scholarly journals The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes

Reproduction ◽  
2011 ◽  
Vol 141 (5) ◽  
pp. 581-593 ◽  
Author(s):  
Valeria S Mouguelar ◽  
Marcelo O Cabada ◽  
Gabriela Coux
Keyword(s):  
Egg Activation ◽  
Binding Motif ◽  
Integrin Subunit ◽  
Oocyte Activation ◽  
Phosphorylated Proteins ◽  
Bufo Arenarum ◽  
Egg Extracts ◽  
Activation Mechanisms ◽  
Mammalian Fertilization ◽  
Signaling Receptors

Integrins are cell adhesion molecules that are thought to be involved in sperm–oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported byXenopus laevisstudies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibianBufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest thatB. arenarumfertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors inB. arenarumoocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.

Activation of amphibian oocytes by sperm extracts

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 303-308 ◽  
Author(s):  
F. Bonilla ◽  
M. T. Ajmat ◽  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
J. Oterino ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Dna Replication ◽  
Egg Activation ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Granule Exocytosis ◽  
Bufo Arenarum ◽  
Sperm Extract ◽  
Cortical Granule Exocytosis

SummaryIn the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.

Role of phospholipase A2 pathway in regulating activation of Bufo arenarum oocytes

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 214-220 ◽  
Author(s):  
M.T. Ajmat ◽  
F. Bonilla ◽  
P.C. Hermosilla ◽  
L. Zelarayán ◽  
M.I. Bühler
Keyword(s):  
Arachidonic Acid ◽  
Ion Channels ◽  
Calcium Release ◽  
Phospholipase A ◽  
Egg Activation ◽  
Specific Cell ◽  
Oocyte Activation ◽  
Regulatory Pathways ◽  
Bufo Arenarum ◽  
Trisphosphate Receptor

SummaryTransient increases in the concentration of cytosolic Ca2+ are essential for triggering egg activation events. Increased Ca2+ results from its rapid release from intracellular stores, mainly mediated by one or both intracellular calcium channels: the inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR). Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of a cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid from phospholipids of the endoplasmic reticulum as well as from the nuclear envelope. Previous studies have suggested that arachidonic acid and/or its metabolites were able to modulate the activity of several ion channels. Based on these findings, we have studied the participation of the phospholipase A2 (PLA2) pathway in the process of Bufo arenarum oocyte activation and the interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization. We found that addition of both melittin, a potent PLA2 activator, and arachidonic acid, the main PLA2 reaction metabolite, was able to induce activation events in a bell-shaped manner. Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs and useful tools for understanding calcium homeostasis in eggs that express both types of receptors.

Experiences with intracytoplasmic sperm injection

1995 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Susan E Lanzendorf
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Meiotic Division ◽  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Cortical Granules ◽  
Specific Sequence ◽  
Mammalian Fertilization ◽  
Pass Through

Mammalian fertilization, whether it takes place within the female reproductive tract or within a laboratory dish, is comprised of many processes which must follow a specific sequence. The spermatozoon must bind to and pass through the zona pellucida, fuse with the oolemma and become incorporated into the cytoplasm of the oocyte. Fusion of the two gametes triggers oocyte activation, resulting in exocytosis of the cortical granules and completion of the second meiotic division of the oocyte. A block in one or more of these processes, due either to abnormalities in the spermatozoon or oocyte, may result in fertilization failure.

Regulation of the Function of αvβ3 in Platelets by a Designed Peptide Targeting the αv Transmembrane Domain.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1504-1504
Author(s):  
Hang Yin ◽  
Joanna S. Slusky ◽  
Bryan W. Berger ◽  
Rustem I. Litvinov ◽  
Gaston Vilaire ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Transmembrane Domain ◽  
Solid Phase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cd Spectroscopy ◽  
Binding Motif ◽  
Integrin Subunit ◽  
Bacterial Membranes ◽  
Tm Domain

Abstract Integrins reside on cell surfaces in an equilibrium between inactive and active conformations. When inactive, the transmembrane (TM) domains of integrin α and β subunits interact, but the domains separate when integrins assume their active conformation. Although this conformational change has not been shown for αvβ3, we hypothesized that a peptide designed to bind to the αv TM domain might activate αvβ3 in platelets by disrupting the TM domain heterodimer of the inactive molecule. To design such a peptide, we used CHAMP (Computed Helical Anti-Membrane Protein) methodology. In the CHAMP method, the αv TM helix was scanned for motifs likely to mediate αvβ3 TM domain interactions. Next, the backbone conformation for an αv-binding peptide was selected based on known structural preferences for the motifs identified in the αv helix. Finally, the sequence for the peptide was designed computationally using a side-chain repacking algorithm. The CHAMP peptide, anti-αv, and its TM helix target, αv-TM, were synthesized by solid phase synthesis. We also synthesized anti-αvmut in which the putative anti-αv-TM binding motif, GXXXG, was mutated to LXXXL. Lys2 dipeptides and short polyethylene glycol sequences were appended to the C- and N-termini of the peptides, respectively, to facilitate their solubility and insertion into membranes. CD spectroscopy revealed that both anti-αv and αv-TM were helical in micelles and phospholipid vesicles. Measurement of Trp fluorescence intensity revealed that anti-αv rapidly inserted into unilamellar POPC/POPG vesicles. Analytical ultracentrifuge and fluorescence resonance energy transfer experiments demonstrated that anti-αv bound to the αv-TM, but not to a homologous αIIb-TM domain peptide. In addition, there was negligible interaction between αv-TM and anti-αvmut. Anti-αv also formed heteromeric complexes with the αv TM domain in bacterial membranes but not with the TM domains of αIIb, α2, β1, or β3. αvβ3 mediates the adhesion of agonist-stimulated platelets to the matrix protein osteopontin (OPN). We found that anti-αV at μM concentrations induced platelet adhesion to OPN. Adhesion was prevented by the divalent cation chelator EDTA, consistent with an integrin-mediated process, but was only minimally affected by pre-incubating the platelets with PGE1, implying that anti-αv-induced adhesion by interacting directly with the αv TM domain. Force spectroscopy using laser tweezers confirmed the specificity of the interaction of anti-αv with platelets: anti-αv induced specific rupture forces between platelets and OPN-coated beads, but not between platelets and fibrinogen-coated beads. Moreover, only non-specific rupture forces were detected between OPN-coated beads and platelets incubated with anti-αvmut. These results demonstrate the successful application of computational methods to design a soluble peptide that specifically recognizes the TM domain of αv in platelets membranes, even when a 400-fold excess of the homologous integrin subunit αIIb is present. Further, because the peptide binds to the site on the αv TM domain that interacts with the β3 TM helix and activates αvβ3, these results strongly support the hypothesis that separation of the αv and β3 TM domains regulates the function of this integrin.

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 495-501
Author(s):  
J. Zapata-Martínez ◽  
M.F. Medina ◽  
M.C. Gramajo-Bühler ◽  
G. Sánchez-Toranzo
Keyword(s):  
Aristolochic Acid ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Egg Activation ◽  
Inositol Triphosphate ◽  
Phosphoinositide Metabolism ◽  
Oocyte Activation ◽  
Rhinella Arenarum ◽  
Different Doses

SummaryRhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation

Zygote ◽  
2010 ◽  
Vol 19 (2) ◽  
pp. 171-180 ◽  
Author(s):  
M.T. Ajmat ◽  
F. Bonilla ◽  
L. Zelarayán ◽  
M.I. Bühler
Keyword(s):  
Calcium Release ◽  
Ryanodine Receptors ◽  
Inhibitory Effect ◽  
Ruthenium Red ◽  
Activation Process ◽  
Dependent Manner ◽  
Oocyte Activation ◽  
Relative Contribution ◽  
Morphological Criteria ◽  
Bufo Arenarum

SummaryCalcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP3Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP3Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP3Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP3Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP3Rs, probably through a CICR mechanism. Both receptors play a role in Ca2+ release mechanisms although their relative contribution to the activation process is unclear.

Nuclei from fertilized mouse embryos have calcium-releasing activity

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1123-1128 ◽  
Author(s):  
T. Kono ◽  
J. Carroll ◽  
K. Swann ◽  
D.G. Whittingham
Keyword(s):  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Nuclear Envelope Breakdown ◽  
Mammalian Embryos ◽  
Sperm Factor ◽  
Physical Stimuli ◽  
Mammalian Fertilization ◽  
Parthenogenetic Embryos

During mammalian fertilization, the sperm triggers a series of intracellular Ca2+ oscillations which initiate oocyte activation and the formation of pronuclei. Oocyte activation can be induced artificially by a variety of chemical and physical stimuli which elevate intracellular calcium. We show that the transfer of nuclei from 1- and 2-cell-stage fertilized mouse embryos to unfertilized oocytes stimulates the completion of meiosis and the formation of pronuclei. Nuclei from embryos that had developed to the 4-cell stage did not stimulate meiotic resumption. The ability to cause oocyte activation was specific to nuclei transferred from fertilized embryos as nuclei from parthenogenetic embryos or cytoplasts from fertilized or parthenogenetic embryos did not induce activation. Nucleus-induced oocyte activation was associated with the generation of intracellular Ca2+ transients, which were seen after nuclear envelope breakdown of the transferred nuclei. Treatment of the oocyte with the intracellular Ca2+ chelator, BAPTA, prior to nuclear transfer inhibited intracellular Ca2+ transients and oocyte activation. The specific Ca(2+)-releasing activity of the nucleus was not caused by sperm-induced protein synthesis since similar activity was present in nuclei originating from embryos exposed to cycloheximide throughout fertilization. The specific ability of nuclei from fertilized embryos to stimulate Ca2+ transients and oocyte activation was also found in nuclei from embryos parthenogenetically activated by the injection of a partially purified cytosolic sperm factor. The results suggest that the fertilizing sperm introduces Ca(2+)-releasing activity which becomes associated with the nucleus of early mammalian embryos.

scholarly journals Cyclin-binding motifs are essential for the function of p21CIP1.

1996 ◽  
Vol 16 (9) ◽  
pp. 4673-4682 ◽  
Author(s):  
J Chen ◽  
P Saha ◽  
S Kornbluth ◽  
B D Dynlacht ◽  
A Dutta
Keyword(s):  
Binding Motif ◽  
Cyclin Dependent Kinase ◽  
Tumor Suppressor P53 ◽  
Binding Motifs ◽  
Cell Extracts ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.

Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa

Development ◽  
1997 ◽  
Vol 124 (11) ◽  
pp. 2267-2274 ◽  
Author(s):  
C. Sette ◽  
A. Bevilacqua ◽  
A. Bianchini ◽  
F. Mangia ◽  
R. Geremia ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Catalytic Domain ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Egg Activation ◽  
Tyrosine Kinase Receptor ◽  
Cortical Granule ◽  
Oocyte Activation ◽  
Cos Cells ◽  
Mouse Eggs

A truncated form of the c-kit tyrosine kinase receptor, corresponding to the phosphotransferase portion of the cytoplasmic catalytic domain and the carboxyterminus (tr-kit), is accumulated during late mouse spermiogenesis. Here we report that tr-kit is specifically localized in the residual sperm cytoplasm, with maximal accumulation in the midpiece of the flagellum, suggesting that it can enter the egg during fertilization. Microinjection of extracts from COS cells expressing a recombinant tr-kit protein into metaphase II-arrested mouse oocytes caused complete oocyte activation, including cortical granule exocytosis, completion of the 2nd meiotic division, formation of a parthenogenetic pronucleus and progression through cleavage stages. No activation above background levels was obtained with extracts from mock-transfected COS cells. Similar results were obtained by microinjection of in vitro synthesized tr-kit mRNA into metaphase II-arrested oocytes. Tr-kit-induced parthenogenetic egg activation was completely inhibited by oocyte preincubation with the Ca2(+)-chelating agent BAPTA-AM or with a specific inhibitor of phospholipase C activity. Tr-kit-induced egg activation was associated with a decrease in activity of mitogen-activated protein kinase, an essential component of the cytostatic factor. These results candidate tr-kit as a putative sperm factor required for triggering activation of mouse eggs at fertilization.

scholarly journals Diagnosis and Treatment of Male Infertility-Related Fertilization Failure

2020 ◽  
Vol 9 (12) ◽  
pp. 3899
Author(s):  
Arantxa Cardona Barberán ◽  
Annekatrien Boel ◽  
Frauke Vanden Meerschaut ◽  
Dominic Stoop ◽  
Björn Heindryckx
Keyword(s):  
Male Infertility ◽  
Current Knowledge ◽  
Oocyte Activation ◽  
Fertilization Failure ◽  
Sperm Abnormalities ◽  
Normal Sperm ◽  
Activation Mechanisms ◽  
Sperm Factor ◽  
Male Factors ◽  
Assisted Oocyte Activation

Infertility affects approximately 15% of reproductive-aged couples worldwide, of which up to 30% of the cases are caused by male factors alone. The origin of male infertility is mostly attributed to sperm abnormalities, of which many are caused by genetic defects. The development of intracytoplasmic sperm injection (ICSI) has helped to circumvent most male infertility conditions. However, there is still a challenging group of infertile males whose sperm, although having normal sperm parameters, are unable to activate the oocyte, even after ICSI treatment. While ICSI generally allows fertilization rates of 70 to 80%, total fertilization failure (FF) still occurs in 1 to 3% of ICSI cycles. Phospholipase C zeta (PLCζ) has been demonstrated to be a critical sperm oocyte activating factor (SOAF) and the absence, reduced, or altered forms of PLCζ have been shown to cause male infertility-related FF. The purpose of this review is to (i) summarize the current knowledge on PLCζ as the critical sperm factor for successful fertilization, as well as to discuss the existence of alternative sperm-induced oocyte activation mechanisms, (ii) describe the diagnostic tests available to determine the cause of FF, and (iii) summarize the beneficial effect of assisted oocyte activation (AOA) to overcome FF.

Sign in / Sign up

Export Citation Format

Share Document