bluuebunny/crossref_metadata_2025_split huggingface dataset:
Storage of messenger RNA in eukaryotes: Envelopment with protein, translational barrier at 5′ side, or conformational masking by 3′ side?
Authors: Spirin Alexander S. 
Published: February 2005

AbstractMessenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA‐binding protein forming mRNPs and belonging to a special p50 family of basic, glycine‐rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA‐specific repressor proteins bound to the 5′‐untranslated regions (5′‐UTR) of mRNAs may be involved. Interactions of the 3′‐untranslated regions (3′‐UTR) with sequence‐specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3′‐UTR–induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible. © 1994 Wiley‐Liss, Inc.
Mouse oocyte maturation is affected by lithium via the polyphosphoinositide metabolism and the microtubule network
Authors: Pesty Arlette , Lefèvre Brigitte , Kubiak Jacek , Géraud Gérard , Tesarik Jan , Maro Bernard 
Published: February 2005

AbstractThe incubation of mechanically denuded mouse oocytes in medium containing LiCl delayed both germinal vesicle breakdown (GVBD) and polar body extrusion in a dose‐dependent and reversible manner. When myo‐inositol alone was added to the culture medium, we observed that it accelerated GVBD and increased the rate of polar body extrusion, whereas, when combined with LiCl, the normal timing of GVBD was recovered. In the same way, when inositol trisphosphate (InsP3) was microinjected into the ooplasma, we observed an important improvement of the rate of GVBD, as compared to control oocytes, and prevention of lithium inhibition. However, neither myo‐inositol nor InsP3 were able to rescue totally the oocytes from the negative effect of lithium on polar body extrusion. Moreover, lithium induced some important changes in microtubule and chromosome organizations. Before extrusion of the first polar body, the reduction of the spindle size or the appearance of short individualized chromosomes dispersed around a large aster of microtubules were often observed, whereas, after polar body extrusion, the spindle appeared smaller and chromosomes were often trapped in the midbody. Thus lithium affects mouse oocyte maturation at two different levels: GVBD and polar body extrusion. Whereas the former seems to be affected via polyphosphoinositide turnover, the latter is InsP3‐independent and seems to be influenced negatively via underdevelopment of microtubular structures. © 1994 Wiley‐Liss, Inc.
Fertilization current in the human oocyte
Authors: Gianaroli Luca , Tosti Elisabetta , Magli Cristina , Iaccarino Mariano , Ferraretti Anna Pia , Dale Brian 
Published: February 2005

AbstractIn this report we show that the first event of activation in the human oocyte, the Fertilization current (FC), is a slow transient outward current of 300 pA, which induces a gradual hyperpolarization of the plasma membrane from –20mV to –60mV, 60–120 min after insemination, followed by a repolarization to –20mV. Activation currents (AC) of 600‐2,500 pA, induced by exposure to the calcium ionophore A23187 or by microinjection of InsP3 into the cytosol, are also outward. The AC are inhibited by preloading oocytes with EGTA suggesting they are calcium dependent. Since AC are 2–10‐fold the amplitude of the FC the fertilizing spermatozoon in the human only activates a portion of the primary elements stored in the oocyte for triggering metabolic depression. Oocyte activation in the human resembles that in the hamster rather than other mammals or invertebrates studied to date. © 1994 Wiley‐Liss, Inc.
Mechanism for procaine‐mediated hyperactivated motility in guinea pig spermatozoa
Authors: Mújica Adela , Neri‐Bazan Leticia , Tash Joseph S. , Uribe Salvador 
Published: February 2005

AbstractHyperactivated motility was studied in guinea pig spermatozoa. In the presence of the local anesthetic procaine, a high number of sperm cells (64%) showed hyperactivation when incubated in minimal culture medium with pyruvate, lactate, and glucose. Hyperactivated motility was dependent on glucose in the medium. Sperm ATP concentration was increased twofold in hyperactivated sperm when compared to procaine‐treated nonhyperactivated cells. cAMP levels were also higher in hyperactivated cells than in control spermatozoa. Thus, in living spermatozoa high levels of ATP appear to be needed to generate hyperactivation. cAMP is present at a high concentration in hyperactivated spermatozoa, therefore a role of cAMP in hyperactivation cannot be excluded. Depletion of external Ca2+ did not inhibit procaine‐induced hyperactivated motility. Hence, procaine canceled the requirement of external Ca2+ for sperm to express hyperactivated motility. © 1994 Wiley‐Liss, Inc.
Identification of two human sperm populations using flow and image cytometry
Authors: Pasteur Xavier , Métézeau Philippe , Maubon Ivan , Sabido Odile , Kiefer Héléagne 
Published: February 2005

AbstractFlow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation.We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion.We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley‐Liss, Inc.
DFP‐sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad, Bufo japonicus
Authors: Takahashi Mika , Tokumoto Toshinobu , Ishikawa Katsutoshi 
Published: February 2005

AbstractThe inhibition of progesterone‐induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD50 on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD50) was set at 1.0], namely, before the beginning of GVBD. DFP‐sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process.These findings raise the possibility that the activities of some endogenous DFP‐sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley‐Liss, Inc.
Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro
Authors: Suzuki Hiroyuki , Yang Xiangzhong , Foote Robert H. 
Published: February 2005

AbstractSurface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh‐like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue‐shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP‐predominant pattern to a MV‐predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley‐Liss, Inc.
Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers
Authors: Assey R. J. , Hyttel P. , Roche J. F. , Boland M. 
Published: February 2005

AbstractA highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)‐stimulated (superovulated) and unstimulated heifers.Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol‐17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy.The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid‐mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH‐stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley‐Liss, Inc.
Correlation of zona‐binding with oocyte maturation and sperm motility in rhesus monkeys by hemizona assay
Authors: Pu X. C. , Ji W. Z. , Yang S. C. , Chen J. C. , Zou R. J. , Shang E. Y. 
Published: February 2005

AbstractThe hemizona assay (HZA) in Rhesus monkeys was employed to study the correlation of zona‐binding ability with sperm motility or with naturally developing oocytes at various maturational stages. Oocytes from unstimulated ovaries were retrieved within 2 hr from monkeys sacrificed for vaccine production (in reproductive season, but with their menstrual cycles not determined). Oocytes were divided into four groups based on their morphological maturation: 1) Oocytes surrounded by more than one cumulus layer (MC); 2) Oocytes retaining intact germinal vesicle nuclei (GV); 3) Oocytes with germinal vesicle breakdown showing distinct perivitelline space (PVS); and 4) Oocytes extruding the first polar body (PBI). The mean numbers of sperm bound to hemizona for PB1, PVS, GV, and MC groups were 132.9 ± 12.0, 71.5 ± 10.1, 36.1 ± 4.0, and 20.1 ± 2.9 (Mean ± SE), respectively. The four groups showed significant differences from each other in sperm/egg binding ability (P < 0.01). The number of bound sperm significantly increased with oocyte maturation. The present study also showed that zona‐binding ability was also affected by sperm motility. For sperm with 67.7% motility and sperm with 31.2% motility, the average numbers of bound sperm were 43.5 ± 2.2 and 25.3 ± 2.9 (Mean ± SE), respectively. There was significantly higher binding ability for sperm with higher motility (P < 0.01). The results suggest that: 1) The rhesus monkey model can serve as a very sensitive model for studying sperm/egg interaction by HZA; 2) Sperm motility positively correlated with sperm/egg binding; and 3) Sperm/egg binding ability increases with oocyte maturation. The binding ability is highest when oocytes matured to the PB1 stage, which is also the best opportunity for fertilization. This is strong evidence for the “zona maturation” hypothesis. © 1994 Wiley‐Liss, Inc.
Targeted disruption of int‐2 (fgf‐3) causes developmental defects in the tail and inner ear
Authors: Mansour Suzanne L. 
Published: February 2005

AbstractThe int‐2 gene (also designated fgf‐3) was originally identified because its transcription is activated by the nearby integration of mouse mammary tumor virus in virus‐induced tumors. Molecular analyses have revealed that the int‐2 gene produces at least four mRNAs, all of which encode a protein that has 40–50% amino acid sequence similarity with the fibroblast growth factors (FGFs). Int‐2 gene expression is localized to a small number of discrete sites in the developing mouse, but has not been detected in any nonneoplastic adult tissue. The expression data and the amino acid sequence similarity with the FGFs suggested several potential roles for int‐2 during normal development.To evaluate these possibilities, we initiated a genetic analysis of int‐2. A gene‐targeting protocol was used to generate embryonic stem (ES) cells that are heterozygous for an insertion of the neor gene into the first proteincoding exon of int‐2. These cells were used to establish a line of mice that carry the gene disruption. Animals that are heterozygous for the int‐2neo allele are normal and fertile. Homozygous mutants survive embryonic development and can be visually identified after 12.5 days of gestation by a short, initially dorsally curled tail. This defect is potentially due to the disruption ofint‐2 expression in the primitive streak/tail bud. Most of the homozygous mutants die at orsoon after birth, but several have survived to adulthood. In addition to the tail phenotype, the surviving homozygotes show, to varying extents, symptoms characteristic of innerear abnormalities. The development of these defects could be attributed to the disruption of normal int‐2 expression in the hindbrain rhombomeres thought to induce inner ear development and/or the otocyst (precursor of the inner ear) itself. Other sites of int‐2 expression are not affected by the mutation. Although the tail phenotype is 100% penetrant, the inner ear phenotype caused by this mutation shows reduced penetrance and variable expressivity.A second int‐2 allele was generated in ES cells by using homologous recombination to introduce a IacZ reporter gene into the int‐2 locus. This allele has also been established in a line of mice. The pattern of β‐galactosidase activity in carriers of the int‐2lacZ allele has been examined in embryos from 8.5–14.5 days of gestation and in neonatal heads using whole mount X‐gal staining. Expression of the lacZ gene mirrors the expected pattern of int‐2 mRNA. Several new sites int‐2 of expression not identified in the initial in situ hybridization studies of int‐2 mRNA have also been identified. Compound heterozygotes (int‐2neo/int‐2lacZ) were found to have the same defects in tail and innerear development as homozygous int‐2neo mice. After staining for β‐galactosidase activity, these mutants should allow an analysis of the int‐2 mutant phenotype at the cellular level. © 1994 Wiley‐Liss, Inc.
Studies on FGF‐2: Nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin
Authors: Rifkin Daniel B. , Moscatelli David , Roghani Monireh , Nagano Yutaka , Quarto Natalina , Klein Sharon , Bikfalvi Andreas 
Published: February 2005

AbstractMultiple forms of FGF‐2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF‐2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF‐2 as the AUG codon is 3′ to the CUG codons. Although the 18 kDa form FGF‐2 is localized primarily in the cytosol, a significant fraction of the HMW FGF‐2 has a nuclear location. The nuclear localization of HMW FGF‐2 is determined by amino acid residues in the amino‐terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino‐terminal extension of HMW FGF‐2. The nuclear localization of HMW FGF‐2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF‐2, or all forms of FGF‐2, we have found that HMW FGF‐2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF‐2. These cells are transformed by what appears to be the intracellular action of HMW FGF‐2. The interaction of FGF‐2 with heparin has also been examined. Contrary to other reports claiming that FGF‐2 required heparin or heparan‐sulfate for interaction with its high‐affinity receptor, we have found that FGF‐2 binds to its receptor in the absence of glycosaminoglycans, and that this binding activates the receptor. © 1994 Wiley‐Liss, Inc.
Transcriptional regulation of the murine k‐fgf gene
Authors: Rizzino Angie , Rosfjord Edward 
Published: February 2005

AbstractEmbryonal carcinoma (EC) cells provide a useful model system for studying the roles of growth factors during early mammalian development. In 1988, we determined that EC cells express a member of the fibroblast growth factor (FGF) family that cannot be detected after EC cells undergo differentiation. Attempts to understand how differentiation regulates the production of FGFs led to the finding that EC cells express the fibroblast growth factor k‐FGF (FGF‐4), whereas there is a large decrease in the steady state levels of k‐FGF mRNA when EC cells differentiate. This suggested that transcription of the k‐fgf gene is repressed when EC cells differentiate. To investigate this possibility, we prepared a series of reporter gene constructs containing various regions of the murine k‐fgf gene. These constructs were transfected into two mouse EC cell lines and one mouse embryonic stem (ES) cell line. We determined that the mouse 5′ flanking region cannot support expression of the reporter gene. In both EC and ES cell lines, expression of the reporter gene is elevated greatly by the addition of a 316 bp region from the third exon of the murine k‐fgf gene. Sequence analysis of the 316 bp region identified one and possibly two conserved octamer binding motifs. These sequences are likely to be involved in regulation of the k‐fgf gene, because differentiation of EC cells is known to reduce the expression of octamer binding proteins, including Oct‐3. To test the possible role of octamer binding proteins, we examined the expression of our reporter gene constructs in F9‐differentiated cells and in PYS‐2 cells. In these cells, the expression of our k‐fgf/reporter gene constructs is very low. However, expression of the reporter gene is elevated significantly when these cells are cotransfected with a construct that contains an Oct‐3 cDNA under the control of a strong viral promoter. To test further the importance of the octamer motifs found in the 316 bp enhancer‐like region, we replaced the 316 bp region in our constructs with a smaller region (48 bp) that contains the downstream octamer motif and flanking sequences. Like the 316 bp region, this 48 bp region elevated the expression of the reporter gene, but it does so at a much lower level. Thus, it appears that the downstream octamer motif may be involved in the regulation of the k‐fgf gene, but that at least one other cis‐regulatory element present in the 316 bp enhancer is likely to be involved in the expression of the k‐fgf gene in EC cells. Lastly, we have identified two potentially important regulatory regions upstream of the transcription start site. One appears to regulate positively the expression of the murine k‐fgf gene in EC cells and in ES cells, and the other appears to regulate negatively the expression of the k‐fgf gene in these cells. © 1994 Wiley‐Liss, Inc.
Modulation of gamete and embryonic microenvironments by oviduct glycoproteins
Authors: H.F. Hunter R. 
Published: February 2005

AbstractStudies on protein molecules in oviduct luminal fluid are viewed historically, and then in terms of more recent studies on a possible involvement of unique glycoproteins in embryonic development. As a caution, however, it is noted that incorporation of such molecules into the vitellus may be nonspecific. The question is raised as to whether oviduct glycoproteins could be acting primarily in a physical sense to stabilize differing chemical environments along the oviduct. Equally or more importantly, glycoproteins might be acting as carrier molecules to present cations and metabolic substrates at appropriate concentrations to the vitelline membrane. This latter possibility is examined in some detail and could be tested by manipulating the composition of the perivitelline fluid. Glycoproteins may also be critically involved in regulating the physiological competence of spermatozoa in the pre‐ and peri‐ovulatory oviduct, in maintaining a coordinated pattern of cilial beat, and in immunosuppressive functions within the oviduct, not least in those associated with the masking of paternal antigens on both spermatozoa and embryos. © 1994 Wiley‐Liss, Inc.