bluuebunny/crossref_metadata_2025 huggingface dataset:
Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3‐quinuclidinyl benzilate, a potent inhibitor of the zona pellucida‐induced acrosome reaction
Authors: R. Ward Cynthia , S. Kopf Gregory , T. Storey Bayard
Published: February 2005

3‐Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)‐inducud acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121–130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157–164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent‐soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent‐soluble fraction maintained intact ZP receptor(s)–G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTPγS binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS–PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a Mr of 72 kDa, which is within the Mr range for muscarinic receptors. A protein with Mr = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)‐catalyzed ADP‐ribosylation of this fraction revealed this protein to be the α subunit of the Gi class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including Gi, may act as part of a sperm receptor complex for the ZP. © 1994 Wiley‐Liss, Inc.
Mammalian cortical granules: Contents, fate, and function
Authors: Hoodbhoy Tanya , Talbot P.
Published: February 2005

Recent studies have extended our knowledge regarding the contents of mammalian cortical granules (CG) and their function in postfertilization events. Cytochemical staining has demonstrated the presence of carbohydrates within mammalian CG, and lectin‐binding studies have shown that these carbohydrates include α‐Dmannose, α‐D‐GalNAc, and galactose residues in the hamster, α‐D‐mannose in the mouse and cat, and β‐D‐Gal(1,3)‐D‐GalNAc in the pig. Following fertilization and artificial activation, mannosylated material is released from CG and can be found on the oolemma and within the perivitelline space (PVS) of hamster oocytes. Fertilized or artificially activated rabbit, mouse, and human oocytes also release mannosylated, fucosylated and sialylated, and fucosylated material, respectively, which localizes to the oolemma. These glycosylated materials are probably of CG origin, although they have not been directly localized to the CG in rabbit, mice, and humans. The function(s) of the glycosylated material released from mammalian oocytes is not known, although it may participate in blocking polyspermy at the level of the plasma membrane, PVS, and/or zona pellucida (ZP), or it may facilitate preimplantation embryonic development. Proteinases, including tissue plasminogen activator, are also released from mammalian oocytes following fertilization and artificial activation, suggesting that they are of CG origin. These proteinases modify the ZP such that it is no longer receptive to sperm, and some proteinases have been suggested to bring about ZP hardening (an increased resistance to denaturing agents) by an unknown mechanism. Mouse ZP may also be hardened by an ovoperoxidase (cross‐links tyrosine residues) cytochemically identified in mouse CG and CG exudate. The phenomena of ZP hardening in mammalian zygotes is not well understood but is likely to function in blocking polyspermic penetration of the ZP and/or in protecting embryos during preimplantation development. Recently, a 75 kD protein (p75) has been immunocytochemically localized to mouse CG and to the PVS of fertilized oocytes and two‐cell embryos. The identity and function of p75 remains to be determined. Heparin binding placental protein may also be a CG component, since it is released from hamster oocytes following fertilization. It has not, however, been directly demonstrated to be a CG component, and its functions in fertilization and/or early embryonic development have yet to be defined. © 1994 Wiley‐Liss, Inc.
Identification of osteopontin (OPN) mRNA and protein in the rat testis and epididymis, and on sperm
Authors: Siiteri Jon E. , Ensrud Kathy M. , Moore Alison , Hamilton David W.
Published: February 2005

We have identified a bone cell adhesion molecule, osteopontin, in the rat testis and epididymis by Northern analysis, RT‐PCR, Western immunoblot analysis, and immunocytochemistry. A polyclonal antibody raised against rat epididymal fluid proteins was used to detect fusion proteins produced by a testis λgt11 cDNA library. Sequence analysis of one of four positive cDNA clones, designated as pREP5, revealed identity with the rat osteopontin (OPN) cDNA. The partial cDNA clone pREP5 encompasses 64% of the 1,457 residues reported by Oldberg et al. (1986; Proc Natl Acad Sci USA 83:8819–8823). Immunoblot analysis with a monoclonal antibody against OPN detects the presence of immunoreactive poly‐peptides in rat testis homogenates as well as in epididymal fluid and sperm extracts. Immunocytochemical localization to the basal and adluminal region of the seminiferous tubule suggests that OPN could be a Sertoli cell product. Indeed, Northern blot analysis of testicular cell preparations demonstated positive hybridization to Sertoli cellenriched RNA, but not to RNA isolated from interstitial cell preparations or to isolated germ cell RNA preparations. OPN is also detected in the rat epididymis and on epididymal spermatozoa. This is the first report on the presence of OPN mRNA and protein in rat testis and epididymis and on the presence of OPN on the surface of epididymal spermatozoa: The characterization of this protein in other tissue suggests that OPN could play a role in testicular cell adhesion during spermatogenesis and/or epididymal maturation, although other potential functions in the male reproductive tract are discussed. © 1995 Wiley‐Liss, Inc.
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

Boar 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

To 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

A 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

Galactosyl 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

The 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

This 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

Ovulation 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

The 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

To 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

We 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

The 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

Phosphorylation 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.
Analysis of the chromosome complement of frozen‐thawed mouse oocytes after parthenogenetic activation
Authors: Bos‐Mikich A. , Whittingham D. G.
Published: February 2005

Frozen‐thawed mouse oocytes were artificially activated with Sr2+ and analyzed cytogenetically at the first cleavage division to examine the behavior of the maternal chromosomes independently of the paternal complement. There was no significant difference in the rate of activation between frozen‐thawed and freshly collected oocytes and the majority of oocytes (>90%) had a normal haploid chromosome constitution. The incidence of second polar body retention in frozenthawed oocytes was low and did not differ significantly from that observed in fresh oocytes and oocytes exposed to dimethylsulfoxide (DMSO) at 0°C or 37°C for extended periods beyond those required for protection. The frequency of aneuploidy was similar for frozen‐thawed and fresh oocytes but oocytes held at 0°C without DMSO or held at 37°C with DMSO for 1 hr showed a 2.5 and 12‐fold increase in the frequency of aneuploidy compared with oocytes subjected to a conventional oocyte/embryo freezing regime. It is concluded that the procedures used in successful oocyte cryopreservation do not increase the incidence of chromosomal abnormalities of maternal origin in the resulting embryos. © 1995 wiley‐Liss, Inc.
Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector
Authors: Tsukui Tohru , Miyake Sanae , Azuma Sadahiro , Ichise Hirotake , Saito Izumu , Toyoda Yutaka
Published: February 2005

Replication‐defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida‐free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter‐fused HTLV‐I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β‐galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula‐blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2‐cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector‐treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley‐Liss, Inc.
Primate reproductive organs reveal a novel pattern of proto‐oncogene c‐mos and transcription factor Oct‐3 mRNA expression
Authors: Heikinheimo O. , Dong K. W. , Lanzendorf S. E. , Gordon K. , Toner J. P. , Gibbons W. E.
Published: February 2005

In mice, expression of the transcription factor Oct‐3 and the proto‐oncogene c‐mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct‐3 and c‐mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT‐PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct‐3 and c‐mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c‐mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c‐mos was the most intense. Weak signal for c‐mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c‐mos expression. In addition to the oocytes, expression of Oct‐3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct‐3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c‐mos seems to be germ cell specific. Therefore, further characterization of c‐mos and Oct‐3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted. © 1995 wiley‐Liss, Inc.
Syndiospecific polymerization of styrene with Cp*TiCl((OCH(R)CH2)2NAr)/MMAO
Authors: Chen Jie , Zheng Zhan‐Jiang , Pan Li , Pan Ding , Li Yue‐Sheng
Published: February 2005

A series of titanium complexes Cp*TiCl((OCH(R)CH2)2NAr) (Cp* = C5Me5, R = H, Ar = Phenyl (2a); R = H, Ar = 2,6‐dimethylphenyl (2b); R = Me, Ar = Phenyl (2c)) was prepared by the reaction of corresponding N,N‐diethoxylaniline derivatives, with Cp*TiCl3 in the presence of excessive triethylamine. All the titanium complexes display higher catalytic activities towards the syndiospecific polymerization of styrene in the presence of modified methylaluminoxane (MMAO) as a cocatalyst, and produce higher molecular weight polystyrenes with higher syndiotacticity and melting temperature than their mother complex Cp*TiCl3. The catalyst activities and polymer yields as well as polymer properties are considerably affected by the steric and electronic effect of the tridentate ligands. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1562–1568, 2005
Techniques and parameters used in compatibility tests between Beauveria bassiana (Bals) Vuill and in vitro phytosanitary products
Authors: Da Silva Roberta Z , Neves Pedro MOJ
Published: February 2005

The objective of this work was to test and compare different techniques used in tests for compatibility between the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuill and phytosanitary products, in order to develop a protocol for in vitro tests. Four modes of contact were studied between B bassiana (CG432) and the fungicides iprodione (Rovral® 500 g litre−1 SC) and azoxystrobin (Amistar® 500 g kg−1 WG), and the insecticide endosulfan (Thiodan® 350 g litre−1 EC), at three rates. The techniques consisted in incorporating the products into the culture medium (IM), combining the conidia into the products mix (MP) and spraying the products before (SB) and after (SA) inoculation of the fungus on Petri dishes. The fungitoxic effect of the products was studied on the basis of parameters such as germination, colony‐forming units (CFUs), vegetative growth and sporulation. The effect of azoxystrobin on conidial germination was significantly higher in the IM technique than in the other techniques. With regard to CFUs, the IM and SB techniques showed the greatest differences relative to the control. Vegetative growth and sporulation were more affected when azoxystrobin was sprayed before the fungus was applied. At the commercial rate, iprodione had a greater effect on the CFU parameter in the IM and MP techniques, and on vegetative growth in the IM technique, than the other techniques used; however, there was no significant difference occurred between the techniques at the commercial rate with respect to germination and sporulation. Endosulfan was more toxic to germination in techniques SB and SA, and to the CFUs parameter in techniques IM and MP. As to vegetative growth and sporulation, regardless of rate, a more pronounced effect was observed in IM than in the other techniques. It can be inferred that there are differences between techniques and that a standardization of the compatibility tests is necessary. Another inference is that these techniques should reflect realistic exposure of the fungus to chemical formulations under field conditions. Copyright © 2005 Society of Chemical Industry
Cinosulfuron: chemical and biological degradability, adsorption and dissipation in flooded paddy field sediment
Authors: Nègre Michèle , Baiocchi Claudio , Gennari Mara
Published: February 2005

Cinosulfuron is a sulfonylurea herbicide largely used in the extensive cultures of flooded rice in North Italy. The degradation of cinosulfuron has been investigated in sterile aqueous solutions at 30 °C at different pH values. It was rapidly degraded at acidic pH (half‐lives 3, 9 and 43 days at pH 4, 5 and 6, respectively) while the half‐life was >1 year at pH 7 and 9. Two degradation products formed by cleavage of the sulfonylurea bridge were identified by LC‐MS. Degradation by selected mixed microbial cultures tested in aerobic and anaerobic conditions was very slow and attributable to chemical hydrolysis due to the acidic pH of the cultural broths. Degradation took place in freshly collected rice field water treated for two years with cinosulfuron but, in this case also, chemical hydrolysis prevailed over microbial degradation. In contrast, in flooded sediment simulating the paddy field environment, the dissipation rate of cinosulfuron was higher than expected from chemical hydrolysis according to the pH of the system, indicating the involvement of soil microflora. Although the herbicide exhibited a reduced affinity for the sediment surfaces demonstrated by the low value of the Kf Freundlich coefficient (0.87 on a micromolar basis), the rapid dissipation observed in the simulated paddy field should prevent its leaching to ground water. Copyright © 2005 Society of Chemical Industry
Magnesium‐doped near‐stoichiometric lithium tantalate crystals for nonlinear optics
Authors: Wirp A. , Bäumer C. , Hesse H. , Kip D. , Krätzig E.
Published: February 2005

We investigate optical damage effects and coercive fields of LiTaO3 crystals and show that these properties may be tailored for applications in nonlinear optics by Mg doping and vapour transport equilibration treatments. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Sensitivity and measurement errors of Makyoh topography
Authors: Riesz F. , Lukács I. E.
Published: February 2005

The sensitivity and measurement errors of both qualitative and quantitative (grid‐projection) Makyoh topography (MT) are analysed using analytic calculations and computer simulations. The essential difference between the two versions is pointed out. For qualitative MT, the sensitivity is defined on the analogy of the modulation transfer function, and a simple analytic expression is derived. For quantitative MT, the measurement errors are studied using numerical simulations. Two regions of imaging exists: at smooth surfaces and small screen‐to‐sample distances, the error is determined by the pixel reading error, while at less smooth surfaces and larger screen‐to‐sample distances, the errors of summation approximation dominates. The summation errors can be decreased by an iterative method. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Cathodoluminescence degradation signatures of AlGaAs‐based high‐power laser arrays
Authors: Pommiès M. , Avella M. , Cánovas E. , Jiménez J. , Oudart M. , Resneau P. , Nagle J.
Published: February 2005

Low‐temperature spectrally resolved cathodo luminescence was implemented on degraded AlGaAs‐based high‐power laser bars to investigate the degradation signatures. We focused on laser bars at different degradation levels and two degradation patterns were described and attributed to two degradation mechanisms. The first one may be due to some locally poorer material quality and induces the formation of dark spots defects in injection channels, the other one (which is correlated to some damages created on the facet during the mounting of the bar) triggers the formation of some characteristic dark defects originating from the facet and extending within the (110) oriented current injection channels. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
A current assessment of photosystem II structure
Authors: Nicholson William V. , Ford Robert C. , Holzenburg Andreas
Published: February 2005

This review covers the recent progress in the elucidation of the structure of photosystem II (PSII). Because much of the structural information for this membrane protein complex has been revealed by electron microscopy (EM), the review will also consider the specific technical and interpretation problems that arise with EM where they are of particular relevance to the structural data. Most recent reviews of photosystem II structure have concentrated on molecular studies of the PSII genes and on the likely roles of the subunits that they encode or they were mainly concerned with the biophysical data and fast absorption spectroscopy largely relating to electron transfer in various purified PSII preparations. In this review, we will focus on the approaches to the three-dimensional architecture of the complex and the lipid bilayer in which it is located (the thylakoid membrane) with special emphasis placed upon electron microscopical studies of PSII-containing thylakoid membranes. There are a few reports of 3D crystals of PSII and of associated X-ray diffraction measurements and although little structural information has so far been obtained from such studies (because of the lack of 3D crystals of sufficient quality), the prospects for such studies are also assessed.
A comparison of simulation techniques and algebraic techniques for verifying concurrent systems
Authors: Lynch Nancy , Segala Roberto
Published: February 2005

Simulation-based assertional techniques and process algebraic techniques are two of the major methods that have been proposed for the verification of concurrent and distributed systems. It is shown how each of these techniques can be applied to the task of verifying systems described as input/output automata; both safety and liveness properties are considered. A small but typical circuit is verified in both of these ways, first using forward simulations, an execution correspondence lemma, and a simple fairness argument, and second using deductions within the process algebra DIOA for I/O automata. An extended evaluation and comparison of the two methods is given.
A single complete rule for data refinement
Authors: Gardiner P. H. B. , Morgan Carroll
Published: February 2005

One module is said to be refined by a second if no program using the second module can detect that it is not using the first; in that case the second module can replace the first in any program. Data refinement transforms the interior pieces of a module — its state and consequentially its operations — in order to refine the module overall.

A method for data refinement is sound if applying it actually does refine the module; a method is complete if any refinement of modules can be realised by its application.

It has been known for some time that there are two methods of data refinement which are jointly complete for boundedly-nondeterministic programs: any refinement can be realised by applying one method then the other. Those two methods are formulated in terms of relations between states. Here it is shown that using predicate transformers, instead, allows a single complete method.
Dynamics, productivity and species richness of tropical rainforests along elevational and edaphic gradients on Mount Kinabalu, Borneo
Authors: Aiba Shin‐ichiro , Takyu Masaaki , Kitayama Kanehiro
Published: February 2005

We studied the dynamics of nine tropical rainforests on Mount Kinabalu, Borneo, at four elevations (700, 1,700, 2,700 and 3,100 m) on various edaphic conditions for four 2‐year periods over 8 years (1995–2003), and examined the relationships with above‐ground productivity. Mean growth rate of stem diameter, basal area turnover rate and estimated recruitment rate (using growth rate and size distribution) correlated with productivity among the nine forests in all periods. These rates based on growth rates of surviving stems appeared to be good measures of stand turnover. However, observed recruitment rate and mortality (and turnover rate as mean of these rates) based on direct observation of recruits and deaths did not correlate with productivity in some periods. These rates may not be useful as measures of stand turnover given small sample size and short census interval because they were highly influenced by stochastic fluctuation. A severe drought associated with the 1997–1998 El Niño event inflated mortality and depressed mean growth rate, recruitment rate and basal area turnover rate, but had little effect on the correlations between these rates (except mortality) and productivity. Across broad elevational and edaphic gradients on Mount Kinabalu, forest turnover, productivity and species richness correlated with each other, but the causal interpretation is difficult given the different histories and species pools among forests at different elevations.
Brain overgrowth in autism during a critical time in development: implications for frontal pyramidal neuron and interneuron development and connectivity
Authors: Courchesne Eric , Pierce Karen
Published: February 2005

While abnormalities in head circumference in autism have been observed for decades, it is only recently that scientists have begun to focus in on the developmental origins of such a phenomenon. In this article we review past and present literature on abnormalities in head circumference, as well as recent developmental MRI studies of brain growth in this disorder. We hypothesize that brain growth abnormalities are greatest in frontal lobes, particularly affecting large neurons such as pyramidal cells, and speculate how this abnormality might affect neurofunctional circuitry in autism. The relationship to clinical characteristics and other disorders of macrencephaly are discussed.