(1/4036) An ultrastructural study of implantation in the golden hamster. II. Trophoblastic invasion and removal of the uterine epithelium.
Sixty six implantation sites from 18 golden hamsters were examined with light and electron microscopy between 4 and 5 1/2 days of pregnancy (post-ovulation). At 4 days some blastocysts began to invade the uterine epithelium, with trophoblastic processes penetrating and engulfing portions of the uterine epithelium. The majority of epithelial cells appeared normal before invasion, although at two implantation sites three or four adjoining epithelial cells were necrotic before penetration by the trophoblast. In general the epithelial cells were degenerating at the time the trophoblast invaded the epithelium. Inclusions, representing portions of the engulfed epithelium, and varying in size and electron density, were present throughout the invading trophoblast cells at 4 1/2 and 5 days of pregnancy. At 5 1/2 days the uterine epithelium had disappeared and the embryo was now almost completely surrounded by blood lacunae. (+info)
(2/4036) Ontogeny of expression of a receptor for platelet-activating factor in mouse preimplantation embryos and the effects of fertilization and culture in vitro on its expression.
Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent ether phospholipid. It is one of the preimplantation embryo's autocrine growth/survival factors. It may act via a G protein-linked receptor on the embryo; however, the evidence for this is conflicting. The recent description of the intracellular form of the PAF:acetlyhydrolase enzyme as having structural homology with G proteins and Ras also suggests this as a potential intracellular receptor/transducer for PAF. This study used reverse transcription-polymerase chain reaction to examine the ontogeny of expression of the genes for these proteins in the oocyte and preimplantation-stage embryo. Transcripts for the G protein-linked PAF receptor were detected in the late 2-cell-stage embryo and in all stages from the 4-cell stage to blastocysts. They were also present in unfertilized oocytes and newly fertilized zygotes but only at relatively low levels. The incidence of expression was generally low and variable in late zygotes and early 2-cell embryos. Expression past the 2-cell stage was alpha-amanitin sensitive. The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote-2-cell stage. New expression of the receptor transcript required activation of the zygotic genome. Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote. Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h. This retardation did not occur if culture commenced at the 2-cell stage. The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue. This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development. The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner. The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial. The effect of IVF and culture may also explain the conflicting literature. (+info)
(3/4036) X inactive-specific transcript (Xist) expression and X chromosome inactivation in the preattachment bovine embryo.
Expression of the X inactive-specific transcript (Xist) is thought to be essential for the initiation of X chromosome inactivation and dosage compensation during female embryo development. In the present study, we analyzed the patterns of Xist transcription and the onset of X chromosome inactivation in bovine preattachment embryos. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of Xist transcripts in all adult female somatic tissues evaluated. In contrast, among the male tissues examined, Xist expression was detected only in testis. No evidence for Xist transcription was observed after a single round of RT-PCR from pools of in vitro-derived embryos at the 2- to 4-cell stage. Xist transcripts were detected as a faint amplicon at the 8-cell stage initially, and consistently thereafter in all stages examined up to and including the expanded blastocyst stage. Xist transcripts, however, were subsequently detected from the 2-cell stage onward after nested RT-PCR. Preferential [3H]thymidine labeling indicative of late replication of one of the X chromosomes was noted in female embryos of different developmental ages as follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts. These results suggest that Xist expression precedes the onset of late replication in the bovine embryo, in a pattern compatible with a possible role of bovine Xist in the initiation of X chromosome inactivation. (+info)
(4/4036) Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras.
During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement. (+info)
(5/4036) In-vitro fertilization and culture of mouse embryos in vitro significantly retards the onset of insulin-like growth factor-II expression from the zygotic genome.
In this study, the effect of in-vitro fertilization (IVF) and culture of mouse embryos in vitro on the normal expression of insulin-like growth factor-II (IFG-II) ligand and receptor was examined. The expression of IGF-II increased in a linear fashion at least up to the 8-cell stage of development. IGF-II expression in embryos collected fresh from the reproductive tract was significantly (P < 0.001) greater than in embryos fertilized in the reproductive tract and cultured in vitro (in-situ fertilized: ISF), and its expression was further reduced (P < 0.001) in IVF embryos at all development stages tested. The expression of IGF-II was significantly (P < 0.001) lower when embryos were cultured individually in 100 microl drops compared with culture in groups of 10 in 10 microl drops of medium. The addition of platelet activating factor to culture medium partially overcame this density-dependent decline of expression. Culture of ISF and IVF zygotes also caused the onset of new IGF-II mRNA transcription from the zygotic genome to be significantly (P < 0.001) retarded, until at least the 8-cell stage of development. This effect was greater (P < 0.05) for IVF than for ISF embryos. Neither IVF nor culture had any obvious effect on IFG-II/mannose-6-phosphate receptor (IGF-IIr) mRNA expression. (+info)
(6/4036) Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa.
Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo. (+info)
(7/4036) Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast.
Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin. (+info)
(8/4036) Trophectoderm differentiation in the bovine embryo: characterization of a polarized epithelium.
Blastocytst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na-K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, beta-catenin, and the tight junction associated protein, zonula occludens protein 1, in pre-attachment bovine embryos, in vitro. Immunocytochemistry and gene specific reverse transcription--polymerase chain reaction methods were used. Transcripts for E-cadherin and beta-catenin were detected in embryos of all stages throughout pre-attachment development. Immunocytochemistry revealed E-cadherin and beta-catenin polypeptides evenly distributed around the cell margins of one-cell zygotes and cleavage stage embryos. In the morula, detection of these proteins diminished in the free apical surface of outer blastomeres. E-cadherin and beta-catenin became restricted to the basolateral membranes of trophectoderm cells of the blastocyst, while maintaining apolar distributions in the inner cell mass. Zonula occludens protein 1 immunoreactivity was undetectable until the morula stage and first appeared as punctate points between the outer cells. In the blastocyst, zonula occludens protein 1 was localized as a continuous ring at the apical points of trophectoderm cell contact and was undetectable in the inner cell mass. These results illustrate that the gene products encoding E-cadherin, beta-catenin and zonula occludens protein 1 are expressed and maintain cellular distribution patterns consistent with their predicted roles in mediating trophectoderm differentiation in in vitro produced bovine embryos. (+info)