Biomedical subjects

R Shalgi

Publications and source records attributed to R Shalgi.

At least 19 recordsLinked to original sources

PubMed2006

Expression and immunolocalization of the calpain-calpastatin system during parthenogenetic activation and fertilization in the rat egg.

Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca(2+)-dependent thiol protease. The calpain-calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain-calpastatin interplay throughout the process. Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110 kDa calpastatin and beta-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static. We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.

Animals↗

PubMed2005

The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat.

Mammalian sperm-egg fusion results in cortical granule exocytosis (CGE) and resumption of meiosis. Studies of various exocytotic cells suggest that filamentous actin (F-actin) blocks exocytosis by excluding secretory vesicles from the plasma membrane. However, the exact function of these microfilaments, in mammalian egg CGE, is still elusive. In the present study we investigated the role of actin in the process of CGE, and the possible interaction between actin and protein kinase C (PKC), by using coimmunoprecipitation, immunohistochemistry and confocal microscopy. We identified an interaction between actin and the PKC alpha isoenzyme in non-activated metaphase II (MII) eggs and in eggs activated by phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). F-actin was evenly distributed throughout the egg's cytosol with a marked concentration at the cortex and at the plasma membrane. A decrease in the fluorescence signal of F-actin, which represents its depolymerization/reorganization, was detected upon fertilization and upon parthenogenetic activation. Exposing the eggs to drugs that cause either polymerization or depolymerization of actin (jasplakinolide (JAS) and cytochalasin D (CD) respectively) did not induce or prevent CGE. However, CD, but not JAS, followed by a low dose of TPA doubled the percentage of eggs undergoing complete CGE, as compared with TPA alone. We further demonstrated that myristoylated alanin-rich C kinase substrate (MARCKS), a protein known to cross-link F-actin in other cell types, is expressed in rat eggs and is colocalized with actin. In view of our results, we suggest that the cytoskeletal cortex is not a mere physical barrier that blocks CGE, but rather a dynamic network that can be maneuvered towards allowing CGE by activated actin-associated proteins and/or by activated PKC.

Actin Cytoskeleton↗

PubMed2005

Strontium-induced rat egg activation.

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]i transient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]i oscillations. Although SrCl2 has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2 (2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2 was optimal for inducing the first [Ca2+]i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]i oscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]i oscillations (8, 15 or 60 min in SrCl2 respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2 caused a delay in CM. Our results demonstrate that SrCl2 is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]i oscillations, CGE and CM.

Animals↗

PubMed2004

Are Src family kinases involved in cell cycle resumption in rat eggs?

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca2+ transients. The pathways, within the egg, leading to the intracellular Ca2+ release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca2+ release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity (PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca2+ elevation, is an SFK-dependent process, while CGE, triggered by either Ca2+ elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm-egg fusion site downstream of the Ca2+ release remains unclear.

Animals↗

PubMed2004

Fyn kinase-tubulin interaction during meiosis of rat eggs.

Prior to fertilization, the spindle of vertebrate eggs must remain stable and well organized during the second meiotic meta-phase arrest (MII). In a previous study we have determined that the completion of meiosis is a Src family kinase (SFK)-dependent event. In the current study we have used the SFK inhibitors, SU6656 and PP2, and demonstrated that inhibition of SFKs caused the formation of a disorganized spindle. The observation that proper organization of an MII spindle is an SFK-dependent process, combined with our previous finding that Fyn kinase is localized at the microtubules (MTs), prompted us to examine the potential role of Fyn in MT signaling. Our results show an association between Fyn and tubulin, the ability of Fyn to phosphorylate tubulin in vitro and stimulation of meiosis completion by injection of a constitutively active form of Fyn (CAF). We suggested that SFKs mediate significant functions during the organization of the MII spindle. In view of CAF injection experiments, and of the pronounced concentration of Fyn kinase at the spindle, we propose that Fyn may play an important role in some aspects of the spindle functions, possibly those involving the MTs.

Animals↗

PubMed2001

PKC in eggs and embryos.

After a spermatozoon enters the egg, rapid processes such as the cortical granules exocytosis (CGE) occur. Other, later processes are observed as well, and they include resumption of the second meiotic division, second polar body (PBII) extrusion and rearrangement of the DNA inside the pronuclei (PN). The above mentioned biochemical processes involve signal transduction pathways that are well known in other cell systems, and require mediation of second messengers like Ca2+ and diacylglycerol (DAG) which are protein kinase C (PKC) activators. The present review, based upon recently published studies, raises the possibility of PKC involvement in fertilization and in early developmental stages of the mammalian embryo.

Animals↗

PubMed2001

Oocyte activation: lessons from human infertility.

During fertilization, the spermatozoon penetrates through the cumulus cells and the zona pellucida that surrounds the oocyte, before it binds and fuses with the oocyte plasma membrane to induce activation. In vitro fertilization (IVF) studies performed in non-human mammals have contributed extensive knowledge regarding the mechanisms by which the spermatozoon activates the meiotic-arrested oocyte to resume meiosis, cleave and develop into an embryo. Although IVF has been used extensively for treating subfertile couples, not all of them were able to benefit from this procedure. In intracytoplasmic sperm injection (ICSI), one viable spermatozoon only is sufficient for successful fertilization of a single oocyte. Moreover, the injected fertilizing spermatozoon bypasses several physiological barriers, compared with IVF, which together could explain the high success rate for this procedure. ICSI has also allowed the identification of sperm components that are required for successful fertilization.

Calcium↗

PubMed2001

Signal transduction pathways in activation of the mammalian egg.

The mature egg arrested at its second metaphase is a rather quiet cell, and possesses a pre-set developmental program. A sequence of biochemical events that leads to the rapid embryonic mitotic divisions initiates at fertilization when the spermatozoon overcomes this second cell cycle arrest. These events induce a transient rise in intracellular Ca2+ concentration ([Ca2+]i) that leads to the cortical granule exocytosis (CGE) and resumption of meiosis. Various treatments can induce parthenogenetic activation as manifested by the extrusion of the second polar body or CGE. Similar to somatic cells, recent studies in mammalian eggs suggest that signal transduction pathways mediate egg activation. The initial increase in ([Ca2+]i) appears to be critical for egg activation. Other messengers such as protein kinase C and protein tyrosine kinases, were suggested as possible inducers of some aspects of egg activation.

Animals↗

PubMed2000

Molecular cloning of rat sperm galactosyl receptor, a C-type lectin with in vitro egg binding activity.

Rat sperm galactosyl receptor is a member of the C-type animal lectin family showing preferential binding to N-acetylgalactosamine compared to galactose. Binding is mediated by a Ca(2+)-dependent carbohydrate-recognition domain (CRD) identical to that of the minor variant of rat hepatic lectin receptor 2/3 (RHL-2/3). The molecular organization of the genomic DNA, cDNA, and derived amino acid sequence of rat testis galactosyl receptor have been determined and in vitro fertilization studies were conducted to ascertain its role. We have determined that the rat testis galactosyl receptor gene generates two mRNA species: one species, designated liver-type, is identical to RHL-2/3; the other, designated testis-type, contains one unspliced intron (86 nt) which alters the reading frame and changes the amino acid sequence of the carboxyl terminus. As a result, the CRD (glutamine-proline-aspartic acid/QPD) and flanked Ca(2+)-binding amino acid sequences were not present in the testis-type protein. Northern and Southern blots demonstrated presence of transcripts with unspliced intron in rat sperm but not liver. Similarly, antibody, raised against a synthetic 12-amino acid peptide (p12) encoded by the unspliced intron, recognized in immunoblots a 54 kDa receptor protein in protein extracts from testis but not from liver. Immunofluorescence and immunogold electron microscopy studies demonstrated that both protein species localized on the plasma membrane surface of the head and tail of rat sperm. Furthermore, capacitated rat sperm preincubated with polyclonal antisera to RHL-2/3 or to the CRD of the liver-type galactosyl receptor showed a statistically significant decrease in the in vitro fertilization rate. We conclude that rat sperm galactosyl receptor may play a role in egg binding and that an undetermined molecular mechanism operates to generate two proteins with identical intracellular amino terminal domain but only one of them displays a CRD and associated Ca(2+)-binding sites at the carboxyl terminal extracellular domain.

Alternative Splicing↗

PubMed2000

Anordiol produces pregnancy loss in the rat--via the embryo or via the uterus?

Anordiol, the dihydroxylated metabolite of anordrin, is an antiestrogen with estrogenic activity that is known to inhibit fertility. The following study was conducted to determine the mechanism of this antifertility effect. Anordiol was administered orally to rats, prior to implantation, on Day 2 of pregnancy. Control animals were treated with the vehicle only. The effectiveness of the agent in terminating pregnancy was determined on Day 14 of pregnancy. Anordiol was 100% effective in abolishing pregnancy at a dose of 0.6 mg/Kg. Administration of smaller doses resulted in a decreased number of implanting embryos, in a dose-dependent manner. An additional dose of anordiol on Day 3 of pregnancy yielded similar results. To determine whether pregnancy impairment by anordiol is exerted via the embryo or via the uterus, reciprocal embryo transfers were performed. Day 5 blastocysts were transferred into the uteri of pseudopregnant rats. In one set of experiments, the donor rats were treated with anordiol, and in the second set the recipient rats were treated. The results indicate that the effects of anordiol administration are exerted via the embryo as well as the uterus.

Abortion, Induced↗

PubMed2000

Expression of cadherin adhesion molecules on human gametes.

The presence of cadherins, Ca(2+)-dependent cell-cell adhesion molecules which may be involved in gamete interaction, was investigated in human gametes. Expression of cadherin molecules was demonstrated using an anti-pan-cadherin antibody and specific antibodies against the three classical cadherins: E- (epithelial), P- (placental) and N- (neural) cadherins. Samples of 48 h old unfertilized oocytes and spermatozoa from in-vitro fertilizing semen samples were lysed and separated by electrophoresis. Localization of cadherins was determined on intact, fixed, permeabilized spermatozoa and oocytes by immunocytochemisty assessed by confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a single band of approximately 120 kDa in spermatozoa (whether 'fresh', capacitated, or frozen-thawed) and oocyte extracts. Oocytes presented all three classical cadherins with the appropriate molecular weights of 120-130 kDa. In sperm lysate we demonstrated the presence of E-cadherin but not N-cadherin. The anti-P antibody detected a 90 kDa peptide as the only immunoreactive antigen. Following immunocytochemistry of human oocytes all cadherin molecules were allocated predominantly to the plasma membrane with only traces in the cytoplasm. In spermatozoa, several staining patterns were observed with each of the pan-cadherin, N-cadherin and E-cadherin antibodies mostly confined to different head regions. We conclude that cadherin molecules are present on plasma membranes of both human spermatozoa and oocytes and may play a role in the intricate recognition process preceding gamete fusion.

Cadherins↗

PubMed1998

Profile of protein kinase C isozymes and their possible role in mammalian egg activation.

Western blot analysis was used to investigate protein kinase C (PKC) profile of rat eggs. The presence of eight PKC isozymes was demonstrated: conventional PKC alpha, beta and gamma; novel PKC delta, epsilon and mu; atypical PKC zeta and lambda. PKC alpha was detected by RT-PCR as well. PKC translocation from the cytosol to the plasma membrane served as a marker for enzyme activation. Immunofluorescence confocal microscopy demonstrated a relatively uniform distribution of PKC alpha, betaI, and betaII throughout the cytosol of metaphase II arrested eggs. PKC accumulation at the plasma membrane was detected 5 min after exposure to 12-O-tetradecanoyl phorbol-13-acetate and increased with time, thus demonstrating activation of these PKCs.

Animals↗

PubMed1998

Expression and immunolocalization of p59c-fyn tyrosine kinase in rat eggs.

Fertilization overcomes meiotic arrest by triggering a series of biochemical events, resulting in activation of the egg. A small group of protein tyrosine kinases (PTKs) have been identified in eggs of invertebrates and lower vertebrates and inhibitor studies have suggested that they play a role in late events of egg activation. A recent study using the sea urchin system demonstrated that Fyn kinase was expressed in eggs and was activated within minutes of fertilization. In the present study, Western blot analysis as well as immune complex kinase assay demonstrated that p59(c-fyn) kinase was expressed in both unfertilized and fertilized rat eggs. Immunofluorescence confocal microscopy demonstrated that Fyn kinase was localized to the egg cortex but also to the polar body and the fertilizing cone which are elevated from the cortical cytoplasm of the activated egg. Surprisingly, Fyn was also found to be highly concentrated over the meiotic and mitotic spindles. To date, Fyn is the first PTK demonstrated to be present in the mammalian egg. Localization of Fyn to the egg cortex as well as the spindle microtubules indicates that this protein kinase may have multiple functions within the egg.

Animals↗

PubMed1998

Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg.

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg.

Animals↗

PubMed1998

Mechanisms leading to cortical reaction in the mammalian egg.

Activation of the mammalian egg results in cortical reaction (CR), which is correlated with an increase in intracellular Ca2+ concentration and PKC activation. The CR is a gradual rather then an "all or none" response, and can be regulated by different concentrations of parthenogenetic activators. To evaluate the biological significance of parthenogenetic induced CR, rat eggs were fertilized or activated by different concentrations of ionomycin and TPA. Cortical granules (CG) were monitored by electron microscopy, while the CG exudate was visualized by Lens culinaris lectin and Texas Red, using light and confocal microscopy. The ability of the CR to trigger a full block to polyspermy was examined in an IVF system. Our study demonstrates the existence of light and dark CG, which differ by number, distribution in the egg cortex, and sensitivity to parthenogenetic activators. Sperm penetration or high concentration of activators, trigger depletion of both light and dark CG, leading to a full CR. Low concentration of activators altered the CG density, the ratio of dark/light CG, and induced partial CR that was sufficient to cause a block to polyspermy. The results imply that Ca2+ rise or PKC activation have different effects on light and dark CG. In recently fertilized or parthenogenetically activated eggs, CG exudate appeared as evenly distributed spots, whereas in more advanced stages of fertilization the exudate was scattered as patchy aggregates. This observation suggests a difference in the dispersion of CG exudate after fertilization as compared to parthenogenetic activation.

Animals↗

PubMed1998

Spontaneous and follicular fluid-induced acrosome reaction in sperm samples from in vitro fertilizing and nonfertilizing normozoospermic patients.

PURPOSE: One of the most challenging and intriguing groups of infertile patients is that of normozoospermic men who have repeatedly not achieved fertilization in vitro. These cases probably present a wide range of gamete disorders manifested at different stages of the fertilization process. The occurrence of spontaneous and follicular fluid (FF)-induced acrosome reactions (ARs) in in vitro fertilizing and nonfertilizing specimens from normozoospermic IVF patients was assessed, and the effect of Percoll and cryopreservation on the incidence of ARs was evaluated. METHODS: Semen samples from 62 normozoospermic (15 in vitro nonfertilizing and 47 fertilizing) patients were analyzed. Spermatozoa were double-stained with FITC-conjugated Pissum sativum lectin, to assess their acrosomal status, and propidium iodide, to evaluate their vitality, using flow cytometry analysis. RESULTS: A lower average of AR incidence was observed in the nonfertilizing than in the fertilizing group with all treatments. Both groups exhibited an increase in the proportion of ARs following incubation with FF. This rise was most prominent when Percoll-separated fractions were used (15.8 and 25.6% AR in the nonfertilizing and fertilizing groups, respectively). Thawed cryopreserved and fresh fertilizing samples exhibited similar AR rates. CONCLUSIONS: That normozoospermic recurrently nonfertilizing compared to fertilizing semen samples have a lower capacity to undergo ARs is suggested.

Acrosome↗

PubMed1998

Replication timing of the various FMR1 alleles detected by FISH: inferences regarding their transcriptional status.

Following the application of two-color fluorescence in-situ hybridization (FISH) to human interphase cells, we examined the replication timing of the fragile-X locus relative to the non-transcribed late replicating alpha-satellite region of chromosome-X, a built-in intracellular reference locus. In this assay, an unreplicated locus is identified by a single hybridization signal (singlet; S), whereas a replicated locus is identified by a duplicated signal (doublet; D). Hence, following simultaneous hybridization with the FMR1 and alpha-satellite probes, male cells with one singlet and one doublet signal per cell (SD cells) indicate S-phase cells where only one of the two loci has replicated. The studied cell samples (lymphocytes and amniocytes) were derived from normal males, fragile-X male patients, and premutation male carriers. Three distinct populations of SD cells were identified among the various samples. The first population had a high frequency of cells showing a doublet FMR1; this pattern, indicating early replication of FMR1, characterized the SD cell population of normal males. The second population had a high frequency of cells showing a singlet FMR1; this pattern, indicating very late replication of FMR1, characterized the SD population of fragile-X patients. The third population had about one half of the cells showing a singlet FMR1 and the other half with a doublet FMR1, indicating somatic variation in the replication timing of FMR1; this pattern was seen in the SD cell population of premutation carriers. The replication status of the FMR1 locus in the cells of patients was altered from late to early in the presence of 5-azadeoxycytidine, an activator of various silent genes. Based on the vast amount of information showing that expressed loci replicate early, whereas unexpressed loci replicate late, we inferred from the replication status of the FMR1 locus that: (1) the normal FMR1 allele is transcriptionally active in lymphocytes and amniocytes; (2) the fully mutated FMR1 allele is transcriptionally silent; (3) the transcriptional activity of the premutated allele is somewhat disturbed; (4) 5-azadeoxycytidine activates the fully mutated FMR1 allele.

Alleles↗

PubMed1998

Early events in mammalian egg activation.

The fertilizing spermatozoon initiates a series of events in the mammalian egg, referred to as 'egg activation'. These biochemical and morphological events include a transient rise in intracellular Ca2+ concentration ([Ca2+]i) that leads to the cortical reaction (CR) and the establishment of the block to polyspermy on the one hand, and to the resumption of meiosis and later embryonic mitotic divisions on the other. The initial increase in [Ca2+]i appears to be critical for the initiation of egg activation. However, another second messenger, protein kinase C (PKC), was also suggested as a possible inducer of some aspects of egg activation. The review focuses on early events during mammalian sperm-egg interaction and discusses possible roles of Ca2+ and PKC in egg activation.

Animals↗