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1.
Biochemical heterogeneity,migration, and pre-fertilization release of mouse oocyte cortical granules
Background
Oocyte cortical granules are important in the fertilization of numerous species including mammals. Relatively little is known about the composition, migration, and pre-fertilization release of mammalian oocyte cortical granules. 相似文献2.
Immunological approaches were used to characterize an antigen that is present within the cortical granules of mouse oocytes and eggs. Immunoelectron microscopy shows a specific localization of the antigen to the cortical granules in the cortex of mouse oocytes and eggs. Following in vitro fertilization, the antigen is present in the perivitelline space and is associated with the zona pellucida. No cortical granules and very little antigen are detected in the two-cell embryo. This antiserum detects a protein of Mr = 75,000 (p75) following immunostaining of egg proteins on Western blots, or immunoprecipitation of metabolically labeled oocyte proteins or radio-iodinated egg proteins. p75 is also present in exudates obtained from A23187-treated eggs, as detected by either radio-iodination of the released egg proteins, or maturation and ionophore activation of metabolically labeled oocytes. Two-dimensional gel electrophoresis of radio-iodinated egg proteins reveals four species of p75 with pIs between 4.9 and 5.3, whereas only the most basic form of p75 is detected in metabolically labeled oocytes. Multiple forms of the radio-iodinated p75 are present in the exudate of ionophore-treated eggs. p75 displays a greater electrophoretic mobility under nonreducing conditions, indicating the presence of intramolecular disulfide bonds, a common characteristic of secreted proteins. We conclude that p75 is synthesized in oocytes, modified and packaged into cortical granules, and released from eggs following fertilization or activation. 相似文献
3.
Background
Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. 相似文献4.
Background
Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.Methodology/Principal Findings
Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.Conclusions
proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway. 相似文献5.
Burkart AD Xiong B Baibakov B Jiménez-Movilla M Dean J 《The Journal of cell biology》2012,197(1):37-44
The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development. 相似文献
6.
The cortical cytoplasm and cortical granules (CGs) of mouse oocytes were analysed by electron microscopy. Oocytes were collected soon and 20h after ovulation from adult young females (3-4 months old). In addition, gametes collected soon after ovulation from 12- to 14-month-old females were used. Ultrastructural analyses were undertaken using the conventional procedures and the alcoholic PTA method. PTA selectively stains the CGs indicating the presence of lysine-rich proteins in these granules. Oocytes from young females showed CGs as dense granules 300-500 nm in diameter linearly arranged under the oolemma. In oocytes recovered 20h after ovulation 24.31% of CGs appeared vacuolated and 38.40% internalized in the cytoplasm. In gametes collected from old females several changes were observed in the cortical cytoplasm: (a) CGs appeared concentrated in some areas while others regions were devoid of granules; (b) groups of CGs appeared internalized in the egg cytoplasm; (c) the CG contents had swollen and changed, showing dense and clear areas; (d) numerous dense structures and vesicles (lysosome-like vesicles) were present; (e) cytoplasmic fragmentation was frequently seen. Fragments contained CGs, dense structures and vacuoles. These changes are closely related to the low fertilization rates shown by these oocytes when they were used for in vitro fertilization procedures. 相似文献
7.
Namhoe Baek Jong-Min Woo Cecil Han Eunyoung Choi Inju Park Do Han Kim Edward M Eddy Chunghee Cho 《Reproductive biology and endocrinology : RB&E》2008,6(1):32
Background
Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library. 相似文献8.
Zhang C Helmsing S Zagrebelsky M Schirrmann T Marschall AL Schüngel M Korte M Hust M Dübel S 《PloS one》2012,7(1):e30684
Background
Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.Results
Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells.Conclusion
The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but also p75NTR-associated cellular responses in PC12 cells. 相似文献9.
10.
Virginie?Barraud-Lange Nathalie?Naud-Barriant Line?Saffar Liliane?Gattegno Beatrice?Ducot Anne-Sophie?Drillet Morgane?Bomsel Jean-Philippe?Wolf
Background
Based on inhibition tests, the alpha6beta1 integrin was suggested to be a sperm receptor, but further experiments using gene deletion techniques have shown that neither oocyte alpha6, nor beta1 integrin subunits were essential for mouse fertilization. 相似文献11.
F J Longo 《The American journal of anatomy》1985,174(3):303-315
The cortices of a number of mammalian eggs are not structurally homogeneous but are polarized. In mouse ova the plasma membrane is a mosaic; the cytoplasm overlying the meiotic spindle is devoid of cortical granules and consists of a filamentous layer containing actin. Functionally, this cortical polarity may be related to the restriction of sperm-egg interaction and fusion to a specific region of the ovum cortex and to dynamic changes of the egg cortex during fertilization, including cortical granule exocytosis, polar body formation, and fertilization cone development. The origin of cortical polarity in mammalian oocytes and its possible relation to components of the cytoskeletal system and meiotic apparatus are discussed and compared with cortical features of eggs of other vertebrates and invertebrates. 相似文献
12.
An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata 总被引:1,自引:0,他引:1
Fine structural studies have been conducted to determine the role of the trypsinlike proteinase during fertilization which is localized within the cortical granules of sea urchin eggs. Eggs were treated with soybean trypsin inhibitor (SBTI), inseminated, and prepared for microscopic examination at various intervals during fertilization. In the presence of SBTI the breakdown of the cortical granules is delayed and the elevation of the vitelline layer is incomplete. Although all the cortical granules in SBTI-treated eggs are dehisced in a manner similar to controls, the rate at which their breakdown is propagated is reduced. Consequently, dehiscence of all the cortical granules in SBTI-treated specimens requires approximately 3 min to complete, vs. 1 min for controls. In addition the fertilization membrane has a blistered appearance owing to its attachment, at multiple loci, to the surface of the zygote. During the early stages of fertilization many of the treated eggs are monospermic, later all are polyspermic. Supernumerary sperm enter treated eggs (zygotes) up to 10 min after the initiation of the cortical granule reaction, apparently at those sites where the fertilization membrane has failed to separate from the zygote's surface. 相似文献
13.
Eric A Evans Hong Zhang Patricia A Martin-DeLeon 《Reproductive biology and endocrinology : RB&E》2003,1(1):54
Background
The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates. 相似文献14.
Background
A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Principal Findings
Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.Significance
These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function. 相似文献15.
Background
The p53 tumor suppressor and its related protein, p73, share a homologous DNA binding domain, and mouse genetics studies have suggested that they have overlapping as well as distinct biological functions. Both p53 and p73 are activated by genotoxic stress to regulate an array of cellular responses. Previous studies have suggested that p53 and p73 independently activate the cellular apoptotic program in response to cytotoxic drugs. The goal of this study was to compare the promoter-binding activity of p53 and p73 at steady state and after genotoxic stress induced by hydroxyurea. 相似文献16.
Background
Degenerative granular structures appear progressively with age in the hippocampus of most mouse strains. We recently reported that these granules contain a neo-epitope that is recognised by IgM antibodies present as contaminants in many commercial antibodies obtained from mouse ascites and mouse or rabbit serum. We hypothesise that these anti-neo-epitope IgMs are in fact natural auto-antibodies that are generated spontaneously during the foetal stage without previous contact with external antigens and whose repertoire and reactivity pattern have been determined through evolution, being remarkably stable within species and even between species.Findings
In the present work we found that mice from the ICR-CD1, BALB/C and SAMP8 strains have anti-neo-epitope IgM antibodies in their plasma at all ages tested and even when maintained under specific opportunistic pathogen-free conditions. Moreover, we determined that these anti-neo-epitope IgMs are also present in rabbit, goat and rat serum. We also found that, in each mouse that presented hippocampal granules, the anti-neo-epitope IgMs contained in its plasma recognised the neo-epitopes in its own granules.Conclusions
This study led to the conclusion that anti-neo-epitope IgMs are widespread natural auto-antibodies contained in the plasma of mice and other species. The presence of these natural auto-antibodies not only explains why they are frequently found as contaminants in commercial antibodies, but also paves the way for a new approach to a treatment and diagnosis of pathological brain processes based on natural IgMs and neo-epitopes.17.
Noriko Hasebe Yuki Fujita Masaki Ueno Kazuhiro Yoshimura Yuji Fujino Toshihide Yamashita 《PloS one》2013,8(12)
Background
The cleavage of β-amyloid precursor protein (APP) generates multiple proteins: Soluble β-amyloid Precursor Protein Alpha (sAPPα), sAPPβ, and amyloid β (Aβ). Previous studies have shown that sAPPα and sAPPβ possess neurotrophic properties, whereas Aβ is neurotoxic. However, the underlying mechanism of the opposing effects of APP fragments remains poorly understood. In this study, we have investigated the mechanism of sAPPα-mediated neurotrophic effects. sAPPα and sAPPβ interact with p75 neurotrophin receptor (p75NTR), and sAPPα promotes neurite outgrowth.Methods and Findings
First, we investigated whether APP fragments interact with p75NTR, because full-length APP and Aβ have been shown to interact with p75NTR in vitro. Both sAPPα and sAPPβ were co-immunoprecipitated with p75NTR and co-localized with p75NTR on COS-7 cells. The binding affinity of sAPPα and sAPPβ for p75NTR was confirmed by enzyme-linked immunosorbent assay (ELISA). Next, we investigated the effect of sAPPα on neurite outgrowth in mouse cortical neurons. Neurite outgrowth was promoted by sAPPα, but sAPPα was uneffective in a knockdown of p75NTR.Conclusion
We conclude that p75NTR is the receptor for sAPPα to mediate neurotrophic effects. 相似文献18.
Background
Proteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue. 相似文献19.
Background
When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.Methodology/Principal Findings
We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.Conclusions/Significance
Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy. 相似文献20.
Yan Xie Minghui Cheng Shan Lu Qilong Yuan Dongyu Yang Ying Chen Chen Pan Yurong Qiu Bo Xiong 《Cell division》2018,13(1):4