Reduced expression of the epidermal growth factor signaling system in preeclampsia.

INTRODUCTION: The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS: Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS: Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION: Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.

Nuclear matrix association: switching to the invasive cytotrophoblast.

Abnormal trophoblast invasion is associated with the most common and most severe complications of human pregnancy. The biology of invasion, as well as the etiology of abnormal invasion remains poorly understood. The aim of this study was to characterize the transcriptome of the HTR-8/SVneo human cytotrophoblast cell line which displays well characterized invasive and non-invasive behavior, and to correlate the activity of the transcriptome with nuclear matrix attachment and cell phenotype. Comparison of the invasive to non-invasive HTR transcriptomes was unremarkable. In contrast, comparison of the MARs on chromosomes 14-18 revealed an increased number of MARs associated with the invasive phenotype. These attachment areas were more likely to be associated with silent rather than actively transcribed genes. This study supports the view that nuclear matrix attachment may play an important role in cytotrophoblast invasion by ensuring specific silencing that facilitates invasion.

Inner cell mass localization of NANOG precedes OCT3/4 in rhesus monkey blastocysts.

The mechanism by which the inner cell mass (ICM) and trophectoderm (TE) become specified is poorly understood. Considerable species variation is evident in the expression of lineage-specific and embryonic stem cell (ESC) regulatory markers. We sought to investigate localization patterns of these markers in rhesus macaque compact morulae and blastocysts. NANOG protein was restricted to the ICM of blastocysts. In contrast to a previous report, the expression of CDX2 was detected in the primate blastocyst, localized specifically to the TE. Unlike the mouse embryo, OCT4 protein was detected using two different antibodies in both the ICM and TE. The ubiquitous pattern of OCT4 expression is consistent with observations in human, cow, and pig embryos. Significantly, lack of restricted OCT4 protein, and ICM localization of NANOG in primate blastocysts, suggests that NANOG may determine inner cell mass fate more specifically during primate development or may be less susceptible to culture artifacts. These results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of NANOG to constitute a complex interactive network. This difference may also underlie observations that regulatory mechanisms in ESC differ between mice and primates.

Expression of heparin-binding EGF-like growth factor in term chorionic villous explants and its role in trophoblast survival.

Heparin-binding EGF-like growth factor (HBEGF) induces trophoblast extravillous differentiation and prevents apoptosis. These functions are compromised in preeclampsia. Because HBEGF is downregulated in placentas delivered by women with preeclampsia, we have examined its expression and cytoprotective activity in term villous explants. Chorionic villous explants prepared from non-pathological placentas collected by cesarean section at term were cultured at either 20% or 2% O2 and treated with the HBEGF antagonist CRM197 or recombinant HBEGF. Paraffin sections were assayed for trophoblast death, proliferation and HBEGF expression using the TUNEL method, immunohistochemistry for nuclear Ki67 expression and semi-quantitative immunohistochemistry with image analysis, respectively. Trophoblast cell death was increased significantly after 8h of culture with CRM197 or by culture for 2h at 2% O2. Exogenous HBEGF prevented cell death due to hypoxia. Proliferative capacity was not affected by culture at either 20% or 2% O2. Contrary to first trimester placenta, term trophoblasts do not elevate HBEGF expression in response to hypoxia. However, low endogenous levels of HBEGF are required to maintain survival. Therefore, HBEGF-mediated signaling significantly reduces trophoblast cell death at term and its deficiency in preeclampsia could negatively impact trophoblast survival.

Regulation of trophoblast invasion - a workshop report.

Trophoblast invasion during placental development helps to establish efficient physiological exchange between maternal and fetal circulatory systems. Trophoblast stem cells differentiate into multiple subtypes, including some that are highly invasive. Signalling to the trophoblast from decidua, uterine natural killer cells and vascular smooth muscle can regulate extravillous trophoblast differentiation. Important questions remain about how these cellular interactions promote trophoblast invasion and the signalling pathways that are involved. New and established biological models are being used to experimentally examine these interactions and the underlying molecular mechanisms.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Protein kinase C stimulates release of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human decidual cells.

OBJECTIVE: Increased concentrations of amniotic fluid matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 have been observed in the context of premature rupture of membranes (PROM) and microbial invasion of the amniotic cavity. However, the source of the stimuli that contribute to the accumulation of these proteins in amniotic fluid remains to be identified. The present study was conducted to investigate MMP-2, MMP-9 and TIMP-1 secretion by decidual cells in response to activated protein kinase C (PKC). METHODS: Decidual cells were isolated from term placentae, grown to confluence and incubated with control media or 10(-11) to 10(-8) mol/l concentrations of phorbol 12-myristate 13-acetate (PMA). Concentrations of MMP-2, MMP-9 and TIMP-1 in the culture supernatant were determined using sensitive and specific immunoassays. Substrate zymography was conducted to confirm MMP-9 assays. RESULTS: PMA induced a concentration-dependent stimulation of release of MMP-9 (control vs. PMA l0(-9) and 10(-8) mol/l; p < 0.01) and TIMP-1 (control vs. PMA 10(-9) and 10(-8) mol/l; p < 0.001), but not MMP-2. A direct positive correlation was observed between MMP-9 and TIMP-1 release (r = 0.645; p < 0.001). Substrate zymography confirmed increased release of MMP-9 in response to PMA (control vs. PMA 10(-8) and PMA 10(-7) mol/l; p < 0.01). CONCLUSIONS: Activation of PKC within the decidua will result in enhanced MMP-9 release, which upon activation could contribute to degradation of matrices within fetal membranes leading to PROM.

Heparin-binding EGF-like growth factor modulation by antiprogestin and CG in the baboon (Papio anubis).

The objectives of this study were to determine whether antiprogestin therapy or the infusion of human CG to mimic blastocyst transit in the baboon alters heparin-binding EGF-like growth factor expression during the window of implantation. During the menstrual cycle, heparin-binding EGF-like growth factor protein accumulation in the glandular epithelium was low in the proliferative phase and increased to maximal expression on d 5 and 10 postovulation. Stromal cells accumulated high levels of heparin-binding EGF-like growth factor in the proliferative phase, which decreased by d 5 postovulation. These transitional changes in both cell types were delayed when cycling baboons were treated with the antiprogestin ZK 137.316 during the luteal phase. The treatment with human CG had no effect on expression of heparin-binding EGF-like growth factor when compared with cycling baboons on d 10 postovulation and was comparable with that observed on d 18 and 22 of pregnancy. However, the superimposition of the antiprogestin with the human CG treatment also decreased expression in the epithelial cells. In summary, heparin-binding EGF-like growth factor accumulation in the epithelial glands is under the influence of progesterone and does not seem to be influenced by the paracrine secretion of trophoblast CG.

Alcohol in pregnancy and neonatal outcome.

Fetal alcohol syndrome (FAS) and other fetal alcohol effects in children are characterized by life-long compromises in growth, health, behaviour and cognitive ability. Most of the structural signs and many behavioural signs of FAS are evident at birth. This review describes the expression of fetal alcohol effects in neonates, including diagnostic criteria, alcohol withdrawal, pregnancy outcome, growth retardation, facial dysmorphology and behavioural outcomes.

Acceleration of trophoblast differentiation by heparin-binding EGF-like growth factor is dependent on the stage-specific activation of calcium influx by ErbB receptors in developing mouse blastocysts.

Heparin-binding EGF-like growth factor (HB-EGF) is expressed in the mouse endometrial epithelium during implantation exclusively at sites apposed to embryos and accelerates the development of cultured blastocysts, suggesting that it may regulate peri-implantation development in utero. We have examined the influence of HB-EGF on mouse trophoblast differentiation in vitro and the associated intracellular signaling pathways. HB-EGF both induced intracellular Ca2+ signaling and accelerated trophoblast development to an adhesion-competent stage, but only late on gestation day 4 after ErbB4, a receptor for HB-EGF, translocated from the cytoplasm to the apical surface of trophoblast cells. The acceleration of blastocyst differentiation by HB-EGF was attenuated after inhibition of protein tyrosine kinase activity or removal of surface heparan sulfate, as expected. Chelation of intracellular Ca2+ blocked the ability of HB-EGF to accelerate development, as did inhibitors of protein kinase C or calmodulin. The absence of any effect by a phospholipase C inhibitor and the requirement for extracellular Ca2+ suggested that the accrued free cytoplasmic Ca2+ did not originate from inositol phosphate-sensitive intracellular stores, but through Ca2+ influx. Indeed, N-type Ca2+ channel blockers specifically inhibited the ability of HB-EGF to both induce Ca2+ signaling and accelerate trophoblast development. We conclude that HB-EGF accelerates the differentiation of trophoblast cells to an adhesion-competent stage by inducing Ca2+ influx, which activates calmodulin and protein kinase C. An upstream role for ErbB4 in this pathway is implicated by the timing of its translocation to the trophoblast surface.

Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line.

During human placentation, extravillous cytotrophoblast cells emerge from chorionic villi contacting the decidua to invade the uterine wall. When isolated from first-trimester placentae, cytotrophoblast cells undergo step-wise differentiation in vitro that recapitulates the phenotypic heterogeneity observed in vivo. We examined a cell line, HTR-8/SVneo, that has been established from human first-trimester cytotrophoblast to determine whether these cells possess some of the unique cytotrophoblast characteristics that have been described previously. Exposure during serum-free culture to hypoxic conditions (2% oxygen concentration) increased HTR-8/SVneo cell proliferation and reduced invasion of a three-dimensional basement membrane (Matrigel). During culture on surfaces coated with individual extracellular matrix proteins, HTR-8/SVneo cells expressed cytokeratin but not the trophoblast-specific major histocompatibility protein, HLA-G. However, HLA-G expression was induced in HTR-8/SVneo cells that contacted Matrigel. Expression of the alpha5 integrin subunit was relatively unaffected by matrix composition, whereas alpha1 was up-regulated and alpha6 was down-regulated after transferring cells to Matrigel. Hypoxia increased alpha6 and decreased both alpha1 and HLA-G expression on Matrigel. HTR-8/SVneo cells retain several important characteristics associated with primary cultures of first-trimester human cytotrophoblast cells, including their altered behavior in response to a changing maternal environment.

Intracellular signaling in the developing blastocyst as a consequence of the maternal-embryonic dialogue.

The success of blastocyst implantation is dependent on signaling between the embryo and the receptive endometrium. Intercellular signaling molecules, which include hormones, growth factors, and cytokines, have been identified that participate in the maternal-embryonic dialogue. These biologically active molecules may target uterine and/or embryonic tissues in a biochemical cascade that coordinates the two developmental programs during implantation. Two notable uterine products are calcitonin and heparin-binding epidermal growth factor-like growth factor, which are both expressed during the receptive phase of the endometrium in humans and in rodent models. We review data that demonstrate the ability of these molecules to accelerate blastocyst differentiation and delineate the respective intracellular signaling pathways that advance the embryonic developmental program. An understanding of the mediators regulating embryonic development in utero and their biochemical mechanisms of the action may provide insights for improvement of embryo culture in vitro prior to blastocyst transfer.

Multiple roles for heparin-binding epidermal growth factor-like growth factor are suggested by its cell-specific expression during the human endometrial cycle and early placentation.

Embryonic expression of the epidermal growth factor (EGF) receptor as well as embryonic and steroid-dependent uterine secretion of its ligand, heparin-binding EGF-like growth factor (HB-EGF), are temporally associated with the period of blastocyst implantation. We examined the temporal cell type-specific expression of HB-EGF in human endometrium during the menstrual cycle by immunohistochemistry and in situ hybridization. Early first trimester implantation sites were also examined to determine HB-EGF protein levels in decidual and fetal tissues. In the endometrial stroma, HB-EGF protein expression increased markedly during the late proliferative phase and then decreased in the early secretory phase. By contrast, luminal and glandular epithelial cells as well as blood vessel endothelium accumulated the protein between midcycle and cycle day 20, with peak expression observed during the period of uterine receptivity for implantation. HB-EGF expression decreased dramatically at the end of the cycle, before menses. Spatiotemporal expression of HB-EGF messenger ribonucleic acid demonstrated a similar pattern. During early pregnancy, HB-EGF immunostaining was noted in the decidua and in both villous and extravillous trophoblast populations. These findings suggest that HB-EGF promotes implantation and trophoblast invasion through paracrine and autocrine signaling as cells penetrate the stroma and displace the arteriole endothelium.

Ethanol elevates c-Myc levels in cultured mouse preimplantation embryos.

A brief exposure to ethanol accelerates the rate of early mouse embryonic development in vitro, increasing blastocyst formation, trophoblast outgrowth, and implantation rates after embryo transfer. The physiological effects of ethanol during preimplantation development are associated with rapid changes in gene expression and apparently arise from the ability of ethanol to elevate cytoplasmic free Ca2+ and alter cellular signaling pathways. The purpose of this study was to examine whether the abundance of c-Myc, a transcription factor that promotes cell proliferation and is required for blastocyst development, is upregulated in mouse blastocysts challenged with ethanol. After exposure of mouse blastocysts to 0.1% (17.5 mM) ethanol, wc determined the levels of: 1) c-Myc mRNA, using reverse transcription and the polymerase chain reaction; and 2) c-Myc protein levels, using specific monoclonal antibodies. Within 10 min of exposure to ethanol, the relative abundance of c-Myc mRNA increased 6-fold, then rapidly returned to baseline levels within 1 hr. As expected, elevation of c-Myc mRNA by ethanol was attenuated in embryos that were first treated with the intracellular Ca2+ chelator, BAPTA-AM. Western blot analysis of solubilized embryos revealed that c-Myc mRNA was translated into a single 62-kD protein that increased in intensity 30 min after treatment with ethanol. Immunocytochemical staining demonstrated that c-Myc was localized exclusively in nuclei and that staining intensity increased significantly after 10 min. Peak levels of c-Myc protein were found 30 min after ethanol exposure and persisted for at least 2 hr. The c-myc proto-oncogene seems to be an immediate early response gene for ethanol that may regulate the transcription of other genes that influence early embryogenesis and growth.

Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development.

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.

Expression of calcitonin receptors in mouse preimplantation embryos and their function in the regulation of blastocyst differentiation by calcitonin.

Calcitonin secretion in the pregnant uterus is tightly regulated by the ovarian hormones, estrogen and progesterone, which limit its expression to a brief period preceding blastocyst implantation. The binding of calcitonin to a G protein-coupled receptor activates adenylate cyclase and elevates cytosolic Ca2+ levels. The acceleration of preimplantation embryonic development that is known to occur upon elevation of intracellular Ca2+ prompted an investigation into calcitonin regulation of blastocyst differentiation. Using reverse transcription and the polymerase chain reaction to estimate the relative abundance of calcitonin receptor mRNA, a 25-fold accumulation of the splice variant, CR-1a, was observed in embryos between the 1-cell and 8-cell stages. Cytosolic free Ca2+ levels were rapidly elevated in embryos at the 4-cell to blastocyst stages after exposure to 10 nM calcitonin. Blastocysts treated for 30 minutes with 10 nM calcitonin differentiated in vitro at an accelerated rate, as assessed by the translocation of alpha5beta1 integrin to the apical surface of trophoblast cells, the corresponding elevation of fibronectin-binding activity and the timing of trophoblast cell migration. Chelation of cytosolic free Ca2+ with BAPTA-AM, but not inhibition of protein kinase A activity by H-89, attenuated the effects of calcitonin on blastocyst development. These findings support the concept that calcitonin secretion within the progesterone-primed uterus and the coordinate expression of CR-1a by preimplantation embryos regulates blastocyst differentiation through receptor-mediated Ca2+ signaling.

Integrin trafficking regulates adhesion to fibronectin during differentiation of mouse peri-implantation blastocysts.

Trophoblast cells of the peri-implantation blastocyst differentiate from a polarized epithelium, the trophectoderm, into invasive cells having an apical surface occupied by integrins that mediate adhesion to the extracellular matrix. Blastocyst differentiation was assessed during serum-free culture using a fibronectin binding assay with intact mouse blastocysts. Fibronectin binding activity became elevated during a 24-h "window" after approximately 72 h of culture. Blastocyst differentiation was unaffected by transcriptional inhibition with alpha-amanitin, however, exposure of cavitating morulae to the drug significantly delayed the onset of maximal fibronectin-binding activity. Inhibition of de novo protein synthesis with cycloheximide delayed development only when added during the first 24 h of blastocyst culture, indicating that proteins required for adhesion to fibronectin were synthesized at least 24 h before blastocyst differentiation was completed. Since blastocyst differentiation did not appear to be regulated temporally by gene expression, the possible role of protein trafficking was investigated using the inhibitor, brefeldin A. Brefeldin A caused a reversible, dose-dependent decrease in fibronectin-binding activity when added to the culture medium between 48 and 72 h of culture. During the period of brefeldin A sensitivity, alpha 5 beta 1 integrin, a major fibronectin receptor, translocated to the apical surface of trophoblast cells, as determined by immunohistochemistry and confocal microscopy. Mouse blastocysts expressed other integrins that recognize the central cell-binding domain of fibronectin, including the alpha v integrins and alpha llb beta 3, but not alpha4 which recognizes the lllCS site. Trafficking of alpha 5 beta 1, and possibly other integrins, to the apical surface of trophoblast cells appears to be a critical step in the differentiation of the mouse blastocyst to an invasive phenotype.

Ethanol-induced intracellular calcium mobilization rapidly alters gene expression in the mouse blastocyst.

The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.

Regulation of blastocoele formation by intracellular calcium release is mediated through a phospholipase C-dependent pathway in mice.

Calcium signaling plays a critical role in the regulation of mouse preimplantation development, in part through the activation of calmodulin. Calcium transients in mouse morulae appear to be generated predominantly through the production of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC). IP3 receptors predominate in mouse embryos as regulators of calcium release. Exposure to the PLC inhibitors ET-18-OCH3 or U73122 resulted in a dose-dependent, reversible inhibition of cavitation, while the inactive analogue U73343 did not alter the rate of cavitation, as compared to that in controls. U73122 inhibited the release of calcium from intracellular stores after exposure to ethanol or lysophosphatidic acid, suggesting that PLC is required for blastocoele formation in the mouse and that calcium signaling may be PLC-dependent. In addition to IP3, PLC activation generates diacylglycerol, which stimulates protein kinase C (PKC) and could also alter the rate of embryonic development. However, activation of PKC with phorbol ester or synthetic diacylglycerol was not sufficient to accelerate development, although embryo culture in medium containing PKC inhibitors did delay cavitation. Exposure to a PKC inhibitor during ethanol-induced calcium signaling did not attenuate the ensuing acceleration of cavitation. These results demonstrate that a PLC-mediated signaling pathway is required for blastocoele formation and that the generation of IP3, but not diacylglycerol, is critical for accelerating preimplantation development.

Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse preimplantation development.

Inositol 1,4,5-trisphosphate can regulate growth and differentiation by modulating the release of intracellular Ca2+ in a variety of cellular systems, and it is involved in oocyte activation. Recent studies suggest that mammalian preimplantation development may also be regulated by the release of Ca2+ from intracellular stores. The rate of cavitation and cell division was accelerated after a transient elevation of intracellular Ca2+ levels was induced in morulae by exposure to ethanol or ionomycin. Embryos exposed to BAPTA-AM, a chelator of intracellular Ca2+, exhibited a brief dose-dependent reduction in basal Ca2+ levels, a temporal inhibition of ionophore-induced Ca2+ signalling and a subsequent delay in blastocoel formation. BAPTA-AM at 0.5 microM did not significantly alter the basal intracellular calcium level, but chelated Ca2+ that was released after ethanol exposure and thereby attenuated the ethanol-induced acceleration of cavitation. BAPTA-AM also inhibited cell division to the 16-cell stage in a dose-dependent manner, which correlated with the inhibition of cavitation. Thimerosal and inositol 1,4,5-trisphosphate significantly elevated the intracellular Ca2+ concentration in mouse morula-stage embryos, providing evidence for the existence of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores. Although caffeine failed to release intracellular Ca2+, ryanodine induced a small biphasic release of Ca2+, suggesting that ryanodine-sensitive Ca2+ stores may also exist in mouse embryos. Morulae exposed to the calmodulin inhibitor W-7 exhibited a dose-dependent delay in blastocoel formation. A 4 hour exposure to 10 microM W-7 did not significantly alter cavitation, but attenuated the ionophore-induced stimulation of blastocoel formation. This finding suggests that the developmental effects produced through Ca2+ signalling are mediated by calmodulin. Our results demonstrate that Ca2+ release in mouse morulae occurs predominantly through the inositol 1,4,5-trisphosphate receptor, and that alteration of intracellular Ca2+ levels can accelerate or delay embryonic growth and differentiation, providing a mechanistic link between the regulation of oocyte and embryonic development.