inhibition of protein synthesis affects histone H1 kinase, but not chromosome condensation activity, during the first meiotic division of pig oocytes
Authors: Kubelka Michal , Rimkevicova Zora , Guerrier Pierre , Motlik Jan 
Published: February 2005

AbstractThe influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase‐stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90‐kDa protein was not seen in cycloheximide‐treated oocytes. On the other hand, the levels of both MPF components, p34cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed. © 1995 Wiley‐Liss, Inc.
Ovulated oocytes collected from PMSG/hCG‐treated and cycling Djungarian or Siberian hamsters (Phodopus sungorus) are structurally similar with no evidence of polar body formation, indicating arrest in meiosis I
Authors: Murray Mary K. , Laprise Shari L. 
Published: February 2005

AbstractThis study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4–5 weeks; n = 70) received 5 IU PMSG followed 50–52 hr later by 10 IU hCG. Ovulated oocytes were collected 14–20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7–12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti‐tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 ± 0.4 (range 1–7). Ovulation occurred in 73% of the PMSG/hCG‐stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 ± 0.8 (range 0–22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre‐ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG‐treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG‐treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f‐actin staining was observed in the region of the ooplasm surrounding the chromosomes. As the interval between hCG injection and the time of collection increased, chromosomes lost their proper alignment and migrated away from the cortex of the oocyte concomitant with a disruption of spindle integrity. This collapse of proper chromosome alignment and disruption of spindle architecture also characterized aging oocytes collected from cycling animals. These data show that in the Djungarian or Siberian hamster (Phodopus sungorus), (1) there is individual animal variation in responsiveness to hCG following PMSG treatment, (2) there are no cytological differences in the quality of oocytes collected from hormonally treated animals when compared to cycling animals, and (3) oocytes are ovulated in meiosis I. © 1995 Wiley‐Liss, Inc.
Cytoskeletal organization in the oocyte, zygote, and early cleaving embryo of the stripe‐faced dunnart (Sminthopsis macroura)
Authors: Merry N. E. , Johnson Martin H. , Gehring C. A. , Selwood Lynne 
Published: February 2005

AbstractOvulation occurs in Sminthopsis macroura approximately 160 hr after administration of 1.3 IU PMSG, and yields significantly more oocytes than does spontaneous ovulation (P = 0.001).Germinal vesicle (GV)‐stage oocytes have a thin cortical rim of microfilaments, which is disrupted by exposure to cytochalasin D. After GV breakdown, the first meiotic spindle forms subcortically and parallel to the oolemma. It rotates during anaphase and telophase to extrude the first polar body. This rotation is associated with a local cortical concentration of microfilaments, which is extruded in the first polar body. The second meiotic spindle is orthogonal to the surface, and extrusion of the second polar body is not associated with obvious local changes in cortical actin, resulting in a polar body containing little polymerized actin. The sites of second polar body emission and sperm entry are always in the half of the oocyte opposite the concentrating yolk mass, and are within 60° of each other in most oocytes. During the concentration and eccentric movement of the yolk, microfilaments condense around it. During yolk expulsion, these microfilaments become continuous with those located subcortically. During early cleavage, the cytocortex of the zygote, but not of the extruded yolk mass, stains heavily for polymerised actin.Multiple sites of pericentriolar material are detectable in the cytoplasm of some secondary unfertilized oocytes which, in the presence of taxol, generate large cytasters and pseudospindle structures. After fertilization, a large aster is formed in association with the sperm entry point and serves as the center of an extensive cytoplasmic network of microtubules which surrounds but does not enter the yolk mass. Taxol treatment generates small cytasters within this meshwork and promotes selective stabilization of some periyolk microtubules opposite to the sperm aster. © 1995 Wiley‐Liss, Inc.
Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary
Authors: Mayerhofer Artur , Garfield Robert E. 
Published: February 2005

AbstractThe present immunocytochemical study examines in the rat ovary the pattern of expression of connexin 43 (Cx43), a subunit of gap junctions. Using a well‐characterized specific antiserum against rat Cx43, immunoreactivity was not detected in the fetal ovary, i.e., prior to follicular formation. However, in the ovary of 20‐day‐old, 35‐day‐old, and adult rats, strong Cx43‐immunore‐activity was associated with the cell borders of the follicular epithelium/granulosa cells of all developmental stages (primordial follicles, preantral and antral secondary follicles). In general, immunoreactivity of the granulosa cells of large antral follicles appeared more intense than the one of smaller follicles. Staining was also seen in oocytes (cytoplasmic staining). Theca cells of large antral follicles, but not of small follicles were immunoreactive. Immunoreactive interstitial cells were not seen in ovaries of 20‐ and 35‐day‐old animals, but staining in these cells was present in adult rats. In large follicles with signs of atresia, granulosa cells lacked Cx43‐immunoreactivity, whereas Cx43‐immunoreactivity in their theca interna strikingly increased. Corpora lutea in the cyclic adult rats were heterogeneously stained, with either no detectable immunoreactivity, staining of cell borders of most luteal cells, or with conspicuous staining of only a few cells. In the pregnant animals on gestation days (GD) 12, 14, and 17, all luteal cells stained strongly for Cx43 at the cell surface. Shortly before delivery (GD 21), however, the staining pattern vanished and only few, presumably luteal cells remained immunoreactive. In Western blots (using homogenates of whole ovaries), the Cx43 antiserum recognized a major band of approximate Mr 43 × 103, together with minor bands, which may reflect the presence of several differently phosphorylated Cx43 forms. This is indicated by treatment with alkaline phosphatase, which reduced the banding pattern to one single band. In summary, the gap junction molecule Cx43 is abundantly expressed in all endocrine compartments of the rat ovary. The staining pattern obtained in the present study indicates that Cx43 and presumably gap‐junctional communication are associated with follicular development, atresia, and the development of the interstitial gland, as well as with the development and regression of the corpus luteum. The heterogeneous staining within the ovary furthermore hints to a contribution of the local intraovarian factors in the regulation of Cx43 expression. © 1995 Wiley‐Liss, Inc.
Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes
Authors: Matsubara Nobumichi , Yanagisawa Masahiro , Nishimune Yoshitake , Obinata Masuo , Matsui Yasuhisa 
Published: February 2005

AbstractTo identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley‐Liss, Inc.
Chromatin organization during mouse oocyte growth
Authors: Zuccotti Maurizio , Piccinelli Anna , Rossi Paolo Giorgi , Garagna Silvia , Redi Carlo Alberto 
Published: February 2005

AbstractWe investigated the changes in the organization of oocyte nuclear chromatin and nucleolar‐associated chromatin throughout folliculogenesis. Zona‐free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive‐chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I–III, 10–40 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4–6‐week‐old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2‐week‐old females; 11.8% NSN vs. 8.2% SN in 56‐week‐old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley‐Liss, Inc.
Localization of microfilaments during oocyte maturation of golden hamster
Authors: Terada Yukihiro , Fukaya Takao , Yajima Akira 
Published: February 2005

AbstractThe localization and changes in microfilaments (MF) during golden hamster oocyte maturation were examined by an immunofluorescein method and confocal laser scanning microscopy (CLSM). We also studied the relationship between the changes in MF and oocyte nuclear and cytoplasmic maturation. During in vivo maturation, generalized submembranous MF were found initially which gradually became more prominent at the site of the first polar body extrusion. However, 43.7% of the in vitro matured metaphase 2 stage oocytes lacked the submembranous MF structure. This fact may partly account for the low fertilization rate of in vitro matured oocytes. MF were not found in the folicular oocytes cultured in cytochalasin‐D‐containing medium, and metaphase‐like chromosomes were located at the center of the oocyte and first polar body extrusion did not occur. Twenty‐five percent of the oocytes, which were arrested at meiosis by hypoxanthine, synthesized submembranous MF structure although the nuclear stage of these oocytes was germinal vesicle. These facts suggest that MF plays a role in nuclear behavior but there are some differences in the changes taking place within the nucleus and MF. MF may play a role in oocyte cytoplasmic maturation although the details of this have yet to be established. © 1995 Wiley‐Liss, Inc.
Effects of different kinases and phosphatases on nuclear and cytoplasmic maturation of bovine oocytes
Authors: Léavesque Johanne T. , Sirard Marc‐André 
Published: February 2005

AbstractPhosphorylation is considered as a common post‐translational modification implicated in the control of various key enzymes. In somatic and germinal cells, important regulators of the cell cycle are controlled by their phosphorylation status, and some act as kinases or phosphatases themselves. Bovine oocytes are blocked in the germinal vesicle (GV) stage until either an LH surge occurs or until oocytes are released from the inhibitory influence of the follicle. Meiotic resumption in vitro is therefore an excellent model for the study of phosphorylation events that occur in the G2/M transition, a control point of the cellular cycle. To better understand this transition, we have modulated, either directly or indirectly, kinases using known effectors (epidermal growth factor, EGF; isobutyl‐methylxanthine‐forskolin, Bx‐Fk; 6‐dimethylaminopurine, 6‐DMAP) or phosphatases (okadaic acid, OA) or cycloheximide, which is known to inhibit maturation through protein synthesis suppression. With this procedure, influence on meiotic resumption and phosphoprotein patterns was verified. Both EGF and OA accelerated nuclear maturation after 9 hr of culture. Only 23% (n = 140) and 9% (n = 111) of oocytes were still at GV stage with EGF and OA, respectively, compared to 41% (n = 105) of control oocytes. The different treatments changed the protein patterns in oocytes. In cumulus cells, the patterns were especially modified by the OA treatment. Characteristic changes that occur in germ cells were also identified. Nuclear maturation was inhibited by modulators of kinase (6‐DMAP, GV = 74%, n = 126; cAMP dependent protein kinase (PKA) stimulators, Bx‐Fk, GV = 71%, n = 129) likewise, phosphoprotein patterns were affected, especially in oocytes. The cycloheximide treatment was able to maintain nearly all oocytes in GV after 9 hr of culture (GV = 92%, n = 131). This analysis allowed the identification of substrates for the different effectors used in this study and also helped in determining the levels of phosphorylation required for nuclear maturation. © 1995 wiley‐Liss, Inc.