Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens.

Retinoic acid (RA) is crucial for mammalian spermatogonia differentiation, and stimulates Stra8 expression, a gene required for meiosis. Certain fish species, including zebrafish, have lost the stra8 gene. While RA still seems important for spermatogenesis in fish, it is not known which stage(s) respond to RA or whether its effects are integrated into the endocrine regulation of spermatogenesis. In zebrafish, RA promoted spermatogonia differentiation, supported androgen-stimulated meiosis, and reduced spermatocyte and spermatid apoptosis. Follicle-stimulating hormone (Fsh) stimulated RA production. Expressing a dominant-negative RA receptor variant in germ cells clearly disturbed spermatogenesis but meiosis and spermiogenesis still took place, although sperm quality was low in 6-month-old adults. This condition also activated Leydig cells. Three months later, spermatogenesis apparently had recovered, but doubling of testis weight demonstrated hypertrophy, apoptosis/DNA damage among spermatids was high and sperm quality remained low. We conclude that RA signaling is important for zebrafish spermatogenesis but is not of crucial relevance. As Fsh stimulates androgen and RA production, germ cell-mediated, RA-dependent reduction of Leydig cell activity may form a hitherto unknown intratesticular negative-feedback loop.

The role of the retinoids in schizophrenia: genomic and clinical perspectives.

Signalling by retinoid compounds is vital for embryonic development, with particular importance for neurogenesis in the human brain. Retinoids, metabolites of vitamin A, exert influence over the expression of thousands of transcripts genome wide, and thus, act as master regulators of many important biological processes. A significant body of evidence in the literature now supports dysregulation of the retinoid system as being involved in the aetiology of schizophrenia. This includes mechanistic insights from large-scale genomic, transcriptomic and, proteomic studies, which implicate disruption of disparate aspects of retinoid biology such as transport, metabolism, and signalling. As a result, retinoids may present a valuable clinical opportunity in schizophrenia via novel pharmacotherapies and dietary intervention. Further work, however, is required to expand on the largely observational data collected thus far and confirm causality. This review will highlight the fundamentals of retinoid biology and examine the evidence for retinoid dysregulation in schizophrenia.

Functions of Intracellular Retinoid Binding-Proteins.

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Not just signal shutoff: the protective role of arrestin-1 in rod cells.

The retinal rod cell is an exquisitely sensitive single-photon detector that primarily functions in dim light (e.g., moonlight). However, rod cells must routinely survive light intensities more than a billion times greater (e.g., bright daylight). One serious challenge to rod cell survival in daylight is the massive amount of all-trans-retinal that is released by Meta II, the light-activated form of the photoreceptor rhodopsin. All-trans-retinal is toxic, and its condensation products have been implicated in disease. Our recent work has developed the concept that rod arrestin (arrestin-1), which terminates Meta II signaling, has an additional role in protecting rod cells from the consequences of bright light by limiting free all-trans-retinal. In this chapter we will elaborate upon the molecular mechanisms by which arrestin-1 serves as both a single-photon response quencher as well as an instrument of rod cell survival in bright light. This discussion will take place within the framework of three distinct functional modules of vision: signal transduction, the retinoid cycle, and protein translocation.

Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium.

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.

Cis-retinoids and the chemistry of vision.

We discuss here principal biochemical transformations of retinoid molecules in the visual cycle. We focus our analysis on the accumulating evidence of alternate pathways and functional redundancies in the cycle. The efficiency of the visual cycle depends, on one hand, on fast regeneration of the photo-bleached chromophores. On the other hand, it is crucial that the cyclic process should be highly selective to avoid accumulation of byproducts. The state-of-the-art knowledge indicates that single enzymatically active components of the cycle are not strictly selective and may require chaperones to enhance their rates. It appears that protein-protein interactions significantly improve the biological stability of the visual cycle. In particular, synthesis of thermodynamically less stable 11-cis-retinoid conformers is favored by physical interactions of the isomerases present in the retina with cellular retinaldehyde binding protein.

Retinoids and their target genes in liver functions and diseases.

Retinoids have been reported to prevent several kinds of cancers, including hepatocellular carcinoma (HCC). Retinoic acid (RA) coupled with retinoic acid receptor/retinoid X receptor heterodimer exerts its functions by regulating its target genes. We previously reported that transgenic mice, in which RA signaling is suppressed in a hepatocyte-specific manner, developed liver cancer at a high rate, and that disruption of RA functions led to the increased oxidative stress via aberrant metabolisms of lipid and iron, indicating that retinoids play an important role in liver pathophysiology. These data suggest that exploring the metabolism of retinoids in liver diseases and their target genes provides us with useful information to understand the liver functions and diseases. Consequently, the altered metabolism of retinoids was observed in liver diseases, including non-alcoholic fatty liver disease. In this review, we summarize the metabolism of retinoids in the liver, highlight the functions of retinoids in HCC, non-alcoholic fatty liver disease, and alcoholic liver disease, and discuss the target genes of RA. Investigation of retinoids in the liver will likely help us identify novel therapies and diagnostic modalities for HCC.

The contributions of oxytocin and vasopressin pathway genes to human behavior.

Arginine vasopressin (AVP) and oxytocin (OXT) are social hormones and mediate affiliative behaviors in mammals and as recently demonstrated, also in humans. There is intense interest in how these simple nonapeptides mediate normal and abnormal behavior, especially regarding disorders of the social brain such as autism that are characterized by deficits in social communication and social skills. The current review examines in detail the behavioral genetics of the first level of human AVP-OXT pathway genes including arginine vasopressin 1a receptor (AVPR1a), oxytocin receptor (OXTR), AVP (AVP-neurophysin II [NPII]) and OXT (OXT neurophysin I [NPI]), oxytocinase/vasopressinase (LNPEP), ADP-ribosyl cyclase (CD38) and arginine vasopressin 1b receptor (AVPR1b). Wherever possible we discuss evidence from a variety of research tracks including molecular genetics, imaging genomics, pharmacology and endocrinology that support the conclusions drawn from association studies of social phenotypes and detail how common polymorphisms in AVP-OXT pathway genes contribute to the behavioral hard wiring that enables individual Homo sapiens to interact successfully with conspecifics. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells.

BACKGROUND & AIMS: New sources of beta cells are needed to develop cell therapies for patients with diabetes. An in vitro, sequential method has been developed to derive pancreatic progenitors, but this technique has not been used for other cell lines. We investigated whether definitive endoderm derived from human embryonic stem (hES) cells might be used to create beta cells. METHODS: Five hES cell lines were induced to form pancreatic progenitors and analyzed for pancreas markers. Cells were incubated with a bone morphogenetic protein (BMP) antagonist, retinoids, a Hedgehog antagonist, or fibroblast growth factor (FGF) and phenotypes were analyzed. RESULTS: Four hES cell lines sequentially generated definitive endoderm, primitive gut, and posterior foregut equivalents, as described previously. However, functional hepatocytes, rather than pancreas progenitors, developed. Consistent with liver development, FGF and BMP signaling pathways were involved in this process; their inhibition disrupted hepatocyte differentiation. During early stages of development, exposure of cells to noggin and retinoid acid, followed by FGF10, generated pancreatic cells (PDX1+; 50%-80%) that coexpressed FOXA2, HNF6, and SOX9. CONCLUSIONS: These findings demonstrate the combined functions of endogenous BMP and supplemented FGF in inducing differentiation of hepatocytes from hES cells and the ability to shift developmental pathways from hepatic to pancreatic cell differentiation. Although additional signals appear to be required for full specification of PDX1(+) early pancreatic progenitors (via PTF1a and NKX6.1 coexpression), these findings indicate the signaling pathways required for differentiation of bipotential progenitors.

BMP action in skeletogenesis involves attenuation of retinoid signaling.

The bone morphogenetic protein (BMP) and growth and differentiation factor (GDF) signaling pathways have well-established and essential roles within the developing skeleton in coordinating the formation of cartilaginous anlagen. However, the identification of bona fide targets that underlie the action of these signaling molecules in chondrogenesis has remained elusive. We have identified the gene for the retinoic acid (RA) synthesis enzyme Aldh1a2 as a principal target of BMP signaling; prochondrogenic BMPs or GDFs lead to attenuation of Aldh1a2 expression and, consequently, to reduced activation of the retinoid signaling pathway. Consistent with this, antagonism of retinoid signaling phenocopies BMP4 action, whereas RA inhibits the chondrogenic stimulatory activity of BMP4. BMP4 also down-regulates Aldh1a2 expression in organ culture and, consistent with this, Aldh1a2 is actively excluded from the developing cartilage anlagens. Collectively, these findings provide novel insights into BMP action and demonstrate that BMP signaling governs the fate of prechondrogenic mesenchyme, at least in part, through regulation of retinoid signaling.

Retinoids induced Pck1 expression and attenuated insulin-mediated suppression of its expression via activation of retinoic acid receptor in primary rat hepatocytes.

Insulin regulates the expression of genes involved in hepatic glucose and lipid metabolism, such as the cytosolic form of phosphoenolpyruvate carboxykinase gene (Pck1). We have reported that lipophilic molecules from rat livers induced Pck1 transcription and attenuated insulin-mediated suppression of its expression in primary rat hepatocytes. After identification of retinol and retinal as the active molecules, the present study was aimed to determine the effects of retinoids on insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. Real-time PCR and reporter gene assays were designed to determine retinoid effects in the absence or presence of insulin on the expression levels of Pck1 mRNA and activation of its promoter constructs, respectively. The lipophilic extract from rat livers specifically induced the expression of Pck1, but not that of two other insulin-suppressed genes, glucose 6-phosphatase catalytic subunit and insulin-like growth factor-binding protein 1. Retinol, retinal, and retinoic acid (RA) induced Pck1 expression dose-dependently in primary hepatocytes. Specific activation of retinoic acid receptor (RAR), but not retinoid X receptor, attenuated insulin-mediated suppression of Pck1 expression. RARα antagonist (Ro41-5253) abolished the retinal-mediated induction of Pck1 expression and attenuation of insulin-mediated suppression of its expression. Disruption of the proximal, but not the distal, RA responsive element in the Pck1 promoter eliminated the RA response of Pck1 promoter reporter constructs in primary hepatocytes. The results of this study demonstrated for the first time that retinoid treatment attenuated insulin-mediated suppression of Pck1 expression in primary rat hepatocytes. It suggests that retinoid metabolism in hepatocytes may modulate hepatic insulin action.

Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse.

The retinoic acid receptors alpha, beta and gamma (RARalpha, RARbeta and RARgamma) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARalpha and RARgamma (or RARbeta and RARgamma) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARalpha and RARbeta, however, are virtually normal, suggesting that RARgamma is essential. In good correlation, we find that RARgamma is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARgamma is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARgamma over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARgamma in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

Retinoids in Embryonic Development.

Retinoids constitute a class of compounds chemically related to vitamin A [...].

Retinoid induction of alveolar regeneration: from mice to man?

The use of retinoids to induce human lung regeneration is under investigation in a number of studies in patients with chronic obstructive pulmonary disease (COPD). Retinoic acid (RA) has complex pleiotropic functions during vertebrate patterning and development and can induce regeneration in a number of different organ systems. Studies of retinoid signalling during lung development might provide a molecular basis to explain pharmacological induction of alveolar regeneration in adult models of lung disease. In this review the role of endogenous RA signalling during alveologenesis is explored and data suggesting that a number of exogenous retinoids can induce regeneration in the adult lung are discussed. Current controversies in this area are highlighted and a hypothesis of lung regeneration is put forward. Understanding the cellular and molecular mechanisms of induction of regeneration will be central for effective translation into patients with lung disease and may reveal novel insights into the pathogenesis of alveolar disease and senescence.

Characterization of concentration gradients of a morphogenetically active retinoid in the chick limb bud.

It has long been suggested that the generation of biological patterns depends in part on gradients of diffusible substances. In an attempt to bridge the gap between this largely theoretical concept and experimental embryology, we have examined the physiology of diffusion gradients in an actual embryonic field. In particular, we have generated in the chick wing bud concentration gradients of the morphogenetically active retinoid TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl] benzoic acid, a synthetic vitamin A compound. Upon local application of TTNPB the normal 234 digit pattern is duplicated in a way that correlates with the geometry of the underlying TTNPB gradient; low doses of TTNPB lead to a shallow gradient and an additional digit 2, whereas higher doses result in a steep, far-reaching gradient and patterns with additional digits 3 and 4. The experimentally measured TTNPB distribution along the anteroposterior axis, can be modeled by a local source and a dispersed sink. This model correctly predicts the site of specification of digit 2, and provides an empirical estimate of the diffusion coefficient (D) of retinoids in embryonic limb tissue. The numerical value of approximately 10(-7) cm2s-1 for D suggests that retinoids are not freely diffusible in the limb rudiment, but interact with the previously identified cellular retinoic acid binding protein. In addition, D affords an estimate of the time required to establish a diffusion gradient as 3 to 4 h. This time span is in a range compatible with the time scale of pattern specification in developing vertebrate limbs. Our studies support the view that diffusion of morphogenetic substances is a plausible mechanism of pattern formation in secondary embryonic fields.

On the association between low resting heart rate and chronic aggression: retinoid toxicity hypothesis.

Low resting heart rate is a strong and consistent predictor of conduct disorder and chronic aggression. Explanations such as fearlessness and low arousal-induced stimulus-seeking have been offered, assuming a causal association between the phenomena, but the origin of low heart rate and its significance for understanding aggression and violence remain obscure. Retinoids (vitamin A and its congeners) play important roles in embryogenesis and neural development. Several lines of evidence also suggest a causal role of retinoids in aggression as well as cognitive and mood disorders. The hypothesis is proposed that retinoid overexpression in utero induces, via a noradrenergic-to-cholinergic switch, alterations in cardiac functioning and hemodynamics resulting in low resting heart rate, brain structural and functional changes, minor physical anomalies, and persistent aggression. Retinoid toxicity occurring early in pregnancy could represent a final common pathway by which various prenatal challenges result in conduct disorder and chronic aggression (e.g., maternal cigarette smoking, alcohol consumption, drug use, exposure to environmental chemicals, stress, trauma or infection). Implications of the model for understanding related aspects of chronic aggression are discussed, as well as strategies for prevention and treatment.

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo.

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

Perinatal Undernutrition, Metabolic Hormones, and Lung Development.

Maternal and perinatal undernutrition affects the lung development of litters and it may produce long-lasting alterations in respiratory health. This can be demonstrated using animal models and epidemiological studies. During pregnancy, maternal diet controls lung development by direct and indirect mechanisms. For sure, food intake and caloric restriction directly influence the whole body maturation and the lung. In addition, the maternal food intake during pregnancy controls mother, placenta, and fetal endocrine systems that regulate nutrient uptake and distribution to the fetus and pulmonary tissue development. There are several hormones involved in metabolic regulations, which may play an essential role in lung development during pregnancy. This review focuses on the effect of metabolic hormones in lung development and in how undernutrition alters the hormonal environment during pregnancy to disrupt normal lung maturation. We explore the role of GLP-1, ghrelin, and leptin, and also retinoids and cholecalciferol as hormones synthetized from diet precursors. Finally, we also address how metabolic hormones altered during pregnancy may affect lung pathophysiology in the adulthood.