Search

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca(2+) downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca(2+) signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca(2+) and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca(2+) influx through either voltage-gated or non-voltage-gated Ca(2+) channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca(2+) signaling and upregulation of adhesion, consistent with involvement of PLC-gamma. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-gamma2, but not PLC-gamma1 nor PLC-beta1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-gamma2, but not PLC-gamma1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-gamma to initiate phosphoinositide signaling and intracellular Ca(2+) mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-gamma could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is down regulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O(2) ( approximately 2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O(2) upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O(2), signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinase-mediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O(2) and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O(2) rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.

Background: Ethanol exposure during gastrulation and early neurulation induces apoptosis within certain embryonic cell populations, leading to craniofacial and neurological defects. There is currently little information about the initial kinetics of ethanol-induced apoptosis, and interest in the ability of endogenous survival factors to moderate apoptosis is growing. Ethanol alters intracellular signaling, leading to cell death in chick embryos, suggesting that apoptosis could occur rapidly and that signaling pathways activated by survival factors might reduce apoptosis., Methods: Pregnant mice were intubated with 1, 2, or 4 g/kg ethanol on day 7.5 of embryogenesis (E7.5) 1, 3, or 6, hours before harvesting gastrulation-stage embryos. Control animals received maltose/dextran. Blood alcohol concentrations (BAC) were determined by gas chromatography. E7.5 embryos isolated from untreated dams were cultured in vitro for 1 or 3 hr with 0 or 400 mg% ethanol and 0 or 5 nM heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). Apoptosis was quantified using fluorescence microscopy to detect annexin V binding and DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)] in whole-mount or sectioned embryos., Results: Both annexin V binding and TUNEL were elevated (p < 0.05) in embryos exposed in utero to 1 g/kg ethanol for 3 hours, increasing linearly with time and ethanol concentration. Apoptosis increased (p < 0.05) in all germ cell layers. Mice treated with 4 g/kg sustained BAC of 400 mg% for nearly 3 hours, significantly increasing apoptosis within the first hour. Cultured embryos exposed to 400 mg% ethanol displayed 2- to 3-fold more TUNEL than vehicle-treated embryos (p < 0.05); however, exogenous HB-EGF prevented apoptosis., Conclusions: Ethanol rapidly produced apoptosis in gastrulation-stage embryos, consistent with induction by intracellular signaling. The ethanol-induced apoptotic pathway was blocked by the endogenous survival factor, HB-EGF. Differences in the expression of survival factors within individual embryos could be partly responsible for variations in the teratogenic effects of ethanol among offspring exposed prenatally.

Experimental models of blastocyst development based on in vitro culture have played a prominent role in advancing our understanding of peri-implantation development, a process that is relatively inaccessible in vivo. Blastocyst culture provides a robust approach for examining embryonic interactions with the microenvironment under highly controlled conditions. Major events that occur in utero can be followed in vitro, including blastocyst expansion, hatching, and adhesion to extracellular matrices. This chapter will describe a method for obtaining and culturing mouse blastocysts. Morphological changes that occur during blastocyst culture will be discussed and related to the corresponding development in utero. Finally, quantitative assays will be detailed for monitoring peri-implanatation development of the trophoblast in vitro.

Objective: Toll-like receptors (TLRs) recognize microbial ligands and host products that are released during tissue damage, the so-called "danger signals." This study was conducted to determine whether changes in TLR-4 and TLR-2 expressions can be detected in the trophoblasts at the placental bed of women with and without preeclampsia., Study Design: Placental bed biopsy specimens were obtained from women with: (1) normal term pregnancies with and without labor (each n = 20); (2) preeclampsia who delivered preterm (n = 15); and (3) preterm labor and intact membranes with and without chorioamnionitis (each n = 15). The expression pattern of TLR-4 and TLR-2 in the trophoblasts was analyzed by double immunohistochemistry., Results: (1) The median percentage of TLR-4 positive interstitial trophoblasts was significantly higher in patients with preeclampsia than in patients with preterm labor without or with histologic chorioamnionitis (P = .0002 and P = .02, respectively). (2) The median percentage of TLR-2 positive interstitial trophoblasts was not different among the study groups (P>.05). (3) TLR-4 positive trophoblasts were also frequently immunoreactive to activated nuclear factor-kappaB, tumor necrosis factor-alpha, and M30 (a specific apoptosis antigen for trophoblast). (4) Lipopolysaccharide treatment inhibited the migration of trophoblast cell lines in vitro., Conclusion: TLR-4 protein expression is increased in interstitial trophoblasts of patients with preeclampsia. We propose that "danger signals" at the feto-maternal interface, which are recognized by trophoblasts through TLR-4, may play a key role in the creation of a local abnormal cytokine milieu. This suggests a novel mechanism that links the activation of the innate immune system through TLR-4 and preeclampsia.

The preimplantation embryo floats freely within the oviduct and is capable of developing into a blastocyst independently of the maternal reproductive tract. While establishment of the trophoblast lineage is dependent on expression of developmental regulatory genes, further differentiation leading to blastocyst implantation in the uterus requires external cues emanating from the microenvironment. Recent studies suggest that trophoblast differentiation requires intracellular signaling initiated by uterine-derived growth factors and integrin-binding components of the extracellular matrix. The progression of trophoblast development from the early blastocyst stage through the onset of implantation appears to be largely independent of new gene expression. Instead, extrinsic signals direct the sequential trafficking of cell surface receptors to orchestrate the developmental program that initiates blastocyst implantation. The dependence on external cues could coordinate embryonic activities with the developing uterine endometrium. Biochemical events that regulate trophoblast adhesion to fibronectin are presented to illustrate a developmental strategy employed by the peri-implantation blastocyst.

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.

Blastocyst implantation and placentation require molecular and cellular interactions between the uterine endometrium and blastocyst trophectoderm. Previous studies showed that histamine produced in the mouse uterine luminal epithelium interacts with trophoblast histamine type-2 receptors (H2) to initiate blastocyst implantation. However, it is unknown whether similar histamine activity is operative in humans. Using a human cell line (HTR-8/SVneo) derived from first-trimester cytotrophoblasts that expresses both histamine type-1 receptor (H1) and H2, we found that histamine promotes cytotrophoblast invasiveness specifically through activation of H1. Stimulation of H1 in human cytotrophoblasts by histamine induced intracellular Ca2+ (Ca(2+)i) transients by activating phospholipase C and the inositol trisphosphate pathway. The enhanced invasion induced by histamine was blocked by pretreatment with H1 antagonist or by chelation of Ca(2+)i. These findings suggest possible differences between rodents and humans in histamine signaling to the trophoblast., (Copyright 2004 Wiley-Liss, Inc.)

Transient elevation of intracellular calcium (Ca2+(i)) by various means accelerates murine preimplantation development and trophoblast differentiation. Several G-protein-coupled receptors (GPCRs), including the lysophosphatidic acid (LPA) receptor (LPAR), induce Ca2+(i) transients and transactivate the EGF receptor (ErbB1) through mobilization of EGF family members, including heparin-binding EGF-like growth factor (HB-EGF). Because HB-EGF accelerates blastocyst differentiation in vitro, we examined whether crosstalk between LPA and HB-EGF regulates peri-implantation development. During mouse blastocyst differentiation, embryos expressed LPAR1 mRNA constitutively, LPAR2 only in late stage blastocysts and no LPAR3. Consistent with a mechanism based on Ca2+(i) signaling, LPA rapidly accelerated the rate of trophoblast outgrowth, an index of blastocyst differentiation, and chelation of Ca2+(i) with BAPTA-AM blocked LPA stimulation. Interfering with HB-EGF signaling through ErbB1 or ErbB4 also attenuated LPA stimulation. We established that mouse blastocysts indeed express HB-EGF and that LPA induces the transient accumulation of HB-EGF on the embryo surface, which was blocked by treatment with either BAPTA-AM or the protein trafficking inhibitor, brefeldin A. We conclude that LPA accelerates blastocyst differentiation through its ability to induce Ca2+(i) transients and HB-EGF autocrine signaling. Transactivation of ErbB1 or ErbB4 by HB-EGF could represent a convergent signaling pathway accessed in the trophoblast by stimuli that mobilize Ca2+(i).

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.

Cytotrophoblasts of the anchoring villi convert during human placentation from a transporting epithelium to an invasive, extravillous phenotype that expresses a distinct repertoire of adhesion molecules. Developing extravillous trophoblasts accumulate heparin-binding EGF-like growth factor (HB-EGF), a multifunctional cytokine, which binds HER1 and HER4 of the human EGF receptor (HER/ErbB) family. HB-EGF is downregulated in placentae of women with preeclampsia, a disorder associated with deficient trophoblast invasion, raising important questions about its physiological impact on cytotrophoblasts. Addition of HB-EGF during explant culture of first-trimester chorionic villi enhanced extravillous trophoblast differentiation and invasive activity. Using a first-trimester human cytotrophoblast line, the potential for autocrine and paracrine regulation of the developing trophoblast was established based on the expression of all four HER isoforms, as well as HB-EGF and related growth factors. HB-EGF did not alter proliferation, but initiated extravillous differentiation, with decreased alpha6 integrin expression, increased alpha1, and elevated cell migration. Function-blocking antibodies against EGF family members reduced basal cell motility and antibody inhibition of either HER1 or HER4 ligation prevented HB-EGF-induced integrin switching. We conclude that HER-mediated autocrine and paracrine signaling by HB-EGF or other EGF family members induces cytotrophoblast differentiation to an invasive phenotype.

Background: Although the ability of ethanol to elicit neural crest cell apoptosis is well documented, the initial target of ethanol in these cells, and the biochemical pathway leading to their apoptosis, have yet to be determined. Recent work in preimplantation mouse embryos demonstrates that ethanol induces a phospholipase-C (PLC)-dependent calcium transient that mediates ethanol's effects. We tested whether a similar effect on calcium and PLC is involved in ethanol-induced neural crest apoptosis., Methods: Chicken embryos were collected and loaded with Fluo-3-AM to assess the effects of ethanol on intracellular calcium levels. Pharmacological agents were used to determine the sources and mechanism of intracellular calcium increases. In separate experiments, embryos were treated in ovo with pharmacological modulators of calcium signaling prior to ethanol exposure, and resulting levels of cell death were assessed by using the vital dye acridine orange., Results: Ethanol exposure caused a localized increase in intracellular calcium levels in embryonic neural folds within 15 sec of ethanol exposure. Ethanol-induced apoptosis was specifically blocked by chelation of intracellular calcium before ethanol exposure. Pretreatment with the PLC inhibitor U73122 blocked ethanol-induced apoptosis as well as the intracellular calcium transient. Depletion of extracellular calcium resulted in a partial block of ethanol-induced apoptosis., Conclusions: Ethanol exposure alters calcium signaling within the neurulation-stage chicken embryo in a PLC-dependent manner. Increases in intracellular calcium and PLC activity are necessary for ethanol's induction of apoptosis within cephalic populations. These effects likely represent an early and crucial event in the pathway leading to ethanol-induced cell death.

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4-5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst.

Key to the maternal-embryonic dialogue during early implantation are the extra embryonic trophoblast cells that play several critical roles including invasion of the maternal decidua. Cytotrophoblast proliferation and differentiation along an invasive pathway is regulated by a positive gradient of oxygen tension. We have employed transcript profiling to characterize the extra-embryonic tissues during early human implantation. We probed a cDNA library isolated from the ectoplacental cone region of stage 7.5 days post coitus mouse embryos with cDNA from hypoxic cultured human trophoblast cells. This identified the pattern of expression of a series of previously unknown genes that correlate to day 20 of human embryonic development. One EST was selected for further analysis since it is identical to the 1588-bp CGI-15 sequence assembled in silico. The human gene is organized into 10 exons 2063 bp in length. The open reading frame contains 1272 bp predicting protein composed of 365 amino acids. This gene is expressed in many tissues including those found in the reproductive tract. Its expression is regulated by oxygen tension and is unaffected by estradiol or progesterone treatment.

Background: Pre-eclampsia is a disorder of pregnancy associated with poor extravillous cytotrophoblast invasion and above-normal rates of apoptosis in the trophoblast. Heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) has strong cytoprotective activity and is an important signalling protein that regulates trophoblast invasion during early placentation. We aimed to establish whether HB-EGF expression is altered in placentae of pre-eclamptic women., Methods: We assessed the expression of HB-EGF mRNA and protein by in-situ hybridisation and immunohistochemical techniques, respectively, in archived placental tissues from pregnancies terminated at around 20 weeks of gestation, and from women delivering between weeks 19 and 35 of gestation with preterm labour, small for gestational age infants, or pre-eclampsia., Findings: HB-EGF mRNA and protein were expressed in villous and extravillous cytotrophoblast cells up to week 35 of gestation in placentae from women who delivered preterm. Similar levels of HB-EGF protein were found in the placentae of women who were not in labour. HB-EGF expression was reduced about five-fold (p=0.0001) in pre-eclamptic pregnancies. Fetal growth retardation, which has been linked with shallow trophoblast invasion and moderate apoptosis, was associated with placentae expressing intermediate levels of HB-EGF., Interpretation: In pre-eclampsia, deficient HB-EGF signalling during placental development could impair trophoblast survival, differentiation, and invasion, leading to poor placental perfusion and hypertension.

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.

Accumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In previous studies of adhesion-competent mouse blastocysts, we have demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. In the present study, we have examined whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. The strengthening of adhesion to FN required integrin ligation, which rapidly elevated cytoplasmic-free Ca(2+). Chelation of intracellular Ca(2+) using BAPTA-AM, or inhibition of the Ca(2+)-dependent proteins, protein kinase C or calmodulin, significantly attenuated the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca(2+) levels with ionomycin upregulated FN-binding activity, demonstrating that Ca(2+) signaling is required and sufficient for strong adhesion to FN. Ca(2+) signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca(2+), which strengthens trophoblast adhesion to ECM through protein redistribution.

Alcohols and aldehydes are metabolized primarily by alcohol (ADH) and aldehyde (ALDH) dehydrogenase isozymes. Although significant progress has been made towards understanding the involvement of these isozymes in the oxidation of alcohol and aldehydes in the body, it is not known how these compounds are handled during fertilization and preimplantation embryogenesis. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used to determine which ADH and ALDH isozymes are expressed at the oocyte, zygote, morula, and blastocyst stages of preimplantation development in the mouse. Transcripts of beta-actin and vimentin, assayed as controls, were detected at all stages, as well as Class III ADH (Adh-2) and Class 3 ALDH (Ahd-4), involved in the detoxification of formaldehyde and aromatic aldehydes, respectively. In contrast, transcripts for the major ethanol oxidizing isozyme, Class I ADH (Adh-1) was not detected during preimplantation development. Cytosolic retinol dehydrogenase (Adh-3) transcripts were marginally detected in oocytes and zygotes. The mRNA for cytosolic retinal dehydrogenase (Ahd-2), microsomal short-chain retinol dehydrogenases (RoDH Type I), and the mitochondrial low-Km acetaldehyde dehydrogenase (Ahd-5) only appeared as maternal transcripts. Microsomal ALDH (Ahd-3), which is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was not expressed until the blastocyst stage. ADH and ALDH enzyme systems may guard mouse preimplantation embryos against the toxic effects of industrial pollutants, such as formaldehyde and TCDD, as well as peroxidatic aldehydes generated during lipid peroxidation. The absence of enzymes to convert ethanol to acetaldehyde, coupled with oocyte expression of the acetaldehyde-degrading enzyme, Ahd-5, may be protective for the early embryo.

Blastocyst implantation in rodents and primates depends on adhesive interactions between trophoblast cells and the endometrial extracellular matrix. As the blastocyst contacts the uterine basal lamina, cells of the trophectoderm become adhesion-competent and conclude their phenotypic conversion from a polarized epithelium to invasive trophoblast cells that anchor the embryo in the uterine wall and eventually infiltrate the endometrium. Trophoblast cells become capable of adhesion to fibronectin as alpha(5)beta(1) integrins traffick to their apical surfaces. While integrin trafficking may be required for trophoblast adhesion to the endometrium, accumulating evidence indicates that, in response to contact with the extracellular matrix, additional molecules must be recruited to the apical surface before strong adhesion is attained. Based on the known interactions of integrins with cytoplasmic regulatory proteins, we propose that extracellular matrix-bound integrins mediate intracellular signaling cascades that strengthen their adhesive activity. We will review some of the known integrin signaling pathways that could regulate trophoblast adhesion and differentiation., (Copyright 2002 S. Karger AG, Basel)

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.

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., (Copyright 2000 Harcourt Publishers Ltd.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

During in vitro culture of murine preimplantation embryos, we have observed that exposure to 0.1% ethanol induces an immediate increase in intracellular calcium levels and subsequently accelerates embryogenesis. If the observed effects of ethanol on developing embryos is mediated by its membrane disordering potency, we hypothesized that the relative membrane disordering potencies of related alcohols would correspondingly effect embryonic intracellular calcium levels and developmental rates. Two-cell embryos were exposed to 0.1% ethanol or 0.05 to 1.0% (w/v) n-butanol, n-propanol, isopropanol, 1,2-propanediol, glycerol, or methanol for 24 hr at 37 degrees C, and development to the blastocyst stage was monitored after 5 days. n-Butanol, n-propanol, isopropanol, and methanol treatment caused a dose-dependent inhibition (p < 0.01) of development to the blastocyst stage, whereas 1,2-propanediol or glycerol neither accelerated nor inhibited development. In a second experiment, 8-cell morulae were treated with 1,2-propanediol or glycerol, and cavitation rates were examined. There was no significant difference from control embryos in the onset of cavitation or the blastocoel expansion rate of 1,2-propanediol- or glycerol-exposed embryos, whereas exposure to 0.1% ethanol accelerate cavitation (p > 0.05). In a third experiment, morulae were exposed to 0.1% or 1.0% of each alcohol and were monitored for changes in intracellular calcium levels using the fluorescent indicator, fluo-3-acetoxymethyl ester. There was an immediate increase in intracellular calcium levels when morulae were treated with 1.0% ethanol or n-butanol, but only ethanol induced an increase (p < 0.05) in the level of intracellular calcium at 0.1%. These data suggest that ethanol is unique in its ability to accelerate embryogenesis and that the membrane disordering potency of ethanol does not directly underlie its effects on intracellular calcium release and the acceleration of preimplantation development.

Alcohol is a known teratogen that causes a broad variety of developmental anomalies, including fetal growth retardation, craniofacial anomalies, and neurological disorders. The etiology of this multiple defect syndrome, known as fetal alcohol syndrome, has been studied in animal models that reproduce many of the attributes of the human disease. These studies show that ethanol is most teratogenic during organogenesis and development of the nervous system. The molecular basis of fetal alcohol effects has been further investigated using embryo and cell culture systems. Recent studies show that signal transduction pathways controlling cell proliferation are perturbed during ethanol exposure. Ethanol can induce the release of intracellular calcium stores, which stimulates the cell cycle, and it also up-regulates the expression of myc proteins associated with cell proliferation. Increased proliferation is advantageous during the preimplantation period, but ethanol interference with terminal differentiation events within developing tissues during organogenesis may underlie alcohol teratogenicity.

In order to understand more about the reproductive physiology of the zebra mussel (Dreissena polymorpha), we have examined the effect of spawning on the kinematics and fertilization ability of their spermatozoa. Sperm morphology was characterized using scanning electron microscopy, differences in sperm kinematics were examined using CASA following manual collection of sperm from the gonad or spawning induced by 5-hydroxytryptamine (5-HT), and the fertilization ability of spawned and manually removed spermatozoa were compared. D. polymorpha sperm have a tail 50 microns long, a midpiece consisting of four spherical mitochondria-rich regions approximately 0.6 micron in diameter, a conical nucleus 2.52 microns in length, and a cylindrical acrosome 0.72 micron in height and width. Kinematic analysis revealed that manually removed sperm exhibit significantly lower motion characteristics as compared to spawned sperm. Treatment with 5-HT had no significant effect on the sperm kinematics of either manually removed or spawned spermatozoa. These data suggest that D. polymorpha spermatozoa have a similar morphology to other bivalve sperm, spawning results in a significant enhancement in sperm kinematics, and 5-HT does not directly alter sperm motion.

Lysolecithin was used to permeabilize embryonic cells to impermeant compounds without compromising embryo viability. Within 2 min of exposure to 0.05% lysolecithin, mouse morulae became permeable to inositol 1,4,5-trisphosphate, leading to the release of calcium from intracellular stores. Although these morulae remained permeable for at least 2 h, they developed normally to the blastocyst stage during subsequent culture in vitro. Lysolecithin permeabilization was then used to potentiate the internalization of alpha-amanitin. Pretreatment with lysolecithin for 2 min markedly accelerated the onset of transcriptional inhibition from 3 h to 10 min after alpha-amanitin addition. The rapid inhibition by alpha-amanitin of mRNA synthesis in lysolecithin-treated embryos provided a precise method for delineating developmentally important transcriptional periods. Using this protocol, we found that mRNAs required for embryonic cavitation were synthesized between 87 and 91 h post-hCG, shortly before blastocoel formation commenced.

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

Implanting mouse blastocysts adhere through their abembryonic surfaces to the endometrial extracellular matrix. Because blastocysts cultured on fibronectin in vitro dissociate to form trophoblast outgrowths, it is unclear whether this adhesion is initially mediated by fibronectin receptors on the apical or basolateral surface of the trophectoderm. Intact blastocysts were examined in a ligand binding assay utilizing the fibronectin cell binding domain attached to fluorescent microspheres. Fibronectin binding activity on the apical surface of the trophectoderm was confined to the abembryonic pole of the blastocyst, where trophoblast differentiation initiates, and was regulated temporally in accordance with blastocyst outgrowth. Soluble fibronectin (IC50 = 0.2 microM) or Gly-Arg-Gly-Asp-Ser-Pro, but not laminin, competitively inhibited fibronectin binding activity. Addition of antibodies against the alpha v, alpha 5, beta 1, or beta 3 integrin subunits also inhibited binding activity. Blastocysts cultured in the absence of an adhesive substratum exhibited fibronectin binding activity only after exposure to immobilized or soluble ligand. Potentiation of binding activity by ligand was unaffected by cycloheximide but was sensitive to brefeldin A inhibition of protein trafficking. These findings suggest that the interaction of fibronectin with the trophectoderm induces a translocation event that up-regulates fibronectin binding beta 1- and beta 3-class integrins on the apical surface.

Objective: To assess the effect of modifications made in the Micro-Cell sperm counting chamber on the motility of washed human spermatozoa., Design: In a 2 x 2 experimental design, human sperm samples were washed with or without protein supplementation, loaded into modified or unmodified Micro-Cell chambers, and assessed by automated semen analysis for changes associated with sperm adhesion to the glass chamber surfaces., Participants: Twenty-one men who were undergoing semen analysis and presented with > or = 50% motile sperm, or normal donors., Main Outcome Measures: Reduced motility and elevated lateral head displacement associated with sperm adhesion to glass surfaces were compared using a doubly repeated measures analysis of variance., Results: Addition of protein to the sperm washing solution partially reversed the adhesion of spermatozoa to the glass of unmodified Micro-Cell chambers. Chambers manufactured to reduce cell adhesion to glass surfaces yielded the highest motility and lowest lateral head displacement, whether the sperm were washed in a solution supplemented with or without protein., Conclusion: These findings indicate that, as in raw semen, kinematics of washed sperm can be measured reliably in the modified Micro-Cell chamber.

Sperm motility patterns of cryopreserved domestic cat ejaculates treated at 37 degrees C with 1 mM caffeine, pentoxifylline, or 2'-deoxyadenosine were analyzed using computer-assisted semen analysis (CASA). The percent motility (MOT), curvilinear velocity (VLC), straight-line velocity (VSL), linearity (LIN), and amplitude of lateral head displacement (ALH) were measured for each group following 15 min of treatment. Motility indices were examined during a 6-h treatment period to determine the effect of each chemical on sperm longevity. Caffeine, pentoxifylline, and 2'-deoxyadenosine each increased (p > .05) the MOT and VCL of the ejaculates compared to the controls. The longevity of the treated and control samples were not significantly different throughout the incubation period. These results, similar to previous findings with cryopreserved epididymal cat sperm, demonstrate that motility stimulants can significantly elevate the MOT and VCL of cryopreserved ejaculated cat sperm without having deleterious effects on longevity.

This study focused on the effects of ethanol on blastocyst outgrowth and implantation in mice. Blastocysts were exposed to ethanol in Ham's F10 medium and then cultured free of ethanol on fibronectin-coated Petri dishes to assess trophoblast cell adhesion and migration. The time necessary for half of the embryos to outgrow was significantly less (P < 0.05) following treatment with 0.1%, 0.2%, 0.4% or 1.0% (w/v) ethanol for either 5 min or 24 h compared with controls. The rate of trophoblast cell migration was determined by measuring the mean area of outgrowing embryos using an image analysis system. Blastocysts exposed to ethanol for 5 min produced a greater (P < 0.05) average outgrowth area than did stage-matched controls. Acceleration of blastocyst cavitation by ethanol is known to be associated with an increase in the intracellular concentration of calcium. Here, treatment with the calcium ionophore A23187 stimulated (P < 0.05) trophoblast outgrowth and accelerated (P < 0.05) the rate of cell migration. In an attempt to correlate the effect of ethanol on outgrowth in vitro with implantation in utero, cultured blastocysts were either not exposed to ethanol or exposed to 0.1% ethanol for 5 min and transferred 24 h later to the uteri of pseudopregnant dams. The implantation rate (39.4%, n = 376) and the rate of development to term (2.45 pups per mouse, n = 20) were higher (P < 0.05) in mice receiving ethanol-treated embryos compared with those receiving control embryos (20.8%, n = 331; 1.16 pups per mouse, n = 18, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the invasive activity of mouse trophoblast cells during embryo implantation in vitro by culturing blastocysts with extracellular matrix (ECM) purified from mouse endometrium obtained on day 4 of pregnancy. Endometrium was dissected from lyophilized mouse uteri, and intact ECM was isolated by sequential precipitation in nonionic detergent and high salt. Electron microscopic examination of the ECM revealed typical collagen fibers plus an amorphous material resembling basement membrane. Electrophoretic analysis of the ECM revealed an enrichment of high molecular weight proteins, and immunoblotting indicated the presence of fibronectin, laminin, entactin, and type IV collagen, but not the intracellular proteins 2',3'-cyclic nucleotide-3'-phosphodiesterase or vimentin. Mouse blastocysts cultured with this ECM attached to it within 3 days, and the trophoblast cells began to migrate through the matrix in a manner resembling trophoblast invasion in utero. Unlike blastocysts cultured on plastic surfaces, the trophoblast did not flatten and become disorganized, but retained a polarized, spherical structure. Fluorescent microscopy with fluorescein isothiocyanate-labeled phalloidin revealed a high degree of microfilament organization and established that actin was absent from the ECM preparation. In the presence of a serum substitute, differentiation continued through yolk sac formation. Without serum components, yolk sac did not form; however, light and electron microscopic examination indicated that the invasive behavior of trophoblast cells persisted and was comparable to that of trophoblasts cultured in the presence of the serum substitute. A three-dimensional model for investigating trophoblast behavior in ECM from the endometrium should be of great value in elucidating the cellular and molecular events surrounding the process of blastocyst implantation.

Objective: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability., Design and Methods: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test., Results: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes., Conclusion: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

We have investigated the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on the motion characteristics and longevity of domestic cat spermatozoa. Freshly collected or cryopreserved domestic cat epididymal sperm were incubated with 0.01-20 mM caffeine, pentoxifylline, or 2'-deoxyadenosine for 15 minutes at 23 degrees C. The percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were determined for each group using computer-assisted semen analysis. Freshly collected domestic cat sperm exhibited a strong forward progressive movement, and treatment with caffeine, pentoxifylline, or 2'-deoxyadenosine did not consistently alter sperm motion. Following cryopreservation, spermatozoa exhibited decreased (P < 0.05) MOT, VCL, VSL, and ALH. Caffeine and pentoxifylline increased (P < 0.05) the MOT, VSL, VCL, and ALH of cryopreserved sperm at 0.01-20 mM, in a dose-dependent manner. 2'-Deoxyadenosine also increased (P < 0.05) both VSL and VCL at 1.0 mM, and MOT, VSL, VCL, and ALH at 10 mM. All treatments shifted the percentage of nonhyperactive sperm to either a transitional or hyperactivated state. The motility indices of cryopreserved samples were examined during a 6-hour incubation to assess the effects of caffeine, pentoxifylline, and 2'-deoxyadenosine on sperm longevity. Compared to untreated control samples, the longevity of stimulated cryopreserved sperm was not reduced. These results indicate that motility stimulants may prove useful for enhancing the fertility of cryopreserved cat sperm by increasing their motility and producing hyperactivated motion.

To extend our previous finding that ethanol stimulates murine preimplantation development, we focused in the current study on the cavitation and expansion of the blastocyst. Ethanol stimulation of blastocyst cavitation and expansion was optimal at a concentration of 0.1% and required only a 5-min exposure. Because intracellular levels of calcium were transiently increased in the ethanol-exposed embryos, we determined the effect of directly increasing calcium on the rates of blastocyst cavitation and expansion. Treatment with the calcium ionophore, A23187, altered development much as did ethanol, indicating that ethanol may stimulate preimplantation development by increasing the intracellular calcium concentration. The relationship between intracellular calcium levels and blastocoel volume suggests that morphogenetic events during preimplantation development, particularly cavitation and blastocyst formation, may be regulated through modulation of intracellular calcium levels.

The two M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3 alpha, but not ZP3 beta, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3 alpha. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1 microgram/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-beta-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3 alpha was an at least 100-fold better antagonist than purified ZP3 beta. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3 alpha macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.

Motion characteristics of epididymal sperm from domestic cats exhibiting a high (> 60%; normozoospermic; n = 21) or low (< 40%; teratozoospermic; n = 6) occurrence of structurally normal spermatozoa were correlated with morphology (MOR) using computer-assisted semen analysis (CASA). Mean values and standard errors for percent motility (MOT), curvilinear velocity (VCL), linearity (LIN), straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were recorded for 3 hours. Average values for percent normal spermatozoa, MOT, VCL, VSL, and ALH were higher (P < 0.01) in samples from normozoospermic cats than from teratozoospermic cats at 0 hours, and there was no difference in motion parameters over the 3-hour incubation period in either group. Strong correlations (P < 0.01) existed between MOR and VCL, VSL, ALH, or MOT, but not LIN, upon regression analysis. We conclude that (1) motion parameters of domestic cat sperm are significantly correlated with morphology and (2) abnormal motion parameters associated with low fertility potential in other species are prevalent in samples from teratozoospermic cats. The correlation between morphology and altered sperm movement found in this study suggests that motion analysis of spermatozoa by CASA may be useful in evaluating fertilization potential in felids.