Sources of all-trans retinal oxidation independent of the aldehyde dehydrogenase 1A isozymes exist in the postnatal testis†.
ABSTRACT: Despite the essential role of the active metabolite of vitamin A, all-trans retinoic acid (atRA) in spermatogenesis, the enzymes, and cellular populations responsible for its synthesis in the postnatal testis remain largely unknown. The aldehyde dehydrogenase 1A (ALDH1A) family of enzymes residing within Sertoli cells is responsible for the synthesis of atRA, driving the first round of spermatogenesis. Those studies also revealed that the atRA required to drive subsequent rounds of spermatogenesis is possibly derived from the ALDH1A enzymes residing within the meiotic and post-meiotic germ cells. Three ALDH1A isozymes (ALDH1A1, ALDH1A2, and ALDH1A3) are present in the testis. Although, ALDH1A1 is expressed in adult Sertoli cells and is suggested to contribute to the atRA required for the pre-meiotic transitions, ALDH1A2 is proposed to be the essential isomer involved in testicular atRA biosynthesis. In this report, we first examine the requirement for ALDH1A2 via the generation and analysis of a conditional Aldh1a2 germ cell knockout and a tamoxifen-induced Aldh1a2 knockout model. We then utilized the pan-ALDH1A inhibitor (WIN 18446) to test the collective contribution of the ALDH1A enzymes to atRA biosynthesis following the first round of spermatogenesis. Collectively, our data provide the first in vivo evidence demonstrating that animals severely deficient in ALDH1A2 postnatally proceed normally through spermatogenesis. Our studies with a pan-ALDH1A inhibitor (WIN 18446) also suggest that an alternative source of atRA biosynthesis independent of the ALDH1A enzymes becomes available to maintain atRA levels for several spermatogenic cycles following an initial atRA injection.
Project description:Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types.
Project description:In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.
Project description:All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.
Project description:Vitamin A (retinol) and its active metabolite, all-trans-retinoic acid (atRA), play critical roles in regulating the differentiation, growth, and migration of immune cells. Similarly, as critical signaling molecules in the regulation of the cell cycle, retinoids are important in cancers. Concentrations of atRA are tightly regulated in tissues, predominantly by the availability of retinol, synthesis of atRA by ALDH1A enzymes and metabolism and clearance of atRA by CYP26 enzymes. The ALDH1A and CYP26 enzymes are expressed in several cell types in the immune system and in cancer cells. In the immune system, the ALDH1A and CYP26 enzymes appear to modulate RA concentrations. Consequently, alterations in the activity of ALDH1A and CYP26 enzymes are expected to change disease outcomes in inflammation. There is increasing evidence from various disease models of intestinal and skin inflammation that treatment with atRA has a positive effect on disease markers. However, whether aberrant atRA concentrations or atRA synthesis and metabolism play a role in inflammatory disease development and progression is not well understood. In cancers, especially in acute promyelocytic leukemia and neuroblastoma, increasing intracellular concentrations of atRA appears to provide clinical benefit. Inhibition of the CYP26 enzymes to increase atRA concentrations and combat therapy resistance has been pursued as a drug target in these cancers. This chapter covers the current knowledge of how atRA and retinol regulate the immune system and inflammation, how retinol and atRA metabolism is altered in inflammation and cancer, and what roles atRA-metabolizing enzymes have in immune responses and cancers.
Project description:Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.
Project description:Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in ( R) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles.
Project description:Substantial evidence exists that during fetal ovarian development in mammals, retinoic acid (RA) induces germ cells to express the pre-meiotic marker Stra8 and enter meiosis, and that these effects are prevented in the fetal testis by the RA-degrading P450 enzyme CYP26B1. Nonetheless, the role of RA has been disputed principally because germ cells in embryos lacking two major RA-synthesizing enzymes, ALDH1A2 and ALDH1A3, remain able to enter meiosis. Here we show that a third RA-synthesizing enzyme, ALDH1A1, is expressed in fetal ovaries, providing a likely source of RA in the absence of ALDH1A2 and ALDH1A3. In ovaries lacking ALDH1A1, the onset of germ cell meiosis is delayed. Our data resolve the conundrum posed by conflicting published data sets and reconfirm the model that meiosis is triggered by endogenous RA in the developing ovary.
Project description:In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present notably in germ cells (GC). In order to determine the contributions of the GC-derived source of ATRA, we have generated mice lacking all RALDH activities in GC. We show that the GC-derived source of ATRA does not perform any specific role despite contributing to two-third of the total amount of ATRA present in the testis. Overall design: Mice carrying loxP-flanked alleles (L2) of Aldh1a1(tm1Nbg), Aldh1a2 and Aldh1a3(tm1Pcn) were crossed with mice bearing the Tg(Stra8-cre)1Reb transgene. Testis were sampled for analysis at the age of 9 weeks.
Project description:Mammalian spermatogenesis is an elaborately organized differentiation process, starting with diploid spermatogonia, which include germ-line stem cells, and ending with haploid spermatozoa. The process involves four pivotal transitions occurring in physical proximity: spermatogonial differentiation, meiotic initiation, initiation of spermatid elongation, and release of spermatozoa. We report how the four transitions are coordinated in mice. Two premeiotic transitions, spermatogonial differentiation and meiotic initiation, were known to be coregulated by an extrinsic signal, retinoic acid (RA). Our chemical manipulations of RA levels in mouse testes now reveal that RA also regulates the two postmeiotic transitions: initiation of spermatid elongation and spermatozoa release. We measured RA concentrations and found that they changed periodically, as also reflected in the expression patterns of an RA-responsive gene, STRA8; RA levels were low before the four transitions, increased when the transitions occurred, and remained elevated thereafter. We found that pachytene spermatocytes, which express an RA-synthesizing enzyme, Aldh1a2, contribute directly and significantly to RA production in testes. Indeed, chemical and genetic depletion of pachytene spermatocytes revealed that RA from pachytene spermatocytes was required for the two postmeiotic transitions, but not for the two premeiotic transitions. We conclude that the premeiotic transitions are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate the two postmeiotic transitions. In combination, these elements underpin the spatiotemporal coordination of spermatogenesis and ensure its prodigious output in adult males.
Project description:Sertoli cells are supporting cells of the testicular seminiferous tubules, which provide a nurturing environment for spermatogenesis. Adult Sertoli cells are polarized so that they can simultaneously support earlier-stage spermatogenic cells (e.g., spermatogonia) basally and later-stage cells (e.g., spermatids) apically. To test the consequences of disrupting cell polarity in Sertoli cells, we perform a Sertoli-specific conditional deletion of Rac1, which encodes a Rho GTPase required for apicobasal cell polarity. Rac1 conditional knockout adults exhibit spermatogenic arrest at the round spermatid stage, with severe disruption of Sertoli cell polarity, and show increased germline and Sertoli cell apoptosis. Thus, Sertoli Rac1 function is critical for the progression of spermatogenesis but, surprisingly, is dispensable for fetal testicular development, adult maintenance of undifferentiated spermatogonia, and meiotic entry. Our data indicate that Sertoli Rac1 function is required only for certain aspects of spermatogenesis and reveal that there are distinct requirements for cell polarity during cellular differentiation.