Fig 2: ALDH enzymes are differentially localized in the neonatal murine testis. Images are representative murine testicular cross-sections displaying IHC analysis of ALDH localization at various neonatal ages. ALDH1A1 is represented in A, E, I, M, Q, and U for 0, 5, 10, 15, 20, and 30 dpp, respectively. ALDH1A2 is represented in B, F, J, N, R, and V, while ALDH1A3 is represented in C, G, K, O, S, and W for the same time points. Finally, ALDH8A1 is displayed in D, H, L, P, T, and X. Negative controls (-ve) are shown in Y-BB. Brown staining indicates an immunopositive reaction. Arrows indicate immunopositive cells, while the respective colors indicate the following cell types: red, Sertoli cells; yellow, Leydig/interstitial cells; dark blue, spermatid; light blue, spermatocyte; green, gonocyte/spermatogonia. Bars = 100 µm.

Fig 3: ALDH1A1 and ALDH1A2 levels do not vary in a manner similar to RA. A represents the cell types entering the testicular environment following synchrony protocol (top row) or control (bottom row). ALDH1A1 (B and D) and ALDH1A2 (C and E) protein levels were quantified in both synchronized (red) and control (blue) animals using a tandem HPLC-MS/MS approach. In the neonatal analysis (B and C), the vertical axis represents ALDH protein/mg of S10 protein. The horizontal axis represents time following neonatal synchrony treatment. Vertical error bars represent the SEM. Student t-tests were used to compare the protein levels in synchronized and control animals (*P < 0.05, **P < 0.01). In the synchronized adult analysis (D and E), 25 animals were collected at 12-h intervals between 42 and 50 days postsynchrony. The midpoint of synchrony was determined and used to bin each sample into three categories (stages II–VII, stages VIII–IX, and stages X–I). ALDH protein/mg of S10 protein was determined and averaged for both the unsynchronized controls (blue) and the three bins (red). The vertical control error bar is representative of the SEM for each category. ALDH1A3 levels were measured in both the adult and neonatal testis, but were at concentrations below the threshold of detection.

Fig 4: Expression of retinoic acid target genes in the infarct zone and effects on cardiofibroblast proliferation. A) Gene expression of retinoic acid target genes in cardiofibroblast (CF) and cardiomyocytes (CM) isolated from left ventricles of infarcted RAR-luciferase reporter hearts 1 week after infarction. RNA was extracted from cells from the infarcted zone and amplified with real time PCR using primers specific for retinol binding protein 1 (RBP1), cytochrome P450 26B1 (CYP26B1), aldehyde dehydrogenase 1A2 (ALDH1A2), and retinoic acid receptors alpha, beta and gamma (RAR a,ß,?). The figure shows test gene expression relative to expression of rpl32, which was similar in both cell types. Data are mean±SD of n = 5 experiments in each group. B) Cardiofibroblasts were isolated from C57BL6 hearts and cultured in medium supplemented with 1 µM all-trans retinoic acid (atRA) and 10 µM EdU, as indicator of cell division. After 96 hours, EdU incorporation was evaluated by flow cytometry. Data are presented as mean±SD of n = 4 in each group.

Fig 5: Expression of retinoic acid target genes and endogenous metabolites after infarction.Wild type C57Bl6 mice were subjected to in vivo ligation of the left coronary artery or sham surgery, and hearts were sampled serially for RNA extraction and amplification of retinoic acid target genes with real time PCR, or proteins were extracted for western blotting. Hearts were also sampled from mice without any surgery (preoperative). Gene expression of retinol binding protein 1 (RBP1) (A), aldehyde dehydrogenase 1A2 (ALDH1A2) (B), and cytochrome p45026B1 (CYP26B1) are shown (C). (n = 6–8 in each group at each time point). D) Representative western blot analysis of retinoic acid transporting and metabolizing proteins one week after induction of infarction or sham operation. Ponceau solution was used as protein loading (control). Histograms show the relative density of RBP1 at 16 kDa and ALDH1A2 at 53–57 kDa in infarcted and sham operated hearts. E) Endogenous retinoic acid metabolite concentrations were evaluated by triple-stage liquid chromatography/tandem mass spectrometry one week after induction of myocardial infarction. The infarcted zone of the left ventricle (INF), the periinfarcted zone (PERIINF) and left ventricles from sham operated hearts (SHAM) were investigated (n = 6). Data are shown as mean±SD.