Swapan K. De

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)

Establishment of a rat placental cell line expressing characteristics of extraembryonic membranes.

A cell line was derived from midgestation chorioallantoic placental explants of the outbred Holtzman rat. The cell line was found to express characteristics of extraembryonic membranes and to grow when introduced into allogeneic hosts. Growth in allogeneic hosts was detected following intraperitoneal injection of the cells but not following subcutaneous injection. The transplanted cells grew as cystic structures free in the peritoneum and as solid masses adhered to various abdominal organs. Cystic structures had a homogeneous morphology consisting of an epithelial-like cell layer surrounding a fluid-filled sac. Solid masses had a heterogeneous morphology, containing parts resembling normal components of the extraembryonic membranes (trophoblast, parietal, and visceral yolk sacs). Biochemical analysis of the placenta-derived cell line and transplanted structures derived from the cell line indicated that the cells had the potential to produce a variety of proteins characteristic of extraembryonic tissues. Cultured cells and both types of in vivo transplants produced the basement membrane protein, laminin. Peritoneal cystic structures also contained alpha-fetoprotein mRNA and very high levels of c-fos mRNA. Solid masses demonstrated elevated alkaline phosphatase activity, a marker of trophoblast cells. Cells grown in vitro expressed elevated c-myc mRNA levels, whereas, c-myc mRNA levels were reduced in the in vivo transplants. The behavior of the cell line in vitro and following in vivo transplantation suggests it contains elements capable of differentiation toward various components of the extraembryonic membranes. The results indicate that the rat placental cell line will be valuable for future studies on the differentiation of trophoblast cells and other components of the extraembryonic membranes.

Stage-specific effects of cadmium on preimplantation embryo development and implantation in the mouse

The effects of cadmium (Cd) exposure during the preimplantation period of pregnancy on the subsequent development and implantation of mouse embryos were examined. Injection of a high dose of Cd (38 mumol Cd/kg body wt.) on day 2 (D1 = vaginal plug), when the embryo is at the two-cell stage, had little effect on the initiation and maintenance of pregnancy when examined on D8. The initiation of implantation (localized sites of increased uterine vascular permeability) in a similarly treated group of mice was assessed in the morning of D5, and these sites were absent in 62% of the animals examined. Thus, Cd treatment on D2 delayed temporarily, but did not prevent implantation and was not embryolethal. In marked contrast, the same dose of Cd administered on D4 caused pregnancy failure in all mice examined on D8. No implantation sites were detected on D5 and the few blastocysts recovered were degenerating. To explore the mechanisms underlying these in vivo stage-specific effects of Cd, preimplantation embryos (two-cell, four-cell, eight-cell and morulae) were exposed in vitro to a high concentration of Cd (50 microM) for 8 h followed by reculture to monitor their potential to develop to the blastocyst stage. Two-cell embryos were remarkably resistant to Cd, but toxicity increased with development, and morulae readily degenerated after Cd exposure. Analysis of the accumulation of 109Cd (50 microM) by preimplantation embryos showed little or none in two-cell embryos, but rapid accumulation and efflux of this metal by blastocysts. Removal of the zona pellucida had no influence on Cd accumulation. Nifedipine (500 nM), a potent voltage-gated calcium channel blocker, and zinc (Zn; 100-fold molar excess) each significantly reduced (approximately 50% in 2 h) Cd accumulation by blastocysts, whereas N-ethylymaleimide (NEM; 20 microM) increased it. These results provide evidence that pregnancy failure after Cd exposure during the preimplantation period reflects a direct embryotoxic effect of Cd, although maternal injury by Cd may also contribute. Resistance to Cd at the two-cell stage (D2) reflects a lack of uptake of this metal, whereas sensitivity to Cd at the blastocyst stage (D4) reflects the ability to accumulate Cd.

Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos

As an approach toward understanding the mechanisms by which cadmium (Cd) exerts its teratogenic effects, the expression and metal regulation of the metallothionein (MT) genes in midgestation mouse embryos were studied by Northern blot and in situ hybridization. Maternal injection of a teratogenic dosage of Cd (50 mumol Cd/kg body wt) did not induce MT mRNA in day 10 (D10) CD-1 mouse embryos, whereas zinc (Zn) (50 mumol/kg was an effective inducer. In contrast, Cd was about 10-fold more potent than Zn at rapidly inducing MT mRNA in D10 embryos incubated in vitro in medium containing micromolar concentrations of these metals. This suggests that following maternal injection, Cd but not Zn is prevented from reaching the D10 embryo and establishes that the embryonic MT genes are not refractory to metal induction, which might have explained the sensitivity of the embryo to Cd. MT mRNA was detected at high levels only in the extraembryonic membranes of D9 embryos exposed to Cd in vivo. On days 9 and 10, no embryonic cell types contained detectable levels of MT mRNA. This mRNA was detected first at low levels in hepatocytes on D11, soon after formation of liver and these levels increased dramatically by D12. Therefore, Cd teratogenicity was not associated with high levels of cell type-specific expression of the MT genes in Cd-sensitive regions of the embryo (neural tube, limb bud), that might have served to target Cd to these cells. Taken together, the results of this study suggest that Cd teratogenicity reflects damage to maternal or extraembryonic tissues. However, the results cannot exclude the possibility that certain cells in the embryo are exceptionally sensitive to low levels of Cd.

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.

Mouse and rat placental cell lines express abundant amounts of laminin

Placental cell lines derived from midgestation placentae of outbred mice and rats were evaluated for the expression of the extracellular matrix protein laminin. The murine cell line, which has not been previously reported, demonstrates morphological characteristics similar to those of the rat cell line. Placental cell lines grow vigorously both in vitro and when transplanted to the peritoneum of allogeneic hosts. When transplanted, placental cells form cyst-like structures (with acellular cores) suspended in the peritoneal fluid, and invade abdominal structures forming solid masses. Using immunohistology, laminin was identified within in vitro cultured cells, within cyst-like structures and their acellular cores, and as a major component of the extracellular matrix of solid masses. Laminin was also identified in the normal rat chorioallantoic placenta. Evaluation of extracts from in vitro cultured placental cells, transplanted placental cells, and the normal chorioallantoic placenta by electrophoresis and immunoblotting demonstrated that laminins were composed of two species with molecular weights of 400,000 (A-chain) and 200,000 (B-chains). Mouse and rat placental cell lines may be valuable for studying laminin biosynthesis and function in the developing placenta.

Trophoendodermal stem cell-derived extracellular matrices : absence of detectable entactin and presence of multiple laminin species

Extracellular matrices (ECM) generated by trophoendodermal stem cells transplanted into the peritoneum of host rats were investigated. Two types of trophoendodermal transplants were studied: (1) free-floating cystic structures, and (2) solid masses adherent to various abdominal organs. Trophoendodermal stem cell ECM obtained from either transplant source was dominated by the presence of laminin similar to Engelbreth-Holm-Swarm (EHS) tumour ECM. However, in contrast to EHS tumour ECM, another ECM component, entactin, was below the level of detection in trophoendodermal stem cell ECM. The laminins present in the two types of trophoendodermal stem cell transplants exhibited distinct differences. Tissues used as sources of one type of laminin were devoid of the other type of laminin. The two species of rat laminin behaved similarly on sodium dodecyl sulphate-polyacrylamide gels and had virtually identical amino acid compositions. The laminins also had similar cruciform patterns when examined by rotary shadowing. Rat laminins differed in their binding to an ion exchange resin: laminin isolated from peritoneal cysts bound to the resin (acidic laminin); laminin isolated from solid masses failed to bind (basic laminin). Acidic rat laminin showed reduced capacity to form laminin-laminin associations when compared with basic rat laminin. Acidic/soluble laminin proved to be a useful reagent in the development of a radio-immunoassay for laminin. Laminin concentrations in the peritoneal fluid of transplant-bearing rats was very high (approximately 400 micrograms/ml) and entirely of the acidic/soluble form. In summary, trophoendodermal stem cell ECM possesses a distinct composition with a lack of detectable entactin, and trophoendodermal stem cells are capable of modulating the characteristics of laminin, depending upon their organization. These features of trophoendodermal stem cell ECM may represent signals responsible for at least some of the unique features of the trophoendodermal stem cell transplants.

Hydrazine sulfate protection against endotoxin lethality: analysis of effects on expression of hepatic cytokine genes and an acute-phase gene☆

Hydrazine sulfate (HS) pretreatment protects mice against the lethal effects of bacterial endotoxin lipopolysaccharide (LPS) through mechanisms yet to be established. The liver was examined as a model organ to determine HS effects on (a) LPS activation of leukocyte (Kupffer cell) interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) genes and (b) subsequent cytokine-mediated induction of the acute-phase response as measured by hepatic metallothionein (MT) gene expression. The utility of this model was documented by in situ hybridization which showed that acute induction by LPS of the IL-1 beta gene occurred in cells found in liver sinusoids, consistent with Kupffer cells, whereas induction of the MT gene occurred in hepatocytes. The cell specific expression of these genes was further verified by Northern blot hybridization to LPS-treated liver RNA which showed that the LPS-mediated increase in hepatic cytokine mRNA levels, unlike that of MT, was not prevented by D-galactosamine (D-GalN) treatment. Northern blot hybridization established that HS pretreatment did not block the acute induction of hepatic cytokine mRNAs (IL-1 beta and TNF-alpha) by LPS nor did it induce these cytokine mRNAs in the absence of LPS. Northern blot hybridization further established that HS did not prevent LPS-mediated activation of hepatocyte MT gene expression. Thus, HS does not prevent LPS from activating liver leukocytes. These results also suggest that HS pretreatment neither prevents the general release of cytokines from LPS activated leukocytes nor the general induction of acute-phase protein gene expression in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Metallothionein Genes in Preimplantation Embryos, Decidua and Placentae

Metallothioneins (MT) are cysteine rich, heavy metal binding proteins whose expression can be regulated by a variety of factors, such as metal ions, glucocorticoids, and cytokines. Although little is known about expression of MT genes during early mammalian development, substantial variations in expression of these genes in the fetal and neonatal period occur which has lead to the concept that MT may play important roles during development, such as maintenance of zinc and copper homeostasis and protection from heavy metal toxicity. However, the precise functions of MT are unknown. We have examined expression and regulation of metallothionein genes from the time of fertilization to midgestation. Utilizing the preimplantation rabbit embryo as a model it was determined that MT is constitutively expressed at low levels in the blastocyst (D6), and zinc can induce MT as early as day-4 of gestation just after the morula to blastocyst transition. The mouse embryo also develops the capacity to respond to metal ions by the late morula stage. Therefore, the preimplantation mammalian embryo can alter expression of the genome in response to changes in the levels of metal ions in the oviductal and uterine milieu just prior to implantation. In mice, MT mRNA is present at low levels in the uterine luminal epithelium just before implantation (D4), but upon implantation, MT gene expression is specifically and dramatically elevated in the deciduum (D5-10). In the midgestation placenta, constitutively high levels of MT mRNA are present in the outer placental spongiotrophoblasts. At this stage of gestation, the visceral yolk sac endoderm also actively expresses the MT genes. These results provide the basis for 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, and perhaps other stress related proteins, play an important role in the establishment and maintenance of normal pregnancy. Studies of the regulation of MT genes in decidua suggest that embryo derived factors are not involved, but rather that these genes may be regulated in an autocrine/paracrine manner, perhaps in response to the pro-inflammatory process of implantation and decidualization.