depicts the mean daily food intake (g) of nursing dams exposed to 21% O2 (Normoxia; N=5) or 12% O2 (Hypoxia; N=4). Hypoxia significantly decreased food intake at each time point, when compared to control (p<0.05). Interestingly, hypoxic dams significantly increased their daily food consumption during the study period, more so than that of the normoxic dams. As a result, hypoxic dam food intake approached that of normoxic dams by the end of the study.
Figure 1 Mean food intake (g/day) of nursing dams exposed to normoxia (21% O2) or hypoxia (12% O2) from delivery to postnatal (PD) 7. Dams were fed ad libitum and individual daily food intake was recorded. Data are reported as mean ± SEM for each group (more ...)
shows plasma ACTH (upper panel) and corticosterone (lower panel) concentrations in pups sacrificed at PD7. Hypoxia from birth significantly increased plasma ACTH when compared to the normoxic control (p=0.025). Normoxic pups reared by dams pair-fed to reduce daily food intake to match hypoxic dams did not exhibit any change in plasma ACTH concentrations (p>0.05). Plasma corticosterone in hypoxic pups was significantly increased when compared to control (p=0.006). There was no effect of decreased maternal food intake on plasma corticosterone concentrations in normoxic pups (p>0.05).
Figure 2 Plasma ACTH (pg/mL) and corticosterone (ng/mL) in control pups (Normoxia; N=10), in pups exposed to 12% O2 (Hypoxia; N=8), and in pups reared by dams pair-fed to match hypoxic dam food intake (Normoxia Pair Fed; N=22). Pups were sacrificed at PD7 and (more ...)
lists selected results from the microarray analysis of adrenal gene expression in pups exposed to hypoxia from birth with food available ad libitum (as compared to Normoxia). Genes were selected based on the putative role(s) their products may play in increasing adrenal steroid production. lists genes that exhibited a significant increase in expression following hypoxia. Many of the genes increased by hypoxia encode proteins having oxidoreductase activity, including Cyp1b1, Gmpr, Xdh, Srd5a1, Cox4b, Dbh, Fmo1, and G6pdx. Other genes that displayed increased expression encode proteins involved in the glycolytic pathway (Eno1 and Aldoc), electrochemical-driven transport (Slc18a2 and Slc7a1), lipid metabolism (Cpt2 and Vldlr), and intracellular signaling (Pde10a and Ass). Hypoxia also increased the expression of genes associated with neural/adrenomedullary function (Nefh, Reln, Anp32a, Uts2, Npy, and Dbh).
Microarray analysis of adrenal gene expression in the 7-day-old rat: effects of hypoxia from birth.
lists significant decreases in adrenal gene expression in pups exposed to hypoxia from birth. The expression of genes encoding proteins with oxidoreductase activity (Cyp2f1, Cyp11b3, Hsd11b2, Cyp4f6, and Cox8h) was decreased. Interestingly, the expression of Cox8h (cytochrome c oxidase subunit; mitochondrial) was decreased nearly eight-fold, the largest fold-change measured. The expression of Ucp1 and Omb5 was also decreased by hypoxia. These genes encode mitochondrial proteins involved in oxidative phosphorylation and steroid metabolism, respectively. Other examples of decreased expression include Mvk (cholesterol biosynthesis) and Pla2g4a and Rgs2 (intracellular signaling), as well as that of Cdh1, Nfasc, Nes, and Tmsb10 (cellular adhesion and cytoskeleton-associated proteins).
lists real-time PCR results, expressed as a percent of control (Normoxia). Calculated delta Ct values are also included in . Real-time PCR was initially used to verify selected microarray results. Twelve genes were selected based on their possible contribution to the functional endpoint of the study (i.e. increased plasma corticosterone). Three of the twelve PCR results did not agree with those indicated by microarray (Gmpr, Rgs2, and Cpt2). This discordance was confirmed by a second PCR assay for each of the three genes. The other nine results from real-time PCR showed trends that agreed with the microarray; however, only two of them reached statistical significance (Cyp1b1 and Nsf).
Real-time PCR analysis of adrenal gene expression: verification of selected microarray results.
The expression of genes that encode proteins involved in adrenal function or the cellular response to hypoxia was also assayed by real-time PCR (), even though these genes were not considered significant by microarray analysis. Interestingly, hypoxia from birth had a tendency to increase the expression of StAR, Cyp11a, Ldlr, and Vegf (p>0.05). The only statistically significant changes in expression were for Cyp21a1 and Cyp1b1 (p<0.05). The effects of maternal food restriction per se (Normoxia vs. Normoxia Pair Fed) on genes involved in adrenal function included significant decreases in StAR, Cyp11a, Cyp1b1, and Cyp21a1 (p<0.05). Comparison of PCR results between Hypoxia and Normoxia Pair Fed treatments showed that the expression of StAR, Cyp21a1, Cyp1b1, and Ldlr were significantly increased (p<0.05). These comparisons provide a more detailed description of the effects of hypoxia per se by accounting for decreased maternal food intake associated with hypoxic exposure.
Real-time PCR analysis of adrenal gene expression: analysis of genes not meeting microarray cutoff criteria.