Protooncogene c-fos Involvement in the Molecular Mechanism of Rat Brain Sexual Differentiation.

Brain sexual differentiation is mediated through testosterone, which acts during the perinatal period in the form of both 5alpha-dihydrotestosterone and estradiol. In order to gain insight into the molecular mechanisms involved, we studied induction of c-fos, an index of functional neuronal activation, in the 2-day-old female rat brain after injection of a masculinizing dose of testosterone. Administration of testosterone resulted in induction of c-fos gene expression in the hypothalamus, as determined by Northern analysis. Following immunocytochemistry, we demonstrated an increase in the number of Fos-positive nuclei in the median and medial preoptic nucleus, the medial preoptic area extending to the lateral preoptic area, and the peri- and paraventricular area. In an effort to see whether testosterone acted as 5alpha-dihydrotestosterone or as estradiol, we injected either steroid and looked at fos induction. Estradiol mimicked the effect of testosterone, while 5alpha-dihydrotestosterone was without effect. Furthermore, injection of an estrogen receptor blocker, clomiphene, together with testosterone, abolished the testosterone-induced increase in Fos-positive nuclei, thus confirming the finding that testosterone induces c-fos by acting through estrogen receptors. Electrophoretic mobility shift assays showed that nuclear extracts from 2-day-old female hypothalamus contain a protein, most probably the estrogen receptor, which binds specifically to oligodeoxynucleotides with the sequence of either vitERE, the consensus estrogen-responsive element (ERE) found in the vittelogenin gene, or fosERE, the ERE found in the 3'-untranslated region of the mouse c-fos gene. This suggests that the effect of testosterone-derived estradiol on c-fos expression is a direct one, mediated by binding of estrogen receptors to an ERE in the c-fos gene-regulatory regions.

In utero radiation-induced apoptosis and p53 gene expression in the developing rat brain.

PURPOSE: This study addressed the question of the role of the p53 gene in prenatal low-dose radiation-induced apoptosis in the neuroepithelium, in an effort to elucidate molecular mechanisms involved in the extreme radiosensitivity of the developing brain. MATERIALS AND METHODS: Pregnant Wistar rats were exposed to a single dose of 10, 20 or 40 cGy of X-rays on day 15 or 17 of gestation. Animals were sacrificed 4 or 24h after exposure. Apoptosis was studied by gel electrophoresis of isolated DNA and in situ by the TUNEL reaction. Expression of the p53 gene was studied by immunocytochemistry and Western analysis, as well as Northern analysis, for the detection of the protein and mRNA respectively. RESULTS: In utero low-dose irradiation led to apoptosis and an increase of p53 gene expression in the developing rat brain. Apoptotic as well as p53 immunopositive cells were detected among proliferating, migratory and post-mitotic neurones in the developing neuroepithelium following prenatal irradiation, even after only l0 cGy. In addition to the p53 protein, p53 mRNA brain levels were also increased following prenatal irradiation. CONCLUSIONS: Low-dose prenatal irradiation of the developing brain led to p53 induction and cell death by apoptosis.

In utero radiation-induced changes in growth factor levels in the developing rat brain.

PURPOSE: To investigate the role of growth factors in the compensatory response to radiation injury during development of the brain. Levels of gene expression in the embryonic rat brain were assessed for IGF-I, IGF-II, BDNF and NT-3. MATERIALS AND METHODS: Pregnant Wistar rats were exposed to a single dose of 10, 20 or 40 cGy X-rays on day 15 or 17 of gestation. Animals were sacrificed 4 or 24 h after exposure. IGF-I, BDNF and NT-3 proteins were detected by immunocytochemistry, while IGF-I and IGF-II mRNA by in situ hybridization, and Northern analysis respectively. RESULTS: In utero low dose X-irradiation led to a decrease in IGF-I gene expression and a compensatory increase in the expression of IGF-II, BDNF and NT-3 in the developing rat brain. IGF-I, BDNF and NT-3 immunopositive cells were detected among proliferating, migratory and post-mitotic neurones in the developing neuroepithelium. CONCLUSIONS: Low dose prenatal irradiation of the developing brain results in down-regulation of IGF-I, which could lead to cell death by apoptosis. On the other hand, IGF-II, BDNF and NT-3 gene expression is increased following irradiation, possibly as a compensatory mechanism.

Aging-related changes in IGF-II and c-fos gene expression in the rat brain.

The protein products of growth factor genes such as IGF-II and cellular oncogenes such as c-fos are believed to be necessary for the support of normal neuronal function. Steady-state levels of c-fos and IGF-II mRNA were determined in the brain of young and old rats, using Northern analysis. Both RNAs were found to be decreased in the brain of aged rats. Age-related decrease was detected in the hippocampus, hypothalamus, striatum, cerebral cortex and cerebellum, for IGF-II mRNA, and in the cerebral cortex and cerebellum for c-fos mRNA. Furthermore, changes in the degree and pattern of DNA methylation were noted at both gene loci, in the aged rat brain. Our results could reflect changes at the genomic level possibly related to the process of aging and the accompanying decline in brain function.

The development of gluconeogenesis in rat liver. Effects of glucagon and ether.

1. Administration of glucagon to foetal rats produced a 10-15-fold increase in hepatic phosphoenolpyruvate carboxykinase activity together with a similar increase in the overall pathway of pyruvate conversion into glycogen in liver slices. 2. Glucagon was without effect on gluconeogenesis in vivo, which remained at approx. 0.1% of the incorporation as measured in newborn animals. 3. The apparent discrepancy between these results was due to the ether anaesthesia that was required for experimentation in vivo. Under conditions when minimal ether was used, the rates of labelling of glycogen from [3-(14)C]pyruvate in vivo were increased 10-20-fold and there was an additional stimulus by glucagon. 4. Ether anaesthesia produced a more reduced redox state of the foetal liver cytosol and lowered the ATP/ADP concentration ratio. 5. It is proposed that these effects are significant in the limitation of gluconeogenesis in the foetal rat liver, so that only with high phosphoenolpyruvate carboxykinase activity, high ATP concentration and a relatively oxidized cytosol redox state will a functional gluconeogenic pathway be present.