The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout (Oncorhynchus clarki lewisi).

To determine if elevated concentrations of waterborne selenium (Se), caused by coal mining, in the Elk River in southeastern British Columbia, may be causing reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory. Eggs from each female were reared separately and the percent mortalities and deformities were related to the selenium content of the eggs. Selenium concentrations in females from the exposed site were highest in the liver (36.6 +/- 22.5 microg/g dry weight, range: 18.3 to 114), followed by the eggs (21.0 +/- 18.3 microg/g, range: 8.7 to 81.3) and the muscle (12.5 +/- 7.7 microg/g, range: 6.7 to 41). Despite these elevated egg Se concentrations, there was no significant effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry deformities or mortalities. Reproductive failure and embryonic terata have been reported at much lower egg Se concentrations in other fish species. The lack of any toxic response in this study may be due to an evolved tolerance to higher tissue Se concentrations in a population of fish living in a seleniferous river system.

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells.

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.

Bisulfite genomic sequencing-derived methylation profile of the xist gene throughout early mouse development.

Differential epigenetic modification by methylation of CpG dinucleotides is a candidate mechanism that may identify the alleles of imprinted genes and result in monoallelic expression of either the maternal or the paternal allele. Determination of the allelic methylation status of imprinted genes in the gametes and during early development is constrained by the limiting quantities of genomic DNA available from these early developmental stages. To circumvent this problem we have used bisulfite genomic sequencing to determine the allelic methylation status of the minimal promoter and a 1-kb region within the Xist gene during preimplantation development. We find that the parental Xist alleles are not differentially methylated in these regions. Our findings are discussed in the context of previous conflicting data obtained using methylation-sensitive restriction enzyme digestion followed by PCR amplification to assay for methylation.

Deoxyadenosine toxicity in an adenosine deaminase-inhibited human CCRF-CEM T-lymphoblastoid cell line causes cell swelling.

The human T-lymphoblastoid cell line CCRF-CEM, pre-treated with 2'-deoxycoformycin, was used as a model for adenosine deaminase deficiency to investigate how 2'-deoxyadenosine exerts its cytotoxic effects. Incubation of these cells with 1 microM or 5 microM deoxyadenosine for 24 and 48 h caused an increase of up to 50% in their modal cell volume as measured by a Coulter Size Distribution Analyzer and this increase in cell volume was accompanied by an increase in their fragility and deformability. The swelling of cells was concomitant with the phosphorylation of deoxyadenosine and its intracellular accumulation as dATP. There was no evidence of osmotic imbalance or of inhibition of the Na+/K(+)-dependent ATPase activity as the intracellular concentrations (and the intracellular:extracellular ratios) of Na+, K+ and Ca2+ were essentially unchanged. Cytochalasin B (20 microM) also caused lymphoblasts to swell over a 6-h period and its effect on cell size was similar to that of either 1 microM or 5 microM deoxyadenosine over 24 or 48 h. Longer time-courses of incubation with cytochalasin B caused severe toxicity leading to the death and lysis of a significant proportion of the cells. Other drugs, such as colchicine, vincristine and vinblastine that are known to affect various components of the cytoskeleton also caused swelling of cells in a concentration- and time-dependent manner but there was no evidence that these effects were additive or synergistic with those of deoxyadenosine. Inhibition of DNA synthesis, either directly by aphidicolin or indirectly by hydroxyurea, was less cytotoxic than the effect caused by deoxyadenosine. We conclude that one of the toxic effects resulting from the excessive phosphorylation of deoxyadenosine and its accumulation as dATP in human T-lymphoblasts is not dependent on inhibition of DNA synthesis but may be caused by the disruption of the cytoskeleton in these cells.

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

Cytoplasmic estrogen receptors in bovine uterus and uterine tube (oviduct).

The effects of progesterone (P) or estradiol benzoate (EB) on unfilled uterine and uterine tubal (oviductal) cytoplasmic estrogen receptors (ER) were studied in nulliparous heifers. Progesterone (4.4 mg/kg of body weight) in oil EB (350 micrograms) in oil, or oil vehicle only (C) were injected IM 24 hours after the end of estrus. The heifers were euthanatized 36 hours later. There was a difference (P less than 0.005) in the concentration of ER among treatment groups, but no difference (P greater than 0.05) between the ER concentrations in the uterus and uterine tube within each treatment group. The affinity constant (KA) of the uterine tubal ER was similar among the treatment groups (P greater than 0.15), and there was no difference (P greater than 0.05) in the KA between the uterus and uterine tubal ER within the P and C treatment groups. The KA of the uterine ER of the EB-treated heifers was greater (P less than 0.001) than that in either P- or C-treated heifers.

Oviduct (uterine tube) transport of ova in the cow.

Normal oviduct (uterine tube) transport of ova was studied in 12 nulliparous heifers oophorosalpingohysterectomized at various times (range, 26 to 85.25 hours) after the end of estrus. The mean oviduct length +/- SEM was 19.7 cm +/- 0.38. The means of left and right oviduct length were not different (P greater than 0.10). The ampulla and isthmus of the oviduct could be indentified grossly by a definite reduction in lumen size and represented 0.66 and 0.34 of the total oviduct length, respectively. The oviduct ipsilateral to ovulation was divided into eight equal segments. Each segment and the uterine horn were flushed with saline solution. An ovum or zygote was recovered from the oviduct of nine heifers. A zygote was surgically recovered from the uterus of one heifer at 76.75 hours after the end of estrus. An ovum or zygote was not recovered from two of the heifers. A significant linear relationship (P less than 0.01) existed between the estimated time after ovulation and the distance the ovum or zygote had traveled. Therefore, the ovum or zygote in these heifers was transported through the oviduct at a constant rate.

Physiochemical properties of oviductal fluid from rabbits exposed to heat stress.

Oviductal fluids (OF) were collected at 24 hour intervals from 27 does. Fluids were collected from all does during estrus and subsequent pregnancy or pseudopregnancy. After collecting OF for 15 days from all does maintained at 21 degrees C and 45% relative humidity (RH), approximately one-half of the does were then subjected to heat stress (33.20+/-0.75 degrees C and 65.4+/-2.3% RH). Oviductal fluid was then collected for 5 days from heat stressed estrous does (HSER), after which all does (both control and HSER) were mated to fertile bucks. Human chorionic gonadotropin was administered to each doe to insure ovulation. Fluids were compared between HSER and heat stressed pregnant rabbits (HSPR) and control estrous (NHER) and pregnant rabbits (NHPR) for volume, pH, various protein concentrations, and total protein content. Fluid volume decreased due both to heat stress and pregnancy. Heat stress increased pH, whereas pregnancy resulted in a decrease. Protein concentration of OF from HSER declined due to heat stress. Protein concentration of OF from NHPR increased prior to breeding, peaking at day 3 of pregnancy, then declined to initial levels. While from HSPR there was also a tendency to have a similar increase, the peak in protein concentration was on day 4 and then decreased on day 5. Total protein content (concentration x volume) of OF from HSER decreased due to heat stress. Total protein content from HSPR and NHPR had similar changes with or without heat stress, although the total protein content from OF of HSPR was less (except for day 3) for the entire 5 day period studied. Relative percentages of albumin decreased and postalbumin increased during the first day of pregnancy (NHPR). These trends were reversed (a relative increase in albumin and a decrease in postalbumin) in OF from HSPR.

Effects of progesterone or estradiol on uterine tubal transport of ova in the cow.

An experiment was designed to determine the effect of progesterone (P) or estradiol benzoate (EB) on uterine tubal transport of ova in the cow. Intramuscular injections of P, EB, or corn oil (C) were administered to heifers 24 hours after the end of estrus. The heifers were euthanatized 60 hours after the end of estrus and the location of the ovum or zygote was determined. Venous serum levels of progesterone and estradiol-17beta were measured by radioimmunoassay. The mean uterine tube (UT) length was 23.9 cm. An ovum or zygote was recovered from 11 of 14 heifers. Serum levels of progesterone and estradiol-17beta were above normal bovine levels following the P and EB treatments, respectively. The mean UT ovum transport rates were 0.42, 0.21 and 0.23 cm/hour in the P, EB and C treatment groups, respectively. The UT ovum transport rate was increased (P<0.05) by the P treatment and EB treatment had no effect (P > 0.05) when compared with the C treatment.