VITAMIN A AND CAROTENOID METABOLISM DURING PREGNANCY AND DEVELOPMENT

To carry out the experiments in objective 1, ML-/-R-/- females be mated with ML-/-R-/- males. At the time of the plug (0.5dpc), the females will be maintained on a vitamin A-sufficient (25 IU/g), or vitamin A-deficient (<0.22 IU/g) or vitamin A-excess diet (220 IU/g). Wild-type, RBP-/- and ML-/- females mated with wild-type, RBP-/- and ML-/- males, respectively, will be used as controls on the same diets during pregnancy. At 14.5dpc, embryos will be collected, genotyped and examined both for their external morphology and by HPLC analysis. Their retinoid status will be also assessed by analyzing the expression levels of LRAT, Stra6, RALDH2 and Cyp26A1 mRNA by real-time PCR. Maternal serum and tissues and placenta will be also collected and analyzed by HPLC. In addition, wild-type, RBP-/-, ML-/- and ML-/-R-/- dams at 14.5 dpc will be fed by gavage with a single dose of radiolabeled retinol (2x106 cpm all-trans[3H]retinol and 6ug of unlabeled all-trans-retinol in a peanut oil). Subsequently, the dams will be sacrificed 1, 5, 10 and 24 hours after the injection. Total [3H]retinoid in maternal and embryonic tissues will be measured by a scintillation counter and HPLC will be also performed on the same tissues to analyze the distribution of retinol and retinyl ester and [3H]retinoid. To carry out the experiment in objective 2, CMO I-/-RBP-/- females will be mated with CMO I+/-RBP-/- males. At 0.5dpc, the females will be placed on a vitamin A-deficient diet and will be administered B-carotene according to one of the following regimens: #1. from 0.5 to 14.5dpc (throughout gestation); #2. from 7.5 to 9.5dpc; #3. from 7.5 to 14.5dpc. A single daily dose of B-carotene (10microg/g of body weight) will be administered to the dams by IP injection. Pregnant females will be sacrificed at 14.5dpc, and embryos will be collected and examined both for their external morphology and in histological sections. Maternal serum, liver, lung and adipose tissue, placenta, yolk sac and whole embryo will be collected to perform retinol, retinyl ester and carotenoids analysis by HPLC. To carry out the experiments in objective 3, wild-type, LDLr knockout (LDLr-/-), SR-BI knockout (SR-BI-/-) and ML-/- mice will be used. LDLr-/- and ML-/- females, maintained on a vitamin A-sufficient diet, will be mated with LDLr+/- and ML+/- males, respectively. Since SR-BI-/- females are not fertile, SR-BI+/- females will be mated with SR-BI-/- males. At 10.5, 12.5, 14.5 and 16.5dpc dams will be supplemented by IP injection with a single dose of radiolabeledB-carotene (106 cpm and 10microg of B-carotene/g of body weight). Dams will be sacrificed 1, 5, 10 and 24 hours after the injection. Wild-type preganant females injected with radiolabeled B-carotene will serve as controls. Maternal and embryonic tissues will be analyzed for their labeled and unlabeled B-carotene content by simultaneous analysis of carotenoids by HPLC analysis and radiolabeled carotenoids counts. B-carotene metabolites will also be identified by GC-MS and LC-MS analysis.