Chorionicity and Heritability Estimates from Twin Studies: The Prenatal Environment of Twins and Their Resemblance Across a Large Number of Traits.

There are three types of monozygotic (MZ) twins. MZ twins can either share one chorion and one amnion, each twin can have its own amnion, or MZ twins can-like dizygotic twins-each have their own chorion and amnion. Sharing the same chorion may create a more similar/dissimilar prenatal environment and bias heritability estimates, but most twin studies do not distinguish between these three types of MZ twin pairs. The aim of this paper is to investigate the effect of chorion sharing on the similarity within MZ twin pairs for a large number of traits. Information on chorion status was obtained for the Netherlands twin register (NTR) by linkage to the records from the database of the dutch pathological anatomy national automated archive (PALGA). Record linkage was successful for over 9000 pairs. Effect of chorion type was tested by comparing the within-pair similarity between monochorionic (MC) and dichorionic (DC) MZ twins on 66 traits including weight, height, motor milestones, child problem behaviors, cognitive function, wellbeing and personality. For only 10 traits, within-pair similarity differed between MCMZ and DCMZ pairs. For traits influenced by birth weight (e.g. weight and height in young children) we expected that MC twins would be more discordant. This was found for 5 out of 13 measures. When looking at traits where blood supply is important, we saw MCMZ twins to be more concordant than DCMZ's for 3 traits. We conclude that the influence on the MZ twin correlation of the intra-uterine prenatal environment, as measured by sharing a chorion type, is small and limited to a few phenotypes. This implies that the assumption of equal prenatal environment of mono- and DC MZ twins, which characterizes the classical twin design, is largely tenable.

[Cognitive impairment due to intensive use and overdoses of gammahydroxybutyric acid (GHB)]. / Cognitieve schade door intensief gebruik en overdoses van GHB.

BACKGROUND: In several countries, including the Netherlands, the use of GHB seems to be increasing. Many recreational users of GHB consider the drug to be harmless and to have no serious side effects. In recent years the number of patients with GHB addition has been increasing steadily. AIM: To draw attention to the possible development of neurotoxicity due to chronic and intensive use of GBH. METHOD: We reviewed the literature using PubMed. RESULTS: Several studies point to an increase in the number of incidents arising from the risky use of GHB or from a GHB overdose. Other drugs, such as ketamine and alcohol, are known to cause neurotoxicity, leading to cognitive impairment. As outlined in this review article, GHB , alcohol and ketamine show clear similarities in their mechanism of action. This suggests that GHB might have almost the same neurotoxic effects as ketamine and alcohol. An overdose of GHB, just like binge-drinking and a high dose of ketamine, may lead to a coma that probably harms the brain, particularly if comas occur repeatedly. CONCLUSION: The risk of neurotoxicity is likely to increase with chronic, intensive use of GHB, which is a feature of GHB-addition. We therefore advocate research into the possible toxic effects of GHB in the long term, involving, for instance, the study of lasting effects on the cognitive functions of GHB users and former users.

The adverse effects of maternal smoking on the human placenta: a review.

Studies of placental pathologies associated with maternal cigarette smoking have led to many interesting observations. For example, maternal smoking impairs human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation. It is likely that chronic exposure to tobacco constituents in early pregnancy can affect placental development directly or indirectly by reducing blood flow, which creates a pathologically hypoxic environment. To understand this process at a molecular level, tissue samples from non-smoking and smoking mothers were studied to determine whether active and/or passive cigarette smoke exposure affects CTB expression of molecules that govern cellular responses to oxygen tension: the von Hippel-Lindau tumor suppressor protein (pVHL), hypoxia-inducible transcription factors (HIFs) and vascular endothelial growth factor-A (VEGF). The results show that maternal smoking dysregulates CTB expression of all three types of molecules. In addition, cell columns and proliferating cells were reduced while there was a corresponding increase in cell islands. All three phenomena were most obvious in the placentas of heavy smokers. Interestingly, a subset of the aforementioned effects can be detected in samples obtained from women who were passively exposed to cigarette smoke during pregnancy. These observations suggest that tobacco constituents exert direct effects on CTB proliferation and differentiation and help to explain the mechanisms by which smoking negatively effects human pregnancy outcome.

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue.

OBJECTIVE: The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S): HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S): Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.

Concordant in situ and in vitro data show that maternal cigarette smoking negatively regulates placental cytotrophoblast passage through the cell cycle.

Maternal cigarette smoking is associated with fetal growth restriction and other pregnancy complications. To investigate possible mechanisms involving the placenta, we studied the morphology of first trimester chorionic villi from mothers who smoked. In mothers who smoked > 20 cigarettes/day, floating villi showed focal defects including an absence of cytotrophoblast stem cells and an abnormal thinning of the syncytium. Anchoring villi displayed a striking increase in the number of cytotrophoblast columns that failed to reach the uterus or degenerated in the intervillous space. Many samples showed a significant reduction in the number of anchoring villi. Also, the number of Ki67-positive cytotrophoblasts was dramatically decreased, indicating that fewer cells were in S phase of the mitotic cycle. Together, these results suggested premature depletion of the cytotrophoblast stem cell population. To test this hypothesis, we exposed anchoring villi from nonsmokers to nicotine in vitro and analyzed the effects on cytotrophoblast passage through the cell cycle. Nicotine (0.23 to 6.0 microM) negatively affected the expression of a number of cell cycle regulators/markers and BrdU incorporation, without discernable effects on apoptosis. These results link abnormal placental development secondary to maternal cigarette smoking to a substantial decrease in the mitotic potential of cytotrophoblasts.

A repertoire of cell cycle regulators whose expression is coordinated with human cytotrophoblast differentiation.

Although placental development depends on careful coordination of trophoblast proliferation and differentiation, little is known about the mitotic regulators that are key to synchronizing these events. We immunolocalized a broad range of these regulators in tissue sections of the maternal-fetal interface (first trimester through term) that contained floating villi (which include cytotrophoblasts differentiating into syncytiotrophoblasts) and anchoring villi (which include cytotrophoblasts differentiating into invasive cells). Trophoblast populations at the maternal-fetal interface stained for 16 of the cell cycle regulators whose expression we studied. The staining patterns changed as a function of both differentiation and gestational age. Differentiation along the invasive pathway was associated with entrance into, then permanent withdrawal from, the cell cycle, as evidenced by the orchestrated expression of cyclins, their catalytic subunits, and inhibitors. Surprisingly, we found coexpression of molecules that regulate different portions of the cell cycle in the syncytium. These data, which constitute one of the few examples to date of in situ localization of an extensive repertoire of mitotic regulators, provide the basis for studies aimed at understanding factors that lead to abnormal placentation.

Upregulation of human cytotrophoblast invasion by hepatocyte growth factor.

Mesenchymal-epithelial interactions exert powerful influences on tissue architecture. At the molecular level, hepatocyte growth factor (HGF; produced by mesenchyme) and its c-met tyrosine kinase receptor (expressed on epithelial cells) participate in this paracrine dialogue. In the present study, anti-HGF immunoreactivity was usually detected in association with the mesenchymal cores of chorionic villi in situ, whereas cytotrophoblasts (CTBs) stained for c-met. In pre-eclampsia, mesenchymal HGF staining was either very low or was not observed, whereas CTB c-met expression was unchanged compared with control samples. These findings suggest that faulty signals emanating from the villus mesenchyme may contribute to the failure of CTB invasion that is associated with pre-eclampsia. In the present study, this hypothesis was tested and it was found that HGF treatment stimulates CTB invasion in vitro by four times. These immunolocalization and functional data indicate that HGF-c-met interactions play a key role in regulating the depth of CTB invasion in normal pregnancy and pre-eclampsia.

Id-2 regulates critical aspects of human cytotrophoblast differentiation, invasion and migration.

During early human placental development, the conceptus attaches itself to the uterus through cytotrophoblast invasion. Invasive cytotrophoblast cells differentiate from precursor villous cytotrophoblasts, but the essential regulating factors in this process are unknown. Basic helix-loop-helix (bHLH) transcription factor dimers are essential regulators of mouse trophoblast development. We therefore examined the importance of this family of factors in the human placenta. In many cell lineages, bHLH factors are sequestered by members of the Id family, HLH proteins that lack the basic DNA binding domain (Inhibitor of DNA binding proteins (Id-1 to Id-4)). During differentiation of some tissues, Id expression declines, allowing bHLH factors to dimerize, bind DNA and trans-activate lineage-specific genes. To begin to study the role of bHLH transcription factors in human placental development, we first characterized Id expression in cytotrophoblast cells. The cells expressed Id-3 constitutively; Id-2 was downregulated, at the mRNA and protein levels, as the cells differentiated in culture and in situ, respectively. In cases when cytotrophoblast differentiation was compromised (in placentas from women with preeclampsia, or in cells grown under hypoxic conditions in culture), Id-2 expression was maintained. To assess the functional relevance of these correlations, we used an adenovirus vector to maintain Id-2 protein expression in cultured cytotrophoblasts. Compared to control (lacZ-expressing) cells, cytotrophoblasts transduced to constitutively express Id-2 retained characteristics of undifferentiated cells: (alpha)1 integrin expression was low and cyclin B expression was retained. Furthermore, invasion through Matrigel was partially inhibited and migration was strikingly enhanced in Id-2-expressing cells. These results suggest that Id-2 and the bHLH factors that it partners play important roles in human cytotrophoblast development.

Complementary expression of HIP, a cell-surface heparan sulfate binding protein, and perlecan at the human fetal-maternal interface.

The human hemochorial placenta is a structure formed by the invasion of cytotrophoblasts into the uterus. Previous studies from our laboratory have demonstrated a role for heparan sulfate proteoglycans (HSPGs) and their binding proteins in interactions between human trophoblastic and uterine cell lines in vitro. In this study, expression of both mRNA and protein of a novel, cell surface, heparin/heparan sulfate interacting protein (HIP), by human trophoblastic cell lines-i.e., JAR, JEG, and BeWo-and by human cytotrophoblast was examined throughout gestation. Immunohistochemistry of the human fetal-maternal interface demonstrated abundant HIP expression in cytotrophoblast cells, with lesser staining in syncytiotrophoblast and little or no staining in surrounding stromal or decidual cells. Staining with antibodies to the basement membrane HSPG, perlecan, demonstrated a pattern of staining complementary to that of HIP. Cytotrophoblasts in the uterine stroma, not affiliated with attached villi, displayed a less intense deposition of perlecan. In vitro binding studies of 125I-perlecan to a 17-amino acid synthetic peptide sequence of HIP, which has a high affinity and specificity for heparin/heparan sulfate, indicates that perlecan binds to the HIP peptide with high affinity (KDapp = 0.6 nM) and in a heparin-inhibitable manner. Furthermore, HIP antibodies inhibited by 61-88% in vitro invasion by trophoblasts in assays using primary cultures of normal human cytotrophoblasts. Consistent with this was the observation that immunohistochemically detectable HIP expression was greatly reduced in pre-eclamptic cytotrophoblasts, a condition in which trophoblast invasion is abnormally shallow. It is suggested that HIP potentiates human cytotrophoblast interactions with HSPGs, in vivo, and facilitates trophoblast invasion processes.

Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts.

HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

Human placental HLA-G expression is restricted to differentiated cytotrophoblasts.

Human placental trophoblasts lie at the maternal-fetal interface, a position in which they could play an important role in maternal tolerance of the fetal semi-allograft. Central to this hypothesis is their unusual MHC class I expression: they suppress class Ia production while expressing HLA-G, a class Ib molecule. We investigated human trophoblast HLA-G protein production in vivo and in vitro. We first used a synthetic peptide corresponding to the variable sequence of the alpha 1 domain to produce mAbs that recognized HLA-G. Ab specificity was demonstrated by immunoaffinity purification of a single protein with the same molecular mass (38 kDa) as HLA-G from choriocarcinoma cells. Use of these Abs to stain tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation showed that HLA-G is expressed only by cytotrophoblasts that invade the uterus. Our previous in vitro studies showed that when early-gestation cytotrophoblast stem cells are cultured, they differentiate rapidly along the invasive pathway, as demonstrated by their expression of stage-specific markers. Here we show they also up-regulate HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to up-regulate HLA-G protein expression. We detected high levels of HLA-G mRNA in cytotrophoblasts isolated from first- and second-trimester placentas, but only trace amounts in term cells. Taken together, these results suggest that HLA-G production is a critical component of cytotrophoblast differentiation along the invasive pathway.

Integrin switching regulates normal trophoblast invasion.

Cells invade extracellular matrices in a regulated manner at specific times and places during normal development. A dramatic example is trophoblast invasion of the uterine wall. Previous studies have shown that differentiation of trophoblasts to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. In the first trimester human placenta, alpha 6 integrins are restricted to cytotrophoblast (CTB) stem cells and downregulated in invasive CTBs, whereas alpha 5 beta 1 and alpha 1 beta 1 integrins are upregulated in differentiating and invasive CTBs. The goal of the present study was to determine whether these changes have functional consequences for CTB invasiveness. Using an in vitro invasion model, we determined first that aggregates of invading first trimester CTBs in vitro undergo the same pattern of integrin switching as was observed in situ, thereby validating the utility of the model. We then showed that antibody perturbation of interactions involving laminin or collagen type IV and their integrin alpha 1/beta 1 receptor inhibited invasion by CTBs, whereas perturbing interactions between fibronectin and the alpha 5/beta 1 fibronectin receptor accelerated invasion. Finally, we report that later gestation CTBs, which display greatly decreased invasive capacity, are also unable to upregulate alpha 1 beta 1 complexes, providing further evidence that this integrin is critical for CTB invasion. This gestational regulation is transcriptional. These data indicate that integrin switching observed during differentiation in situ has significant functional consequences for CTB invasion. The data suggest further that differentiating CTBs upregulate counterbalancing invasion-accelerating and invasion-restraining adhesion mechanisms. We propose that this contributes to regulating the depth of CTB invasion during normal implantation.

Human trophoblast invasion. Autocrine control and paracrine modulation.

Development of the human placenta involves rapid invasion of the uterine wall by fetal trophoblasts, a process with certain similarities to tumor cell invasion. Unlike tumor invasion, however, this unique interaction between genetically dissimilar trophoblast and uterine cells is closely regulated and limited both temporally and spatially by mechanisms that are largely unknown. We have used a combination of two experimental approaches to study this process: immunolocalization using tissue sections to investigate trophoblast invasion in vivo, and a cell culture model that allows manipulation of the invasion process in vitro. The results show that invading trophoblasts express activated forms of metalloproteinases, adhesion molecules and the novel class I histocompatibility antigen, HLA-G, in a highly regulated manner during invasion. The behavior of cytotrophoblasts in vitro, removed from the influences of uterine cells, closely parallels their behavior in vivo, suggesting the existence of autocrine control mechanisms. However, studies examining the effect of growth factors and cytokines on trophoblast invasion suggest that molecules of uterine origin can modify this process. Thus, we hypothesize that the intrinsic invasiveness of these cells is controlled, at least in part, by the specialized environment of the uterus. Future studies will concentrate on identifying these factors and the specific trophoblast functions they modify.

Association of monocytes and neutrophils with early events of blastocyst implantation in mice.

The distribution and activation of monocytes and neutrophils in the mouse uterus were examined early in the process of blastocyst implantation. Implantation regions, detected by increased capillary permeability at sites of blastocyst apposition to the uterine luminal epithelium, could be distinguished at about 21:00 h on day 4 of pregnancy (day 1 = day of vaginal plug), and were also examined at 01:00, 05:00 and 09:00 h on day 5. Serial sections containing the implanting blastocyst (implantation sites), and random sections of inter-implantation regions were examined by immunohistochemistry using interleukin-1 beta as a marker for monocytes-macrophages and lactoferrin as a marker for neutrophils in the uterine stroma. At implantation sites, interleukin-1 beta-positive cells were transiently abundant within endometrial capillaries. In interimplantation regions only a few interleukin-1 beta-positive cells were dispersed at the myometrial-stromal junction. Northern blot hybridization to RNAs from implantation and interimplantation regions showed that the abundance of interleukin-1 beta and interleukin-1 alpha mRNAs was much lower than that found in the uterus during acute inflammation. However, these cytokine mRNAs were more abundant in implantation regions. On the evening of day 4 and the early morning of day 5, lactoferrin-positive neutrophils were detected juxtaposed to the basolateral surface of the antimesometrial epithelial cells surrounding the implanting blastocyst. They were primarily at the myometrial-stromal junction in interimplantation regions. Metallothionein gene expression was examined as a marker for uterine responses to inflammatory reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and distribution of inflammatory cells in the mouse uterus during the preimplantation period.

Northern blot analysis of mouse uterine RNA showed that IL-1 (alpha and beta), and TNF-alpha mRNA were abundant on day (D) 1 of pregnancy, reduced on D2, and remained basal throughout the remainder of the preimplantation period (D3 and D4). Elevated IL-1 beta and TNF-alpha mRNA levels on D1 were accompanied by increased levels of immunoreactive protein in uterine cytosol preparations as determined by ELISA. In situ hybridization detected IL-1 beta mRNA in cells located in the endometrial stroma and concentrated in subepithelial regions on D1. Immunocytochemical localization of IL-1 beta and TNF-alpha identified cells scattered throughout the endometrial stroma, but more concentrated in the subepithelial region on D1. On D3 and D4, cytokine-immunopositive cells decreased in number and became located predominantly at the endometrial-myometrial junction. Histochemical localization of peroxidase as a marker predominantly for eosinophils showed an abundance of these cells in the D1 uterus. The distribution of peroxidase-positive cells in the uterus followed the same temporal and spatial changes as cytokine-immunopositive cells during the preimplantation period. These data document the occurrence of an inflammatory response in the uterus on D1 of pregnancy, and demonstrate that as the preimplantation period progresses the distribution of inflammatory cells changes from the subepithelial region of the endometrial stroma to the periphery of the uterus at the endometrial-myometrial junction. Mechanisms regulating the uterine inflammatory response on D1 were investigated. Cytokine mRNA levels were not significantly elevated during the estrous cycle or after treatment of adult ovariectomized mice with estradiol-17 beta. In contrast, mating with vasectomized males resulted in an inflammatory response on D1 of pseudopregnancy similar to that on D1 of normal pregnancy, whereas mechanical stimulation of the uterine cervix failed to elicit such a response. These results strongly suggest a role for some factor(s) in the ejaculate, other than spermatozoa, in the initiation of a uterine inflammatory response after mating, but an effect of the act of mating cannot be excluded.

Lactoferrin in the mouse uterus: analyses of the preimplantation period and regulation by ovarian steroids.

Uterine expression of lactoferrin (LF) during the preimplantation period and its regulation by the ovarian steroids estradiol (E2) and/or progesterone (P4) in ovariectomized adult mice were examined. Immunoblot detection of LF in uterine cell lysates demonstrated the presence of this protein from days 1-8 of pregnancy [day 1 (D1) = day of vaginal plug]. Immunoprecipitation of 35S pulse-labeled uterine proteins showed that the relative rate of LF synthesis was high on D1, but below the level of detection by D4. Immunolocalization of LF in uterine sections showed intense luminal and glandular epithelial staining on D1 and D2, and progressively decreased staining through D4. Immunoreactive protein was also detected in cells, primarily concentrated in the stroma. The relative number of these cells was greatest on D1 and decreased progressively to a low number by D4. These cells were morphologically similar to neutrophils, which are known to contain LF protein, but little or no LF mRNA. Northern blotting showed that uterine LF mRNA levels were very high on D1 and D2 of pregnancy and decreased to low, but detectable, levels by D4. In situ hybridization to uterine sections showed that LF mRNA was highly abundant only in glandular and luminal epithelial cells, and followed the same pattern as immunolocalization on D1-D4 in epithelial cells. These results document two sources of LF in the preimplantation mouse uterus: neutrophils and epithelial cells. The synthesis of LF in the uterus reflects the abundance of epithelial LF mRNA, which is high on the first 2 days of pregnancy. Neutrophils that contain LF are also abundant in the uterine stroma during this time. E2 and/or P4 regulation of uterine LF was examined. LF mRNA was rapidly induced by E2 in ovariectomized adult mice, and this mRNA was localized exclusively to epithelial cells. P4 had little effect on uterine LF mRNA levels, but antagonized the prolonged induction of this gene by E2. E2 induced the accumulation of immunoreactive LF in uterine epithelial cells and the appearance of numerous immunopositive neutrophils distributed throughout the uterine stroma. P4 also antagonized these effects. Thus, E2 regulates LF gene expression in uterine epithelial cells and causes the recruitment of neutrophils into the uterus. These results suggest that LF may play an important role in early pregnancy and that uterine LF gene expression is regulated by a balance between estrogen and P4.

Metallothionein gene expression and metal regulation during preimplantation mouse embryo development (MT mRNA during early development).

In order to provide information concerning gene expression and regulation in the preimplantation mammalian embryo, and to explore the roles of metallothionein (MT) during this period of development, the constitutive and metal-induced MT mRNA levels in mouse ova, preimplantation embryos, and oviducts were determined. These results were correlated with the effects of transient exposure to high levels of metals (zinc (Zn) or cadmium (Cd] on the continued development of preimplantation embryos into blastocysts in culture. RNA from preimplantation mouse embryos at different stages of development (Days 1 through 4 of gestation; D1 = vaginal plug) was analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) to specifically amplify MT-I and MT-II mRNA transcripts. MT-I mRNA in ova, preimplantation embryos, and oviducts was detected using in situ hybridization. This mRNA in the oviduct was also analyzed by Northern blotting. The results establish that the mouse MT genes are coordinately and constitutively expressed at low basal levels in ova and preimplantation mouse embryos. In unfertilized (ova), fertilized (one-cell) eggs, and two-cell embryos, the MT-I gene was not detectably responsive to metal ions, whereas in later cleavage stage embryos (four- and eight-cell) the MT-I gene was detectably responsive to metals in some blastomeres of some of the embryos. In contrast, after the third cleavage this gene was highly metal-inducible in essentially all cells of the embryo (morula/blastocyst). Surprisingly, the appearance of metal responsiveness of the MT genes during development correlated with decreased Zn toxicity and increased Cd toxicity; two-cell embryos were Zn-sensitive and Cd-resistant, whereas eight-cell and older embryos were Zn-resistant and Cd-sensitive. In the oviduct, MT-I mRNA was not abundant in total RNA, but was detected specifically in the epithelial cells of the isthmus region and was elevated in these cells on D3 and D4 of gestation. In the oviduct, only isthmus epithelial cells responded to metals (Zn or Cd) by increased accumulation of this mRNA. These studies suggest that preimplantation mouse embryo develops the capacity to respond to metals in the environmental milieu by induction of MT gene expression at about the third cleavage. Whether the lack of responsiveness of these genes before this stage reflects transcriptional repression or attenuated metal ion influx and/or enhanced efflux remains to be determined. Sensitivity and resistance of preimplantation embryos to acute metal toxicity involve mechanisms other than MT gene expression in preimplantation mouse embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

Endotoxin induction of murine metallothionein gene expression.

Bacterial endotoxin-lipopolysaccharide (LPS) rapidly induced hepatic metallothionein (MT) mRNA levels in the LPS-sensitive CD-1 strain of mice. This LPS effect was severely attenuated in the LPS-resistant C3H/HeJ strain of mice, but could be mimicked by injection of human recombinant interleukin-1 alpha (IL-1 alpha) or human recombinant tumor necrosis factor (TNF-alpha). In the CD-1 strain, LPS induction of MT gene expression occurred in each of 10 organs examined (liver, kidney, pancreas, intestine, lung, heart, brain, ovary, uterus, and spleen). Solution hybridization with probes specific for MT-I or MT-II mRNA established that these genes were co-induced in each of the organs and that the liver and kidney contained the highest absolute levels of these mRNAs, whereas in the intestine and spleen they were 10-20-fold lower. LPS and cytokine induction of hepatic MT gene expression occurred in hypophysectomized mice, which suggests a lack of significant involvement of glucocorticoids. Several recombinant cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, interferon-gamma (IFN-gamma), as well as poly(rI.rC) were effective inducers of hepatic MT-I and MT-II genes. As an attempt to determine which of these cytokines may mediate LPS effects on MT gene expression in vivo, CD-1 mice were injected with LPS or various cytokines, and RNA from liver, ovary, and uterus was extracted at various times postinjection and analyzed by Northern blotting using probes specific for IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and MT mRNA. In each organ examined, LPS, IL-1 alpha, or IL-1 beta injection caused a rapid, coordinate, transient increase in the levels of each of the cytokine mRNAs which peaked by 1 h and declined to low levels by 4 h. In contrast, levels of MT mRNA did not reach a peak until 4-6 h postinjection. TNF-alpha had minimal effects on expression of cytokine and MT genes in organs other than liver. IL-6 had no effect on hepatic cytokine mRNA levels, and induced MT mRNA only in the liver which suggests a direct effect of IL-6 on hepatic MT gene expression. These data suggest that the acute effects of LPS on MT gene expression may include complex paracrine interactions between a variety of cytokines and the cells expressing MT genes in each organ, and tissue-specific cytokine effects on the MT genes.

Cell type-specific expression of transforming growth factor-beta 1 in the mouse uterus during the periimplantation period.

Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-beta 1 (TGF beta 1) in the mouse uterus during the periimplantation period. The polyclonal antisera anti-LC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF beta 1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF beta 1 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF beta 1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-beta 1 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-beta 1 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-specific metallothionein gene expression in mouse decidua and placentae.

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.