Regulation of fertilization-initiated [Ca2+]i oscillations in mammalian eggs: a multi-pronged approach

The calcium ([Ca(2+)](i)) oscillations associated with mammalian fertilization and required to induce egg activation occur during M-phase stages of the cell cycle. The molecular mechanisms underlying this regulation remain unproven and may be multi-layered. Type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R-1), which mediate [Ca(2+)](i) release during fertilization, have emerged as key regulatory units because they contain multiple phosphorylation consensus sites and undergo changes in cellular location and mass prior to and following fertilization. Hence, control of IP(3)R-1 function together with regulation of PLCzeta activity, the putative sperm factor, may combine to impart cell cycle and species-specific [Ca(2+)](i) oscillations characteristic of mammalian fertilization.     

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.     

Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation

Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca(2+)]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or injected with several Ca(2+)-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting. Exposure to ethanol or ionomycin, which induce a single [Ca(2+)]i rise, failed to signal down-regulation of IP3R-1. However, [Ca(2+)]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modifies the IP3R without stimulating production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl(2) failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very specific proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1.     

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.     

Ca(2+) oscillations induced by a cytosolic sperm protein factor are mediated by a maternal machinery that functions only once in mammalian eggs

At fertilization in mammals, the sperm activates the egg by inducing a series of oscillations in the intracellular free Ca(2+) concentration. There is evidence showing that this oscillatory event is triggered by a sperm-derived protein factor which diffuses into egg cytoplasm after gamete membrane fusion. At present the identity of this factor and its precise mechanism of action is unknown. Here, we studied the specificity of action of the sperm factor in triggering Ca(2+) oscillations in mammalian eggs. In doing so, we examined the patterns of Ca(2+) signaling in mouse eggs, zygotes, parthenogenetic eggs and maturing oocytes following the stimulation of bovine sperm extracts which contain the sperm factor. It is observed that the sperm factor could induce Ca(2+) oscillations in metaphase eggs, maturing oocytes and parthenogenetically activated eggs but not in the zygotes. We present evidence that Ca(2+) oscillations induced by the sperm factor require a maternal machinery. This machinery functions only once in mammalian oocytes and eggs, and is inactivated by sperm-derived components but not by parthenogenetic activation. In addition, it is found that neither InsP(3) receptor sensitivity to InsP(3) nor Ca(2+) pool size are the determinants that cause the fertilized egg to lose its ability to generate sperm-factor-induced Ca(2+) oscillations at metaphase. In conclusion, our study suggests that the orderly sequence of Ca(2+) oscillations in mammalian eggs at fertilization is critically dependent upon the presence of a functional maternal machinery that determines whether the sperm-factor-induced Ca(2+) oscillations can persist.     

Initiation and organization of events during the first cell cycle in mammals: applications in cloning

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.     

Sperm factor induces intracellular free calcium oscillations by stimulating the phosphoinositide pathway

Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.     

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.     

Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.     

Isoforms of the inositol 1,4,5-trisphosphate receptor are expressed in bovine oocytes and ovaries: the type-1 isoform is down-regulated by fertilization and by injection of adenophostin A.

Mammalian fertilization is characterized by the presence of long-lasting intracellular calcium ([Ca2+]i) oscillations that are required to induce oocyte activation. One of the Ca2+ channels that may mediate this Ca2+ release is the inositol 1,4, 5-trisphosphate receptor (IP(3)R). Three isoforms of the receptor have been described, but their expression in oocytes and possible roles in mammalian fertilization are not well known. Using isoform-specific antibodies against IP(3)R types 1, 2, and 3 and Western analysis, we determined the isoforms that are expressed in bovine metaphase II oocytes and ovaries. In oocytes, all isoforms are expressed, but type 1 is present in overwhelmingly larger amounts and is likely responsible for the majority of Ca2+ release at fertilization. In ovarian microsomes, all three isoforms appear well expressed, suggesting the participation of all IP(3)R isoforms in ovarian Ca2+ signaling. We then investigated whether the reported cessation/reduction in amplitude of fertilization-associated [Ca2+]i oscillations, which is observed as pronuclear formation approaches, corresponded with down-regulation of the IP(3)R-1 isoform. Fertilization resulted in approximately 40% reduction in the amount of receptor by 16 h postinsemination. In addition, injection of adenophostin A, a potent IP(3)R agonist that elicits high-frequency [Ca2+]i oscillations in mammalian oocytes, induced similar reduction in receptor numbers. Together, these data show that 1) the three IP(3)R isoforms are expressed in bovine oocytes; 2) IP(3)R-1 is likely to mediate most of the Ca2+ release during fertilization; 3) its down-regulation may explain the decline in amplitude of sperm-induced [Ca2+]i rises as fertilization progresses toward pronuclear formation; and 4) agonists of the IP(3)R induce down-regulation of the type-1 receptor in oocytes similar to that evoked by fertilization.     

A hypothesis on p34cdc2 sequestration based on the existence of Ca(2+)-coordinated changes in H+ and MPF activities during Xenopus egg activation [corrected]

The entry into, and exit from, mitosis are controlled by a universal M-phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic inter-phase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca(2+)-dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi-regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling.     

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.     

Phosphorylation changes associated with the early cell cycle in Xenopus eggs

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.     

Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division.

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, sperm nuclei remain condensed until the first mitosis. During mitosis of the first cleavage division sperm nuclei decondense, subsequently recondense and are passively displaced to the daughter blastomeres. In the 2-cell embryos sperm nuclei form interphase nuclei which are able to replicate DNA and to condense into discrete chromosomes during the following mitotic division. These observations suggest that the mitotic cytoplasm of 1-cell embryos creates similar conditions for the transformation of sperm nuclei into male pronuclei as the cytoplasm of metaphase II oocytes.     

Maturation-associated increase in IP3 receptor type 1: role in conferring increased IP3 sensitivity and Ca2+ oscillatory behavior in mouse eggs

Maturation of mouse oocytes is accompanied by an increase in sensitivity to inositol 1,4,5-trisphosphate (IP(3))-mediated release of intracellular calcium. To test the hypothesis that the maturation-associated 1.5- to 2.0-fold increase in the mass of the type 1 IP(3) receptor (IP(3)R-1) confers this increase in IP(3) sensitivity, we employed RNA interference to prevent this change in IP(3)R-1 protein level. Microinjection into germinal vesicle (GV)-intact oocytes of dsRNA corresponding to the IP(3)R-1 sequence resulted in a >90% reduction in the amount of maternal IP(3)R-1 mRNA and prevented the maturation-associated increase in the mass of the IP(3)R-1 protein. These injected oocytes matured to metaphase II, and there was no effect on the maturation-associated increases in p34(cdc2)/cyclin B kinase and MAP kinase activities or the global pattern of protein synthesis. IP(3)-induced cortical granule exocytosis was significantly decreased in these eggs when compared with controls previously injected with enhanced green fluorescent protein (EGFP) dsRNA. Following insemination, the IP(3)R-1 dsRNA-injected eggs displayed significantly fewer Ca(2+) transients than controls, and the duration of the first Ca(2+) transient was about half that of controls. These results support the hypothesis that the maturation-associated increase in the mass of IP(3)R-1 confers the increase in IP(3)-sensitivity that is observed following oocyte maturation and is necessary for the proper Ca(2+) oscillatory pattern following insemination.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Mammalian fertilization: from sperm factor to phospholipase Czeta

In mammalian eggs, the fertilizing sperm evokes intracellular Ca2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor (SF), is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP3) underlies the generation of [Ca2+]i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP3, or as an activator of a PLC(s) pre-existing in the egg. This review discusses the role of SF as the molecule responsible for the production of IP3 and the initiator of [Ca2+]i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCzeta, a novel sperm specific PLC.     

Calcium oscillations in Xenopus egg cycling extracts

Cell cycle in various types of cells and in early embryos is often accompanied by transient changes in the concentration of free cytosolic calcium. In the present study, using fluorescent indicator fura-2, we demonstrate that Ca(2+) oscillates cyclically with an amplitude of about 100 nM and a period of mitotic cycle in cell-free Xenopus egg cycling extracts. It peaks in early metaphase just preceding mitotic reactivation of Cdc2 kinase and MAPK and reaches a minimum in interphase. The source of Ca(2+) in the extracts is a particulate fraction containing egg intracellular Ca(2+) stores, since the addition of a calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), induced a transient increase in Ca(2+). The inclusion of heparin, an IP3 receptor antagonist, or ultrafiltration of the extracts prevented Ca(2+)-releasing activity of IP3. The depletion of Ca(2+) in the extracts by the calcium chelator BAPTA resulted in the blockade of cell cycle at different stages, depending on the time of drug administration. The addition of BAPTA late in interphase blocked cell cycle at mitotic entry in prophase, whereas its application in anaphase or telophase blocked the extracts in early interphase. BAPTA administration in metaphase before transition to anaphase brought about a metaphase-like arrest in the cycling extracts. Inhibition of IP3-induced calcium release by heparin also arrested cell cycle progression in the cycling extracts.     

Muscarinic signalling affects intracellular calcium concentration during the first cell cycle of sea urchin embryos

The existence of a response to acetylcholine (ACh) and cholinomimetic drugs in sea urchin eggs and zygotes was investigated in two sea urchin species: Paracentrotus lividus and Lytechinus pictus. The calcium sensitive fluorescent probe, Fura-2 dextran, was employed to investigate the regulation of cytosolic free calcium concentration ([Ca(2+)](i)) by cholinomimetic drugs in unfertilised and fertilised eggs of both the sea urchin species. Exposure to cholinomimetic agonists/antagonists, either extracellularly or intracellularly, had no effect either on resting [Ca(2+)](i) levels in the unfertilised sea urchin egg, or on the transient [Ca(2+)](i) increase at fertilisation. However, following fertilisation, extracellular application of ACh receptors agonists, such as ACh and carbachol, predominantly muscarinic agonist, but not nicotine, induced a significant increase in [Ca(2+)](i), which was partially inhibited by atropine. As a consequence of exposure after fertilisation to the agonists of ACh receptors, chromatin structure was transiently affected. The hypothesis is proposed that muscarinic receptors may be involved in the (presumably Ca(2+)-dependent) modulation of the nuclear status during the first cell cycles.