Retinoic acid metabolism and signaling pathways in the adult and developing mouse testis

As a first step in investigating the role of retinoic acid (RA) in mouse testis, we analyzed the distribution pattern of the enzymes involved in vitamin A storage (lecithin:retinol acyltransferase), RA synthesis (beta-carotene 15,15'-monoxygenase and retinaldehyde dehydrogenases) and RA degradation (cytochrome P450 hydroxylases) as well as those of all isotypes of receptors transducing the RA signal [RA receptors (RARs) and rexinoid receptors (RXRs)]. Our data indicate that in adult testis 1) cytochrome P450 hydroxylase enzymes may generate in peritubular myoid cells a catabolic barrier that prevents circulating RA and RA synthesized by Leydig cells to enter the seminiferous epithelium; 2) the compartmentalization of RA synthesis within this epithelium may modulate, through paracrine mechanisms, the coupling between spermatogonia proliferation and spermatogenesis; 3) retinyl esters synthesized in round spermatids by lecithin:retinol acyltransferase may be transferred and stored in Sertoli cells, in the form of adipose differentiation-related protein-coated lipid droplets. We also show that RARalpha and RXRbeta are confined to Sertoli cells, whereas RARgamma is expressed in spermatogonia and RARbeta, RXRalpha, and RXRgamma are colocalized in step 7-8 spermatids. Correlating these expression patterns with the pathological phenotypes generated in response to RAR and RXR mutations and to postnatal vitamin A deficiency suggests that spermiation requires RXRbeta/RARalpha heterodimers in Sertoli cells, whereas spermatogonia proliferation involves, independently of RXR, two distinct RAR-mediated signaling pathways in both Sertoli cells and spermatogonia. Our data also suggest that the involvement of RA in testis development starts when primary spermatogonia first appear.     

Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males

Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that Rdh10 loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of Rdh10 in postnatal mouse testes and found that an RDH10 deficiency in Sertoli cells, but not in germ cells, results in a mild germ cell depletion phenotype. A deficiency of RDH10 in both Sertoli and germ cells in juvenile mice results in a blockage of spermatogonial differentiation, similar to that seen in vitamin A-deficient animals. This defect in spermatogenesis arises from a complete deficiency in juvenile testicular RA synthesis and can be rescued by retinoid administration. Thus, in juvenile mice, the primary, but not exclusive, source of RA in the testes is Sertoli cells. In contrast, adult Rdh10-deficient mice exhibit phenotypically normal spermatogenesis, indicating that during development a change occurs in either the cellular source of RA or the retinaldehyde dehydrogenase involved in RA synthesis.     

Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment

Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos. Morphogenetic RA gradients are thought to be set up by tissue-specific actions of retinaldehyde dehydrogenases (RALDHs) and catabolizing enzymes. According to the species, two enzymatic pathways (β-carotene cleavage and retinol oxidation) generate retinaldehyde, the substrate of RALDHs. Placental species depend on maternal retinol transferred to the embryo. The retinol-to-retinaldehyde conversion was thought to be achieved by several redundant enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 [Rdh10(Trex) allele; Sandell LL, et al. (2007) Genes Dev 21:1113-1124] as responsible for a homozygous lethal phenotype with features of RA deficiency. We report here the production and characterization of unique murine Rdh10 loss-of-function alleles generated by gene targeting. We show that although Rdh10(-/-) mutants die at an earlier stage than Rdh10(Trex) mutants, their molecular patterning defects do not reflect a complete state of RA deficiency. Furthermore, we were able to correct most developmental abnormalities by administering retinaldehyde to pregnant mothers, thereby obtaining viable Rdh10(-/-) mutants. This demonstrates the rescue of an embryonic lethal phenotype by simple maternal administration of the missing retinoid compound. These results underscore the importance of maternal retinoids in preventing congenital birth defects, and lead to a revised model of the importance of RDH10 and RALDHs in controlling embryonic RA distribution.     

Regional Restriction of Alcohol/Retinol Dehydrogenases along the Mouse Gastrointestinal Epithelium

The gastrointestinal tract is a major site of alcohol dehydrogenase (ADH) activity in humans and rodents. Because class IADH (ADH-I) and class IV ADH (ADH-IV), but not class III ADH (ADH-III), function as retinol dehydrogenases in vitro and may thus participate in retinoid signaling needed for epithelial differentiation, the aim of this study was to determine the localization of these enzymes along the gastrointestinal tract. Specific antibodies were used to examine the tissue distribution of all three known classes of mouse ADH by Western blotting, and cellular localization was determined by immunohistochemistry. ADH-I was detected primarily in the intestine, liver, kidney, adrenal, and uterus, with detection of ADH-III in all tissues examined, and detection of ADH-IV primarily in the esophagus, stomach, adrenal, skin, ovary, and epididymis. Along the gastrointestinal tract, ADH-III was not specifically localized, whereas ADH-I was localized exclusively in the villus epithelium of the small intestine and absorptive epithelium of the large intestine, with ADH-IV being localized exclusively in the basal and suprabasal epithelial cells of the esophagus and gastric pit surface epithelium of the stomach. The ADH localization patterns observed are consistent with ADH-I and ADH-IV, but not ADH-III, functioning physiologically in retinol metabolism needed for epithelial differentiation. Our results further suggest that the functions of ADH-I and ADH-IV are regionally restricted to the lower and upper components, respectively, of the gastrointestinal epithelium, a finding that may relate to the different efficiencies of these two enzymes for retinol oxidation, as well as to the different susceptibilities of the upper and lower digestive tracts for ethanol-induced cancers.     

The Interaction of Ethanol and Vitamin A as a Potential Mechanism for the Pathogenesis of Fetal Alcohol Syndrome

The mechanism of the fetal embryopathology resulting from ethanol ingestion during pregnancy is not established. This review summarizes recent research on the interaction of ethanol and vitamin A in models that explore if an interaction between these two compounds might potentially be the mechanism for fetal alcohol syndrome. The rationale for this hypothesis includes the known facts that: (1) in adults, ethanol ingestion alters vitamin A metabolism and tissue distribution; (2) there are many phenotypic similarities between fetal alcohol syndrome and malformations of both vitamin A toxicity and deficiency; and (3) the vitamin A metabolite, retinoic acid (RA), is a potent mediator in embryogenesis and differentiation. One interaction that could possibly alter fetal development is that the synthesis of RA from retinol, catalyzed by alcohol dehydrogenase, might be competitively inhibited by ethanol leading to RA deficiency. Controversy over this hypothesis continues. Another model demonstrates in vivo effects of pregnant rat mother's ethanol consumption on retinol, retinyl ester, RA content, RA receptor (RAR) binding, and the levels of RAR expression in developing fetal organs. The variable responses in this model still need clarification, and specific defects resulting from specific RAR changes have not yet been identified. In a quail embryo model, ethanol treatment mimics vitamin A deficiency, and RA appears to prevent the adverse effects of ethanol. Finally, RA and ethanol reverse or block each other's effects in studies on isolated neuroblastoma cells. Taken together, these experiments show definite interactions between ethanol and vitamin A. Further studies are needed to determine if any of these mechanisms significantly contribute to prenatal ethanol consumption embryopathy; but, clearly this hypothesis is gaining experimental support.     

Structural Features of the NAD-Dependent In Situ Retinoic Acid Supply System in Esophageal Mucosa

Background: We previously reported that an NAD-dependent in situ retinoic acid supply system, which comprises some isoforms of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) and provides retinoic acid from retinol via a 2-step oxidation process, exists in the rat esophagus. Herein, their isoforms responsible for the pathway and its localization in the rat esophagus was examined. Methods: The expressions of mRNAs of various isoforms of ADH and ALDH were examined in the fraction mainly comprising mucosal layer of the rat esophagus by RT-PCR. Expression levels of Class IV ADH and ALDH 1A1 were compared between the fractions and that mainly comprising muscle layer of the rat esophagus by quantitative PCR. The catalytic activities producing retinoic acid from retinal were compared between the 2 fractions and its optimum pH was also determined. Results: Classes I, III, and IV ADHs and ALDHs 1A1 and 3A1 were predominant isoforms in the rat esophageal mucosa. The expression levels of mRNA of Class IV ADH and ALDH 3A1 were significantly higher in the mucosal than in the muscle layer. Consistently, the catalytic activities producing retinoic acid from retinal were significantly higher in the former than the latter. The optimum pH of the process was 9.0. Conclusions: Considering the affinities for retinol and retinal of ADHs and ALDHs expressed in the rat esophagus, the NAD-dependent in situ retinoic acid supply system in the rat esophagus is thought to comprise Class IV ADH and ALDH 1A1. In the rat esophagus, the system exists predominantly in the mucosal layer.     

Computational Modeling of a Putative Fetal Alcohol Syndrome Mechanism

Fetal alcohol syndrome (FAS) refers to a pattern of birth defects occurring in a subpopulation of children born to women who consume alcohol during pregnancy. The significant medical, social, and economic impact of FAS is increasing. Particularly hard-hit are African-American and native-American women and children. Over the past two decades, basic and clinical research produced voluminous data on ethanol effects on developing organisms. In 1991, Duester and Pullarkat proposed that competition of ethanol with retinol at the alcohol dehydrogenase (ADH) binding site formed the basis of the FAS mechanism. This competition adversely affects the developing fetus caused by deregulation of retinoic acid (RA) homeostasis essential for proper fetal tissue development. Stated concisely, the FAS hypothesis is:

• 1 Class I ADH catalyzes the rate-limiting step in oxidation of retinol (ROH) to RA, and ethanol (ETOH) to acetic acid, thus establishing competition for ADH between ROH and ETOH.
• 2 RA is required as a signal molecule for cell differentiation critical for normal fetal morphogenisis.
• 3 ADH binds ingested ETOH, thus deregulating RA homeostasis leading to improper RA signal transduction.

Preliminary results from molecular modeling studies of ROH-ADH and ETOH-ADH structures, and physiologic pharmacokinetic modeling confirm the hypothesis with remarkable fidelity.     

Polymorphism of ADH and ALDH Genes among Four Ethnic Groups in China and Effects upon the Risk for Alcoholism

The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) that metabolize ethanol are polymorphic. Different alleles encode subunits of the enzymes that differ in their rate of metabolizing ethanol. These polymorphisms are distributed differently among populations and have been shown to influence the risk for alcoholism in some Asian populations. We have examined the allele frequencies at the ADH2, ADH3 , and ALDH2 loci in four populations from China (Han, Mongolian, Korean, and Elunchun) and in alcoholics within each population. The four populations differ in allele frequencies, with the Elunchun having a much lower frequency of ADH2 *2 alleles, and the Mongolian and Elunchun having a much lower frequency of ALDH2 *2 alleles. Within each population, alleles at one or more of these three loci are protective against alcoholism, although the populations differ in which loci play significant roles. The protective allele at each locus ( ALDH2 * 2 , ADH2 * 2 , and ADH3 * 1 ) encodes a subunit that either metabolizes ethanol to acetaldehyde more rapidly or slows the conversion of acetaldehyde to acetate. Taken as a whole, data demonstrate that genetic differences in the enzymes that metabolize alcohol can substantially affect the risk for alcoholism.     

Estrogen directly induces expression of retinoic acid biosynthetic enzymes, compartmentalized between the epithelium and underlying stromal cells in rat uterus

Estrogen (E2) has been shown to induce the biosynthesis of retinoic acid (RA) in rat uterus. Here we examined whether E2 could directly induce the enzymes involved in this process by using the ovariectomized rat. A retinol dehydrogenase that we have previously described, eRolDH, and the retinal dehydrogenase, RalDH II, were found to have markedly increased uterine mRNA levels within 4 h of E2 administration, independent of the prior administration of puromycin. eRolDH and RalDH II and their mRNAs were also increased in uteri of rats during estrus. This indicated that RA biosynthesis in rat uterus is directly controlled by E2 and provides a direct link between the action of a steroid hormone and retinoid action. We also examined the cell-specific localization of RalDH II by immunohistochemistry. The enzyme was observed in the stromal compartment, particularly in cells close to the uterine lumenal epithelium. eRolDH was observed only in the lining epithelial cells. Taken together with the previous observations of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two also being expressed in the lumenal epithelium, we propose that RA production is compartmentalized, with retinol oxidation occurring in the lumenal epithelium and subsequent oxidation of retinal to RA occurring in the underlying stromal cells.     

A Hypothetical Mechanism for Fetal Alcohol Syndrome Involving Ethanol Inhibition of Retinoic Acid Synthesis at the Alcohol Dehydrogenase Step

Ethanol acts as a teratogen causing brain, craniofacial, and limb abnormalities in those suffering from fetal alcohol syndrome. Normal embryonic development of the vertebrate nervous system and limbs has recently been shown to be governed by retinoic acid, the active form of vitamin A. Retinol dehydrogenase is an enzyme needed to convert vitamin A (retinol) to retinoic acid, a molecule that specifies embryonic pattern formation by controlling gene expression. Ethanol acts as a competitive inhibitor of the retinol dehydrogenase activity attributed to mammalian alcohol dehydrogenase (ADH), an enzyme that uses both retinol and ethanol as substrates. An hypothesis is presented in which many of the abnormalities observed in fetal alcohol syndrome may be caused by high levels of ethanol acting as a competitive inhibitor of ADH-catalyzed retinol oxidation in the embryo or fetus. This would presumably result in a reduction of retinoic acid synthesis in embryonic tissues such as the nervous system and limbs that require critical levels of this molecule to specify spatial patterns.     

Activities of ethanol-metabolizing enzymes in liver diseases

The activities of hepatic alcohol (ADH), aldehyde (ALDH), and lactate dehydrogenases were measured in 69 patients with various liver diseases (15 controls, 20 with alcoholic and 8 with non-alcoholic fatty liver, 13 with alcoholic cirrhosis, 2 with alcoholic hepatitis, 3 with cryptogenic and 3 with primary biliary cirrhosis, and 5 with acute or chronic hepatitis). The specific activities of all these enzymes were decreased in both alcoholic and non-alcoholic liver diseases. The activities of ADH and low-Km ALDH were significantly decreased both in alcoholic (ADH, 7.22 mU/mg protein, p less than 0.001; low-Km ALDH, 5.00 mU/mg protein, p less than 0.001) and in other liver diseases (ADH, 10.70 mU/mg protein, p less than 0.001; low-Km ALDH, 6.80 mU/mg protein, p less than 0.005) when compared with controls (ADH, 20.87 mU/mg protein; low-Km ALDH, 14.41 mU/mg protein). The hepatic protein content was significantly (p less than 0.001) increased in alcoholic fatty degeneration but not in alcoholic cirrhosis or other liver diseases. The results suggest that in man alcohol- and acetaldehyde-metabolizing enzymes are not induced by chronic alcohol consumption. On the contrary, the hepatic activities of these enzymes appeared to be lower in alcoholic than in non-alcoholic liver diseases. Consequently, in addition to liver injury alcohol may also directly affect the synthesis or breakdown of alcohol-metabolizing enzymes.     

Complementary deoxyribonucleic acid cloning and enzymatic characterization of a novel 17beta/3alpha-hydroxysteroid/retinoid short chain dehydrogenase/reductase.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) convert androgens and estrogens between their active and inactive forms, whereas retinol dehydrogenases catalyze the conversion between retinol and retinal. Retinol dehydrogenases function in the visual cycle, in the generation of the hormone retinoic acid, and some also act on androgens. Here we report cloning and expression of a complementary DNA that encodes a new mouse liver microsomal member of the short chain dehydrogenase/reductase (SDR) superfamily and its enzymatic characterization, i.e. 17betaHSD9. Although 17betaHSD9 shares 88% amino acid identity with rat 17betaHSD6, its closest homolog, the two differ in substrate specificity. In contrast to other 17betaHSD, 17betaHSD9 has nearly equivalent activities as a 17betaHSD (with estradiol approximately = adiol) and as a 3alphaHSD (with adiol approximately = androsterone). It also recognizes retinol as substrate and represents in part the NAD+-dependent liver microsomal dehydrogenase that uses unbound retinol, but not retinol complexed with cellular retinol-binding protein. Thus, this enzyme has catalytic properties that overlap with two subgroups of SDR, 17betaHSD and retinol dehydrogenases. Inactivation of estrogen and a variety of androgens seems to be its most probable function. Because of its apparent inability to access retinol bound with cellular retinol-binding protein, a function in the pathway of retinoic acid biosynthesis seems less obvious. These data provide additional insight into the enzymology of estrogen, androgen, and retinoid metabolism and illustrate how closely related members of the SDR superfamily can have strikingly different substrate specificities.     

Estrogen suppresses retinaldehyde dehydrogenase 1 expression in the anterior pituitary glands of female rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development. RA is also a regulating factor of pituitary function. RA is synthesized from retinoids through oxidation processes. The oxidation of retinal to RA is catalyzed by the retinaldehyde dehydrogenases (RALDHs), including RALDH1, RALDH2 and RALDH3. Recently, we demonstrated that RALDH1 is expressed in the anterior pituitary glands of adult male rats. However, the expression of RALDH1 in the female pituitary gland and the regulation of RALDH1 expression have not been determined. Therefore, we examined the expression of RALDH1 mRNA in the pituitary glands of adult female rats. By in situ hybridization with digoxigenin-labeled cRNA probes and quantitative real-time PCR analysis, we found that the expression level of RALDH1 was significantly lower in estrus as compared to proestrus, metestrus and diestrus. RALDH1 mRNA levels increased after ovariectomy and decreased remarkably after a 1-week treatment with 17beta-estradiol implants. Estradiol (0.01-100 microg per rat) dose-dependently decreased the expression of RALDH1 in the pituitary glands after 24 hours of subcutaneous administration. These results clearly show that RALDH1 mRNA expression is suppressed by estrogen. We speculate that the generation of RA is regulated by estrogen and that RA plays a role in the estrus cycle through paracrine and/or autocrine mechanisms in the anterior pituitary gland of female rats.     

Ethanol Oxidation and Acetaldehyde Production in Vitro by Human Intestinal Strains of Escherichia coli under Aerobic,Microaerobic, and Anaerobic Conditions

Background: Many human colonic facultative anaerobic and aerobic bacteria are capable of alcohol dehydrogenase (ADH)-mediated ethanol oxidation. In this bacteriocolonic pathway for ethanol oxidation intracolonic ethanol is first oxidized by bacterial ADHs to acetaldehyde, which is further oxidized by either colonic mucosal or bacterial aldehyde dehydrogenases to acetate. The produced acetaldehyde is a highly toxic and carcinogenic agent. This study was aimed to investigate the ethanol oxidation capability and acetaldehyde formation of Escherichia coli IH 50546 and IH 50817. These intestinal E. coli strains expressed either high (IH 50546) or low (IH 50817) ADH activity. Methods: Strains were cultured for 48 h on agar plates supplemented with ethanol under aerobic, microaerobic (6% O₂), and anaerobic conditions. Results: Under aerobic conditions both E. coli     

Expression Patterns of Class I and Class IV Alcohol Dehydrogenase Genes in Developing Epithelia Suggest a Role for Alcohol Dehydrogenase in Local Retinoic Acid Synthesis

Vitamin A (retinol) regulates embryonic development and adult epithelial function via metabolism to retinoic acid, a pleiotrophic regulator of gene expression. Retinoic acid is synthesized locally and functions in an autocrine or paracrine fashion, but the enzymes involved remain obscure. Alcohol dehydrogenase (ADH) isozymes capable of metabolizing retinol include class I and class IV ADHs, with class III ADH unable to perform this function. ADHs also metabolize ethanol, and high levels of ethanol inhibit retinol metabolism, suggesting a possible mode of action for some of the medical complications of alcoholism. To explore whether any ADH isozymes are linked to retinoic acid synthesis, herein we have examined the expression patterns of all known classes of ADH in mouse embryonic and adult tissues, and also measured retinoic acid levels. Using in situ hybridization, class I ADH mRNA was localized in the embryo to the epithelia of the genitourinary tract, intestinal tract, adrenal gland, liver, conjunctival sac, epidermis, nasal epithelium, and lung, plus in the adult to epithelia within the testis, epididymis, uterus, kidney, intestine, adrenal cortex, and liver. Class IV ADH mRNA was localized in the embryo to the adrenal gland and nasal epithelium, plus in the adult to the epithelia of the esophagus, stomach, testis, epididymis, epidermis, and adrenal cortex. Class III ADH mRNA, in contrast, was present at low levels and not highly localized in the embryonic and adult tissues examined. We detected significant retinoic acid levels in the fetal kidney, fetal/adult intestine and adrenal gland, as well as the adult liver, lung, testis, epididymis, and uterus—all sites of class I and/or class IV ADH gene expression. These findings indicate that the expression patterns of class I ADH and class IV ADH, but not class III ADH, are consistent with a function in local retinoic acid synthesis needed for the development and maintenance of many specialized epithelial tissues.