Retinoic Acid Synthase Research Articles

Seasonal changes in vitamin A metabolism-related factors in the oviduct of Chinese brown frog (Rana dybowskii)

The oviduct of the Chinese brown frog (Rana dybowskii) expands during pre-brumation rather than the breeding period, exhibiting a special physiological feature. Vitamin A is essential for the proper growth and development of many organisms, including the reproductive system such as ovary and oviduct. Vitamin A is metabolized into retinoic acid, which is crucial for oviduct formation. This study examined the relationship between oviducal expansion and vitamin A metabolism. We observed a significant increase in the weight and diameter of the oviduct in Rana dybowskii during pre-brumation. Vitamin A and its active metabolite, retinoic acid, notably increased during pre-brumation. The mRNA levels of retinol binding protein 4 (rbp4) and its receptor stra6 gene, involved in vitamin A transport, were elevated during pre-brumation compared to the breeding period. In the vitamin A metabolic pathway, the mRNA expression level of retinoic acid synthase aldh1a2 decreased significantly during pre-brumation, while the mRNA levels of retinoic acid α receptor (rarα) and the retinoic acid catabolic enzyme cyp26a1 increased significantly during pre-brumation, but not during the breeding period. Immunohistochemical results showed that Rbp4, Stra6, Aldh1a2, Rarα, and Cyp26a1 were expressed in ampulla region of the oviduct. Western blot results indicated that Aldh1a2 expression was lower, while Rbp4, Stra6, RARα, and Cyp26a1 were higher during pre-brumation compared to the breeding period. Transcriptome analyses further identified differential genes in the oviduct and found enrichment of differential genes in the vitamin A metabolism pathway, providing evidences for our study. These results suggest that the vitamin A metabolic pathway is more active during pre-brumation compared to the breeding period, and retinoic acid may regulate pre-brumation oviductal expansion through Rarα-mediated autocrine/paracrine modulation.

Evidence for RA-dependent meiosis onset in a turtle embryo.

Meiotic entry is one of the earliest sex determination events of the germ cell in higher vertebrates. Although advances in meiosis onset have been achieved in mammals, birds and fish, how this process functions in reptiles is largely unknown. In this study, we present the molecular analysis of meiosis onset and the role of retinoic acid (RA) in this process in the red-eared slider turtle. Our results using Stra8 as a pre-meiosis indicator show that in the female embryonic gonad, meiosis commitment starts around stage 19. Additionally, signals of the meiosis marker Sycp3 could be detected at stage 19 and become highly expressed by stage 23. No expression of these genes was detected in male embryonic gonads, suggesting the entry into meiosis prophase I was restricted to female embryonic germ cells. Notably, RA activity in fetal gonads is likely to be elevated in females than that in males, as evidenced by the higher expression of RA synthase Aldh1a1 and lower expression of RA-degrading enzyme Cyp26a1 in female gonads prior to meiotic entry. In addition, exogenous RA treatment induced the expression of Stra8 and Sycp3 in both sexes, whether in vivo or in vitro. Together, these results indicate that high levels of RA in the embryonic female gonads can lead to the initiation of meiosis in the turtle.

Brown Rice Inhibits Development of Nonalcoholic Fatty Liver Disease in Obese Zucker (fa/fa) Rats by Increasing Lipid Oxidation Via Activation of Retinoic Acid Synthesis

BackgroundWhite rice and its unrefined form, brown rice, contain numerous compounds that are beneficial to human health. However, the starch content of rice can contribute to obesity, a main risk factor for nonalcoholic fatty liver disease (NAFLD). ObjectivesWe investigated the effect of rice consumption on NAFLD and its underlying molecular mechanism. MethodsWe randomly divided 7-week-old male obese Zucker (fa/fa) rats, an animal model of NAFLD, into 3 groups (n = 10 each) fed 1 of 3 diets for 10 weeks: a control diet (Cont; AIN-93G diet; 53% cornstarch), a white rice diet (WR; AIN-93G diet with cornstarch replaced with white rice powder), or a brown rice diet (BR; AIN-93G diet with cornstarch replaced with brown rice powder). Liver fat accumulation and gene expression related to lipid and vitamin A metabolisms, including retinoic acid (RA) signaling, were analyzed. ResultsHepatic lipid values were significantly decreased in the BR group compared with the Cont group, by 0.4-fold (P < 0.05). The expression of genes related to hepatic fatty acid oxidation, such as carnitine palmitoyltransferase 2, was approximately 2.1-fold higher in the BR group than the Cont group (P < 0.05). The expression of peroxisomal acyl-coenzyme A oxidase 1 and acyl-CoA dehydrogenase medium chain was also significantly increased, by 1.6-fold, in the BR group compared with the Cont group (P < 0.05). The expression of VLDL-secretion-related genes, such as microsomal triglyceride transfer protein, was also significantly higher in the BR group (2.4-fold; P < 0.05). Furthermore, aldehyde dehydrogenase 1 family member A1, an RA synthase gene, was 2-fold higher in the BR group than the Cont group (P < 0.05). ConclusionsBrown rice prevented development of NAFLD in obese Zucker (fa/fa) rats. The beneficial effects of pregelatinized rice on NAFLD could be manifested as increased fatty acid oxidation and VLDL secretion, which are regulated by RA signaling.

Retinoic acid and androgen influence germ cells development and meiotic initiation in juvenile orange-spotted grouper, Epinephelus coioides

Meiosis is essential for germ cells development for all sexually reproducing species. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in mammals, birds and tetrapods. Here, we investigated the effects of RA on meiotic initiation and sex determination in protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Expression profile investigations of meiotic marker genes during gonadal development indicated that germ cells undergone meiosis approximately at 180 days after hatching in the orange-spotted grouper. RA synthase inhibitor treatments on juvenile orange-spotted groupers resulted in impeded germ cells development and delayed meiotic initiation with simultaneous down-regulation of vasa, dazl, sycp3 and rec8, which was rescued by exogenous RA administration. Additionally, exogenous androgen treated fish showed a delayed meiotic initiation consistent with decreased sycp3 and rec8 expression and were directed to a spermiogenesis fate. Our results imply that meiotic initiation in the orange-spotted grouper is strongly influenced by RA and androgen, and the regulation of meiotic initiation may involve in the spermatogenesis induced by exogenous androgen.

Retinoic acid triggers meiosis initiation via stra8-dependent pathway in Southern catfish, Silurus meridionalis

Existing studies demonstrated that retinoic acid (RA) regulates meiotic initiation via stra8-independent pathway in teleosts which lack stra8 in their genomes. However, stra8 was recently identified from several fish species including Southern catfish (Silurus meridionalis). To explore the existence of stra8-dependent pathway in RA mediated meiotic initiation in fishes, in the present study, the genes encoding RA synthase aldh1a2 and catabolic enzyme cyp26a1 and cyp26b1 were cloned from the Southern catfish. By immunohistochemistry, Aldh1a2 signal was observed in gonads of both sexes during the meiotic initiation period. By real-time PCR, differentially expressed gene was observed for cyp26a1, but not for cyp26b1, in gonads during the meiotic initiation. Administration of exogenous RA or inhibition of endogenous RA degradation by either KET (RA catabolic enzyme inhibitor) or cyp26a1 knockdown using CRISPR/Cas9 induced advanced meiotic initiation in the ovaries as demonstrated by increased Stra8/stra8 expression and appearance of oocytes. In contrast, treatment with RA synthase inhibitor DEAB resulted in delayed meiotic initiation and Stra8/stra8 expression in the ovaries, which was rescued by exogenous RA administration. These results indicated that (1) RA triggers the onset of meiosis via stra8-dependent pathway in stra8 existing teleosts, as it does in tetrapods; (2) exogenous RA can rescue the endogenous RA deficiency; (3) Cyp26a1, instead of Cyp26b1, is the key catabolic enzyme involved in meiosis initiation in teleosts. Taken together, RA might trigger meiotic initiation via stra8-dependent and -independent pathway in different teleosts.

Exopolysaccharides Produced by Leuconostoc mesenteroides Strain NTM048 as an Immunostimulant To Enhance the Mucosal Barrier and Influence the Systemic Immune Response.

Leuconostoc mesenteroides strain NTM048 has been shown to have intestinal IgA-inducing ability. In this study, we investigated the immunostimulant potency of an exopolysaccharide secreted from strain NTM048 (NTM048 EPS) in vitro and in vivo in a murine model. NTM048 EPS ranges in size from 10 to 40 kDa and is speculated to be mainly composed of glucose and fructose. The in vitro study revealed that NTM048 EPS induced total and antigen-specific IgA production by Peyer's patch cells and influenced Th1 and Th2 cell-mediated response in splenocytes. Oral administration of NTM048 EPS dose-dependently induced fecal IgA production accompanied by the up-regulation of retinoic acid synthase and transforming growth factor-β receptor genes in Peyer's patch cells. Flow cytometric analysis of the splenocytes revealed an increase of the CD3+ T-cell population and the ratio of CD4+ T-cells/CD8+ T-cells. These results indicate that NTM048 EPS could enhance the mucosal barrier and influence the systemic immune response.

Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts.

The LIM Protein fhlA is Essential for Heart Chamber Development in Zebrafish Embryos

Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology. However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog of human FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restricted to the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression of the cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulated expression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns at the 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domain in embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes in the number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause between heartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals. Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed the expression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthase inhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterations in the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RA signaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducing the number of cardiac cells.

Identification of a retinoic acid‐responsive neural enhancer in the Ciona intestinalis Hox1 gene

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent.

Retinoic Acid-Mediated Gene Expression in Transgenic Reporter Zebrafish

Retinoic acid-mediated gene activation is important for normal vertebrate development. The size and nature of retinoic acid make it difficult to identify the precise cellular location of this signaling molecule throughout an embryo. Additionally, retinoic acid (RA) signaling is regulated by a complex combination of receptors, coactivators, and antagonizing proteins. Thus, in order to integrate these signals and identify regions within a whole developing embryo where cells can respond transcriptionally to retinoic acid, we have used a reporter transgenic approach. We have generated several stable lines of transgenic zebrafish which use retinoic acid response elements to drive fluorescent protein expression. In these zebrafish lines, transgene expression is localized to regions of the neural tube, retina, notochord, somites, heart, pronephric ducts, branchial arches, and jaw muscles in embryos and larvae. Transgene expression can be induced in additional regions of the neural tube and retina as well as the immature notochord, hatching gland, enveloping cell layer, and fin by exposing embryos to retinoic acid. Treatment with retinoic acid synthase inhibitors, citral and diethylaminobenzaldehyde (DEAB), during neurulation, greatly reduces transgene expression. DEAB treatment of embryos at gastrulation phenocopies the embryonic effects of vitamin A deprivation or targeted disruption of the RA synthase retinaldehyde dehydrogenase-2 in other vertebrates. Together these data suggest that the reporter expression we see in zebrafish is dependent upon conserved vertebrate pathways of RA synthesis.

Experimental induction of jaw, gill and pectoral fin malformations in Japanese flounder, Paralichthys olivaceus, larvae

The phenotypes of skeletal malformations induced in pharyngeal arches and pectoral fins of Japanese flounder larvae by retinoic acid (RA), disulfiram, 2,2′-dipyridyl and azetidine-2-carboxylic acid were characterized, RA controls gene expression essential for pharyngeal and pectoral fin development; disulfiram is an inhibitor of RA synthase, 2,2′-dipyridyl and azetidine-2-carboxylic acid are inhibitors of collagen synthesis. In larvae exposed to RA at shield stage for 1 h, the Meckel's cartilage did not form in mandible arch. Exposure to RA from hatching period shifted the growth direction of the pharyngeal cartilages posteriorly. Disulfiram did not affect the cartilage formation when given before hatching, even though it shortened the trunk. However, disulfiram exerted teratogenic effects when given after hatching time, inducing bending pharyngeal cartilages and S-shaped pectoral fin plate. 2,2′-Dipyridyl given from hatching also caused bending of pharyngeal cartilages and S-shaped pectoral fin plates. Azetidine-2-carboxylic acid reduced the size of cartilages, without causing remarkable malformation. Thus, it was demonstrated that both RA and inhibitor of its synthesis, and inhibitors of collagen synthesis exert specific teratogenic effects on both pharyngeal and pectoral skeletons of flounder larvae.

Retinoic acid in the anteroposterior patterning of the zebrafish trunk.

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.

Characteristic properties of retinal oxidase (retinoic acid synthase) from rabbit hepatocytes

Retinal oxidase (retinoic acid synthase) (EC 1.2.3.11) was purified electrophoretically, as a single protein band, from rabbit liver cytosol. The characteristic properties, enzymatic reaction mechanism, substrate specificity and kinetic parameters for retinals and molecular oxygen of the retinal oxidase were investigated. The K m values for all- trans-retinal of the retinal of the retinal oxidase was the lowest than those for the other retinal derivatives. The retinal oxidase is a metalloflavoenzyme containing 2 FADs as the coenzyme, and 8 irons, 2 molybdenums,2 disulfide bonds and 8 inorganic sulfurs. Its relative molecular mass was determined to be 270 kDa by gel filtration HPLC on a TSKgel G3000sw XL column. Its minimum molecular mass was estimated to be 135 kDa by SDS-PAGE. The optical spectrum of the retinal oxidase showed absorption peaks at 275, 340 and 450 nm, and shoulders at 420 and 473 nm, in the oxidized form. The molecular extinction coefficients of the oxidase at selected wavelengths were determined. Circular dichroism spectra of the retinal oxidase were measured in the ultraviolet and visible regions. These spectra showed positive absorption in the visible region. The amino-acid composition was determined. The activity of the oxidase was not affected by any cofactors, such as NADP +, NAD +, NADPH and NADH, and it did not occur under anaerobic conditions. The oxidase was not inhibited by BOF-4272, a potent inhibitor of xanthine dehydrogenase, or rat anti-xanthine dehydrogenase IgG. Experiments on retinoic acid formation under 18O 2 or H 2 18O demonstrated that the oxygen of waer was incorporated into retinoic acid by the retinal oxidase, but not molecular oxygen.

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.2.2. The maximum pH of the reaction in the liver microsomes was 7.6.3.3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined.4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG.7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.