Intracellular pH of the preimplantation mouse embryo: Effects of extracellular pH and weak acids

Although intracellular pH (pHi), is a regulator of numerous biological processes, it has received relatively little attention with regard to the physiology of the mammalian preimplantation embryo. Interestingly, there is some controversy as to whether the early embryo can recover from an acid load. The significance of this is that two constituents of mouse embryo culture media are pyruvate and lactate. These carboxylic acids are utilised by the early mouse embryo for energy production. However, as weak acids, pyruvate and lactate may induce perturbations in the pHi and thus alter the physiology of the embryo. The aims of this study were therefore to measure the pHi of the mouse preimplantation embryo and to determine the effect of lactate on pHi at different developmental stages. The pHi was measured using the ratio-metric fluorophore carboxy-seminaphthorhodafluor-1-acetoxymethylester (SNARF-1) in conjunction with confocal microscopy. The pHi increased significantly with development from the zygote to the morula stage. Furthermore, at concentrations greater than 5 mM, lactate caused the pHi of the zygote to become significantly more acidic. It was demonstrated that facilitative transport in association with a smaller passive component was responsible for the movement of lactate into the zygote. Metabolic studies revealed that, through their acidifying effect, weak acids caused a reduction in glycolytic activity in the early embryo. In contrast, the pHi of the compacted embryo remained unchanged by the presence of lactate in the external media. Furthermore, incubation with weak acids did not affect the rate of glycolysis in the morula. These data suggest that, by the generation of a transporting epithelium at compaction, the embryo develops the ability to regulate pHi against an acid load. Mol. Reprod. Dev. 50:434–442, 1998. © 1998 Wiley-Liss, Inc.     

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

对与小鼠胚胎发育相关的印记基因Mcts2的研究

目的：对与小鼠胚胎发育相关的印记基因Mcts2表达模式及生物学功能做初步的分析。方法：采用切片原位杂交,全胚胎原位杂交,Northern blot和real-time PCR对该基因进行了表达谱的分析。结果：切片原位杂交结果显示Mcts2基因在E13.5和E15.5胚胎中的脑、舌、心脏、肺脏、肝脏、肾脏等重要脏器中都有普遍表达。全胚胎原位杂交结果显示Mcts2基因在E10.5胚胎中的前脑、前肢、尾芽中出现较强的信号,其他部位信号较弱。Northern和Real-time PCR实验分析了Mcts2基因在E12.5,E15.5,E18.5胚胎和新生小鼠的脑、心脏、肺脏、肝脏和肾脏中的表达谱,发现Mcts2基因在这几个主要发育时期都有普遍表达,在E15.5胚胎中表达信号最为强烈。结论：Mcts2基因在小鼠胚胎的发育的各主要时期的重要脏器中都有普遍的表达,提示该基因在小鼠胚胎发育过程中起到了重要的作用。     

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures

A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed. The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation. This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices.     

Risk assessment of mouse hepatitis virus infection via in vitro fertilization and embryo transfer by the use of zona-intact and laser-microdissected oocytes

The aim of this study was to estimate the risk of mouse hepatitis virus (MHV) transmission by the in vitro fertilization and embryo transfer (IVF-ET) procedure. In addition, resistance to infection of zona-intact and laser-microdissected oocytes was compared. For this purpose, infectious mouse hepatitis virus, a common viral pathogen in mouse facilities, was used. Oocytes having an intact or laser-microdissected zona pellucida were incubated for fertilization in media containing MHV-A59 and resulting embryos were transferred to the oviduct of specific pathogen-free (SPF) Swiss recipients. The oocytes were divided into three experimental groups: 1) zona-intact oocytes continuously exposed to MHV in fertilization (HTF), culture (KSOM), and embryo transfer (M2) media; 2) zona-intact oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2; and 3) laser-microdissected oocytes exposed to MHV in HTF medium and transferred after a standard washing procedure with virus-free KSOM and M2. Respective serum samples of embryo recipients and their offspring were tested for MHV antibodies using ELISA. In experiment 1, 10 out of 14 embryo recipients seroconverted to MHV and only their offspring (8 of 19) received maternal antibodies. In experiments 2 and 3, MHV antibodies were detected neither in the recipients nor in the offspring. These results indicate, for the first time, that even if the zona pellucida is partially disrupted by laser microdissection, the transmission of MHV-A59 can be avoided by correctly performed washing steps in the IVF-ET procedure.     

The migratory pathway of neural crest cells into the skin of mouse embryos

The migration of neural crest derived melanoblasts into the skin of mice was studied by the ectoderm-mesoderm recombination technique. Dorsolateral skin from albino and black mouse embryos at the time of initial melanoblast invasion was separated into ectoderm and mesoderm components, recombined with each other, and grown in the chick embryo coelom for a sufficient period to allow melanin formation. Recombined skin from embryos 11 days old formed pigment only when the mesodermal component was from a genetically black embryo. The black ectoderm-albino mesoderm recombinations failed to produce pigment in all cases. At this critical age when melanoblasts were first entering the skin, they were present exclusively in the mesodermal component. Skin recombinations made from 12-day mouse embryos showed a spread of melanoblasts into the ectodermal component, and by 13 and 14 days both dermal mesoderm and epidermal ectoderm were populated by melanoblasts.     

Expression of type VIII collagen during morphogenesis of the chicken and mouse heart.

The expression of type VIII collagen is restricted, in adult mammals, to specialized extracellular matrices and to a select subset of blood vessels. We have examined the distribution of type VIII collagen in sequential stages of mouse and chicken embryos and found a temporal and spatially restricted pattern of expression during cardiogenesis. Type VIII collagen was first detected by immunocytochemistry on Day 11 in the developing mouse embryo and at stage 19 in the chicken embryo. The distribution of this protein was rapidly modulated during cardiac morphogenesis. Initially (Day 11 in the mouse embryo), type VIII collagen was associated with cardiac myoblasts. From Days 15 to 18, the immunoreactive component was progressively diminished in the myocardium; however, this collagen was observed in the subendocardial layer of the atrioventricular canal and later in the cardiac jelly (or the myocardial basement membrane, an area associated with the formation of cardiac valves). On Day 17, type VIII collagen was also detected in the subendothelium (intima) and tunica media of large vessels. Neonatal and adult hearts contained low to undetectable levels of type VIII collagen. The presence of type VIII collagen was confirmed by immunoblot analysis of heart extracts at different stages of development. A major 185-kDa component, as well as polypeptides of 68 and 15 kDa, reacted with anti-type VIII collagen IgG. Exposure of heart extracts to hyaluronidase or reducing agent eliminated immunoreactivity of the 185-kDa component but not that of the 68- and 15-kDa polypeptides. Type VIII collagen therefore appears to be associated with a hyaluronidase-sensitive component of the extracellular matrix during a temporally restricted stage of embryonic cardiogenesis. The contribution of this collagen to cardiac morphogenesis might reside, in part, in its ability to influence the differentiation of the myocardium and formation of the cardiac valves.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture

A growth-factor-like substance capable of inducing nontransformed mouse AKR-2B, rat NRK, and EGF-receptorless mouse NR6 cells to form progressively growing colonies in soft agar was identified in acid/ethanol extracts of 17-day mouse embryos. This "mouse embryo factor" (MEF) is similar to previously described transforming growth factors in that it is capable of stimulating DNA synthesis and conferring a reversible transformed morphology on nontransformed cells in vitro. Passage of crude embryo extracts over a Bio-Gel P-60 column gave a major peak of soft agar growth-stimulating activity in the 15,000 molecular weight range with a minor peak at about 22,000. This biological activity was sensitive to treatment with either trypsin or dithiothreitol, but was unaffected by heat (56 degrees C for 30 minutes or 100 degrees C for 3 minutes), indicating that the activity is due to a heat-stable polypeptide(s) with disulfide bonds. Separation of these polypeptide(s) by chromatography on carboxymethyl cellulose revealed two peaks of soft agar growth-stimulating activity which did not cochromatograph with a peak of epidermal growth factor receptor-competing activity. The similarities of this mouse embryo-derived growth factor to previously identified transforming growth factors suggest that both fetal development and neoplastic transformation may be affected by similar mechanisms.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.     

DNA synthesis in the heterokaryons of non-dividing differentiated cells and culture cells with various proliferative potentials

Resident peritoneal mouse macrophages (non-dividing differentiated cells) were fused with mouse embryo fibroblasts (cells with a limited lifespan), NIH 3T3 and C3H 10T 1/2 cells ('immortal' cell lines) and SV 3T3 cells (a malignant cell line). DNA synthesis was investigated in the resultant heterokaryons. No inhibitory effect upon the transition of NIH 3T3 and mouse embryo fibroblasts nuclei to the S-phase was observed. C3H 10T 1/2, NIH 3T3 and SV 3T3 cells induced the reactivation of DNA synthesis in the macrophage nuclei in the heterokaryons. At the same time, no replication was detected in the macrophage nuclei after fusion with mouse embryo fibroblasts.     

Retinoic acid-inducible gene-I mediates late phase induction of TNF-alpha by lipopolysaccharide

LPS is the known component of bacterial pathogens that stimulates a number of proinflammatory factors. However, the mechanism of the induction of these factors by LPS has not been fully elucidated. We show here that LPS induces retinoic acid-inducible gene-I (RIG-I) in vitro and in vivo as a result from autocrine secretion of IFN-beta in macrophages. TIR-domain-containing adapter-inducing IFN-beta-deficient mouse embryo fibroblast (trif(-/)(-)) fail to show expression of RIG-I following LPS stimulation. Interference of RIG-I expression short interfering RNA represses the expression of LPS-induced TNF-alpha, whereas over-expression of RIG-I leads to the activation of TNF-alpha promoter and the induction of TNF-alpha expression. LPS- and IFN-beta-induced TNF-alpha are suppressed in RIG-I-deficient mouse embryo fibroblasts (rig(-/)(-)). Thus, RIG-I plays a key role in the expression of TNF-alpha in macrophages in response to LPS stimulation, mainly for the late phase LPS-induced expression of TNF-alpha.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Low molecular weight RNA species from chromatin.

Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.     

Spatiotemporal expression of connexin 39 and -43 during myoblast differentiation in cultured cells and in the mouse embryo

Connexin39 (Cx39) and connexin43 (Cx43) are known to be expressed during development of skeletal muscles. Here we have compared the expression pattern of both connexins during differentiation of established C(2)C(12) mouse myoblasts and in the mouse embryo. Cx43 is highly abundant in undifferentiated myoblasts, but no Cx39 protein was detected in these cells. Upon differentiation into myotubes, Cx39 expression increased. The consecutive expression of these connexins was also observed in the mouse embryo. Cx39 and Cx43 were found in different plaques in accordance with the notion that Cx43 is exclusively expressed in myoblasts and Cx39 in myotubes. Thus, differentiating C(2)C(12) cells in culture can serve to study the involvement of gap junctions in myogenesis, since expression of corresponding Cx39 and Cx43 proteins appears to be very similar as in the mouse embryo.     

Rab23 regulates Nodal signaling in vertebrate left–right patterning independently of the Hedgehog pathway

Asymmetric fluid flow in the node and Nodal signaling in the left lateral plate mesoderm (LPM) drive left–right patterning of the mammalian body plan. However, the mechanisms linking fluid flow to asymmetric gene expression in the LPM remain unclear. Here we show that the small GTPase Rab23, known for its role in Hedgehog signaling, plays a separate role in Nodal signaling and left–right patterning in the mouse embryo. Rab23 is not required for initial symmetry breaking in the node, but it is required for expression of Nodal and Nodal target genes in the LPM. Microinjection of Nodal protein and transfection of Nodal cDNA in the embryo indicate that Rab23 is required for the production of functional Nodal signals, rather than the response to them. Using gain- and loss-of function approaches, we show that Rab23 plays a similar role in zebrafish, where it is required in the teleost equivalent of the mouse node, Kupffer?s vesicle. Collectively, these data suggest that Rab23 is an essential component of the mechanism that transmits asymmetric patterning information from the node to the LPM.