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Search for other papers by N. Dekel in
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Search for other papers by R. Shalgi in
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Summary. Oocytes were exposed to GnRHa to induce their maturation both in vivo, by administration of the hormone to hypophysectomized rats, and in vitro, in cultures of intact ovarian follicles. Mature oocytes obtained under both these conditions were then exposed in vitro to a sperm suspension for fertilization. Fertilization of control groups of oocytes, isolated from intact or hypophysectomized PMSG-primed hCG-induced ovulators, was 88·3 ± 3·3% (n = 331) and 90·0 ± 2·8% (n = 427), respectively, as compared to 82·8 ± 3·2% (n = 413) for oocytes isolated from hypophysectomized PMSG-primed GnRHa-induced ovulators. Fertilization rate in oocytes treated by GnRHa in vitro was 78·5 ± 3·1% (n = 247) as compared to 79·3 ± 4·1% (n = 261) in LH-treated oocytes. These results demonstrate that fertilizability of oocytes undergoing maturation in response to GnRHa is similar to that of oocytes induced to mature by LH. No differences could be detected in the proportions of abnormal oocytes (polyspermic, fragmented and dead) and the zygotes obtained after fertilization of GnRHa- or LH-treated oocytes showed similar ability to cleave.
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Summary. A gonadotrophin-releasing hormone analogue (GnRHa) was administered to hypophysectomized immature rats. Postovulatory mature oocytes obtained under these conditions were exposed in vitro to a sperm suspension for fertilization. Developmental ability of the fertilized ova was studied by transfer of the 2-cell stage embryos to oviducts of foster mothers. The potential of oocytes, undergoing maturation in response to GnRHa, to develop into 2-cell embryos was similar to that of oocytes stimulated by hCG (76·4% and 83·1% respectivey). The 2-cell stage embryos obtained from such oocytes were equally able to implant in the uteri of foster mothers (25·7% and 21·2% respectively) and subsequenty develop into live embryos (15·3% and 15·2%, respectively, at Day 20 of pregnancy).
Keywords: GnRH; oocyte maturation; rat; embryos
Search for other papers by J. Seligman in
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Search for other papers by R. Shalgi in
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Summary. Thiol (SH) oxidation to disulphides (SS) is thought to be involved in sperm chromatin condensation and tail structure stabilization, which occur during maturation of spermatozoa. Previously developed procedures, using the fluorescent labelling agent monobromobimane (mBBr), enabled us to study the thiol-disulphide status of spermatozoa. Electrophoretic separation of labelled sperm proteins from the caput and cauda regions showed that during maturation thiol oxidation occurs in many protein fractions from the tail and that the magnitude of oxidation differs between proteins. Among the protein bands, one major band (MPB), probably a dense fibre constituent, is quantitatively prominent. N-Ethylmaleimide (NEM) or mBBr alkylation (of intact spermatozoa) changes the mobility of the caput MPB, but not that of the cauda MPB. The results indicated that the altered mobility of MPB is mainly due to a change in its shape, possibly resulting from the alkylation of a few critical SH groups. Epididymal fluid proteins contain both SH and SS. The thiol and disulphide content of the various epididymal proteins appears similar, although some diminution in fluorescence is seen in epididymal fluid proteins from the cauda region as compared with those from the caput region. The prominent changes in thiol status occur in the spermatozoa.
Keywords: thiols; epididymis; sperm maturation; rat
Search for other papers by E. Skutelsky in
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Search for other papers by R. Shalgi in
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The lectin-binding patterns of mammalian zonae pellucidae were investigated to determine whether differences reflected their characteristic carbohydrate distribution patterns. Ovaries isolated from rodents (mouse, rat and hamster), rabbits, cats, dogs and pigs were fixed with glutaraldehyde and embedded in paraffin wax. Sections 5 μm, were deparaffinized, rehydrated and labelled with ten different biotinylated lectins as probes and avidin–biotin– peroxidase complex as visualant. The zonae pellucidae of all animals studied exhibited species-specific variations in lectin-binding patterns, whereas the lectin binding of their granulosa cells and follicular fluids were identical. Phylogenetically close species, such as the rodents and rabbits demonstrated high similarity in zona pellucida saccharides, expressed in binding of succinylated wheatgerm agglutinin and peanut agglutinin. Lectins such as Dolichos biflorus agglutinin, which binds only to mouse, Griffonia simplicifolia (GS-I), which binds to mouse and rat but not hamster and rabbit and soybean agglutinin, which binds only to rodents, reflect characteristic differences between phylogenetically related mammals.
Search for other papers by D Ben-Yosef in
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Search for other papers by R Shalgi in
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At fertilization of the mammalian egg, the spermatozoon initially binds to and then fuses with the egg plasma membrane. This critical event activates specific biochemical pathways within the egg. Activation of the egg induces resumption of meiosis and the start of rapid embryonic mitotic divisions on the one hand, and cortical granule exocytosis leading to modification of the zona pellucida and a block to polyspermy on the other. It has been shown in different systems that changes in intracellular ion concentrations can serve as second messengers of signal transduction mechanisms. The use of specific fluorescence probes, combined with the image analysis technique, facilitates the measurement of their dynamics in real time in the living cell and, thereby, assessment of their role in activation of the mammalian egg. This review focuses on the dynamics of intracellular Ca2+ and pH and their role in transducing the sperm signal to downstream cell cycle regulators.
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Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca2+-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 110kDa calpastatin and β-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.
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Search for other papers by R Shalgi in
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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.
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The thiol–disulfide status in proteins of human spermatozoa categorized as normozoospermic, teratozoospermic and asthenozoospermic was examined. Washed spermatozoawere incubated with or without dithiothreitol (DTT) to reduce disulfides (SS) to thiols (SH), and then labelled with the specific fluorescence thiol labelling agent monobromobimane (mBBr). The SH and SS in intact labelled spermatozoa were evaluated by fluorescence microscopy and by flow cytometry analysis; mBBr-labelled spermatozoa were solubilized and sperm proteins analysed by gel electrophoresis (SDS-PAGE for non-basic, whole sperm proteins and acid urea-PAGE for sperm nuclear basic proteins). Microscopy and flow cytometry showed that normozoospermic samples (having normal sperm count, morphology and motility) contained both SH and SS, with more SS than SH. Heterogeneity in the proportion of SH/(SH plus SS) was observed among spermatozoa within the ejaculates. The total SH plus SS was similar among the ejaculates, with some variability in SH/(SH plus SS) noted among them. SDS-PAGE of solubilized normozoospermic cells showed differences in the SH and SS content of the protein bands. Acid urea-PAGE of basic proteins isolated from normozoospermic samples showed protamines P1 and P2 and traces of non-protamine basic proteins. P1 and P2 contained SH and SS, with variability in SH/(SH plus SS) observed among the samples. Teratozoospermic samples (in which > 90% of the spermatozoa exhibited abnormal morphology) were similar in thiol–disulfide status to normozoospermic samples, but contained non-protamine basic proteins in addition to protamines. Spermatozoa in asthenozoospermic samples (in which > 90% of the spermatozoa were immotile) contained lower amounts of SH than did those of normozoospermic samples; the total (SH plus SS) in the asthenozoospermic samples was similar to that in the normozoospermic samples, as shown by microscopy, flow cytometry and gel electrophoresis. The significantly lower SH/(SH plus SS) was evident in most sperm proteins, including the protamines, of the asthenozoospermic samples. This 'over oxidation' of sperm thiols may result from an abnormal maturation in the epididymis or from an effect of the seminal plasma.
Search for other papers by A Talmor-Cohen in
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Search for other papers by R Tomashov-Matar in
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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.
Search for other papers by R Tomashov-Matar in
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Search for other papers by R Shalgi in
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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.
