Age-dependent effects of developmental lead exposure on performance in the Morris water maze.

The neurobehavioral toxicity of developmental exposure to lead (Pb) was investigated by conducting tests of spatial learning in the Morris water maze. Female Long-Evans rats were exposed to 0 or 250 ppm Pb acetate in the diet beginning 10 days prior to breeding and continued throughout gestation and lactation. Pups were weaned onto the same diets as the dams at postnatal day 20 (PN20). Increased levels of Pb were detected in the hippocampus of the 250 ppm Pb acetate group relative to controls. The highest concentration of Pb measured in the hippocampus was at PN21 with decreasing levels at older ages. In the Morris Water Maze, a statistically significant (p < 0.03; female rats) or near significant (p < 0.07; male rats) increase in the time required to find the hidden platform (escape latency) was observed when Pb-treated rats were tested in a reference memory paradigm. This effect was only observed when rats were tested at PN21 and not at older ages. No significant effects of developmental Pb exposure were measured when rats were tested in a working memory paradigm of the Morris water maze at any age. These initial studies indicate an impairment of performance in the swim task in PN21 rats exposed to Pb during development. The age-dependent effect of Pb in this learning paradigm is consistent with previous studies in experimental animals and with the observation that children are more susceptible to Pb-induced cognitive deficits than adults. The Morris water maze may be useful for studying the effects of Pb on learning and memory, and their neurochemical basis.

Structural analysis of the ras transgene in MMTV/v-Ha-ras transgenic mice.

Transgenic animals are becoming increasingly important in laboratory animal research. In cancer research, the role of specific genes in tumorigenesis can be directly tested in live animals by using transgenic animal technology. Since DNA-damaging carcinogens are not required to initiate tumors in transgenic animals, these models are particularly useful in the analysis of genetic alterations associated with tumorigenesis. Southern blot analysis was used to assess the copy number, structure, and methylation status of the ras trans-gene in MMTV/v-Ha-ras transgenic mice. The results indicate that MMTV/v-Ha-ras transgenic mice carry about 20 copies of the ras transgene. The integrated ras transgene is maintained without major rearrangements in normal tissues and in mammary tumors. The ras transgene is methylated in liver, hypomethylated at a single site in normal mammary tissue, and hypomethylated at two sites in mammary tumors. Transgenic animals provide a new model to assess genetic alteration in tumorigenesis.

Linoleic acid controls neonatal tissue-specific stearoyl-CoA desaturase mRNA levels.

The mouse genome contains two stearoyl-CoA desaturase (SCD) structural genes (SCD1 and SCD2) that are expressed in a tissue-specific manner. Brain SCD2 mRNA levels are about 2-fold higher in pups nursed by mothers fed a control diet (5% corn oil (CO), (essential fatty acid (EFA) adequate)), compared with brain SCD2 mRNA levels in pups nursed by mothers fed an EFA-deficient (EFAD) diet (5% coconut oil (COCO)). In contrast to brain, control pup hepatic SCD1 mRNA levels are reduced to < 1.0% of the EFAD pup hepatic SCD1 mRNA levels. EFA status does not alter SCD1 or SCD2 transcription initiation sites. CCAAT/enhancer-binding proteins (C/EBP) have been implicated in the transcriptional control of key genes in energy metabolism. Both the SCD1 and SCD2 gene promoters contain C/EBP transcription factor consensus-binding sites. Neonatal mouse liver expresses C/EBP-alpha, C/EBP-beta and C/EBP-delta mRNAs. In contrast neonatal mouse brain expresses high levels of C/EBP-delta, but little C/EBP-alpha or C/EBP-beta mRNA. EFA intake has no effect on tissue-specific C/EBP isoform mRNA levels suggesting that C/EBP isoform function is controlled at the translational or post-translational level.

Dietary fat promotes mammary tumorigenesis in MMTV/v-Ha-ras transgenic mice.

The purpose of this study was to investigate the influence of dietary fat on mammary tumorigenesis in MMTV/v-Ha-ras transgenic mice. Female MMTV/v-Ha-ras transgenics were fed diets providing 0, 5 or 25% of calories from corn oil (CO). The mammary tumor incidence was 7% (0% CO), 36% (5% CO) and 52% (25% CO). Ras mRNA levels were increased in mammary tumors in the 25% CO group. The ras transgene was hypomethylated in mammary tumors, but not in liver or nontransformed mammary tissue. Mammary tumors expressed apolipoprotein E mRNA. Alterations in gene structure and expression in transgenic mice may suggest mechanisms by which dietary fat promotes mammary tumors.

Quaking phenotype influences brain lipid-related mRNA levels.

Although lipids compose almost 80% of myelin, the influence of quaking on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been previously reported. Understanding the influence of quaking on myelin-specific and lipid-related mRNAs will be useful in determining the mechanism of the quaking defect. Stearoyl CoA desaturase (SCD) catalyzes a key step in the biosynthesis of oleic acid (C18:1, n-9), a major fatty acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levels are all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levels are normal. These results indicate that lipid-related mRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected. The quaking defect influences gene expression in multiple cell types of glial lineage in the developing CNS.

Postnatal dietary fat influences mRNAS involved in myelination.

The synthesis and composition of myelin in the developing mouse central nervous system can be influenced by diet. Postnatal maternal fat intake altered nursing pup brain and liver fatty acid composition. Peak (day 21) proteolipid protein (PLP) and myelin basic protein (MBP) mRNA levels were reduced when pups were nursed by mothers fed a fat-free or 5% coconut oil diet. This effect was reversed by feeding a corn oil based diet. Oleic acid accounts for about 30% of myelin fatty acids. mRNA levels of stearoyl CoA desaturase (SCD), the rate-limiting step in oleic acid synthesis, increase in neonatal mouse brain. Postnatal maternal fat-free feeding reduced day 21 pup brain SCD and LDL receptor, but not apolipoprotein (Apo E) E mRNA levels. In contrast to brain, nursing pup hepatic SCD mRNA levels were induced, LDL receptor mRNA levels were unaffected and Apo E mRNA levels were reduced by postnatal maternal fat-free feeding. Myelin-specific mRNA levels are developmentally regulated and influenced by dietary fat. Neonatal brain SCD and LDL receptor mRNA levels are also altered by neonatal fat intake. The neonatal response to dietary fat is tissue-specific at the mRNA level.

Hormonal regulation of insulin-like growth factor gene expression in pig uterus.

The present studies were undertaken to define potential regulation of endometrial insulin-like growth factor-I (IGF-I) synthesis and secretion by steroid hormones and to correlate conceptus- and serum-derived estrogens with these events. Four experimental groups of gilts were studied: 1) prepubertal gilts administered estradiol (E) and/or progesterone (P4) daily for up to 15 days; 2) mature gilts ovariectomized (ovx) on day 2 of the estrous cycle, and then administered E, P4, or E plus P4 from days 4-11 after estrus; 3) unilaterally pregnant gilts on day 12 or 16 after the onset of estrus; and 4) cyclic gilts administered E on day 11 and hysterectomized 1, 6, 12, or 24 h after E. IGF-I mRNA was measured by blot hybridization of total cellular RNAs, while a specific RIA was used to quantitate levels of tissue and uterine luminal fluid (ULF) IGF-I. In prepubertal gilts, E or P4 increased uterine IGF-I mRNA levels and IGF-I protein. Mature, ovx gilts treated with E, P4, or E plus P4 had increased levels of endometrial IGF-I mRNAs and protein and luminal IGF-I. The magnitude of E or P4 induction was comparable and was not additive. Gravid horns on day 12 had higher amounts of IGF-I in ULF compared to day 12 nongravid horns or day 16 gravid and nongravid horns. However, no significant differences in the amounts of endometrial IGF-I mRNA and tissue IGF-I content between horns on each day were detected. Acute treatment of cyclic gilts (day 11) with E increased levels of endometrial IGF-I mRNAs and luminal IGF-I 1 h after treatment. The effect of E was transient and was reversed within 24 h of E injection. IGF-II and IGF-binding protein-2 mRNAs were regulated by E and P4 distinct from IGF-I mRNAs. These results indicate that E and P4 are involved in the normal regulation of uterine IGF-I synthesis and/or secretion. However, the relative contributions of serum and conceptus-derived estrogens in these processes during the preimplantation period in the pig remain to be defined.

Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase.

Antileukoproteinase (ALP) is a low mol wt mucosal secretory protein which, in human tissues, inhibits the activities of the neutral serine lysosomal proteinases elastase and cathepsin-G. In this study a number of recombinant cDNA clones corresponding to porcine ALP (pALP) were isolated from a cDNA library prepared from porcine endometrial poly(A)+ RNAs. The combined nucleotide sequences of the cDNA clones, representing the entire pALP mRNA sequence, are approximately 600 nucleotides long and encode a protein of 114 amino acids. The deduced amino acid sequence of pALP is 68% similar in primary structure to that of human ALP, is cysteine and proline rich, and exhibits a two-domain structure which, in the human protein, is involved in binding trypsin/cathepsin-G and elastase, respectively. However, pALP appears to lack the internal signal sequence of the corresponding human protein. Northern blot analysis of uterine RNAs using pALP cDNAs as probe demonstrated a single mRNA species approximately 0.8 kilobase in length. Uterine expression of pALP mRNA was highest in mid- and late pregnancy and very low or undetectable in early pregnancy. Estrogen and progesterone increased the levels of uterine pALP mRNA in prepubertal gilts, but not to the levels obtained at mid- and late gestation. pALP mRNA was also abundant in adult pig lung, where its expression was constitutive. Lower levels of pALP were found in fetal and neonatal lung and small intestine and in maternal cervix, spleen, and small intestine. Our study on the molecular cloning and analysis of pALP mRNA represents the first report on the porcine proteinase inhibitor and extends the identification of pregnancy-associated uterine proteins, which may play important functions in embryo or fetal development. The control of expression of pALP mRNA, which is distinct from those of other porcine uterine proteins studied to date, should provide additional insights into the mechanisms of regulation of uterine secretory activity.