Dissecting Germ Cell Metabolism through Network Modeling

Metabolic pathways are increasingly postulated to be vital in programming cell fate, including stemness, differentiation, proliferation, and apoptosis. The commitment to meiosis is a critical fate decision for mammalian germ cells, and requires a metabolic derivative of vitamin A, retinoic acid (RA). Recent evidence showed that a pulse of RA is generated in the testis of male mice thereby triggering meiotic commitment. However, enzymes and reactions that regulate this RA pulse have yet to be identified. We developed a mouse germ cell-specific metabolic network with a curated vitamin A pathway. Using this network, we implemented flux balance analysis throughout the initial wave of spermatogenesis to elucidate important reactions and enzymes for the generation and degradation of RA. Our results indicate that primary RA sources in the germ cell include RA import from the extracellular region, release of RA from binding proteins, and metabolism of retinal to RA. Further, in silico knockouts of genes and reactions in the vitamin A pathway predict that deletion of Lipe, hormone-sensitive lipase, disrupts the RA pulse thereby causing spermatogenic defects. Examination of other metabolic pathways reveals that the citric acid cycle is the most active pathway. In addition, we discover that fatty acid synthesis/oxidation are the primary energy sources in the germ cell. In summary, this study predicts enzymes, reactions, and pathways important for germ cell commitment to meiosis. These findings enhance our understanding of the metabolic control of germ cell differentiation and will help guide future experiments to improve reproductive health.     

Retinoic acid in alveolar development, maintenance and regeneration

Recent data suggest that exogenous retinoic acid (RA), the biologically active derivative of vitamin A, can induce alveolar regeneration in a rat model of experimental emphysema. Here, we describe a mouse model of disrupted alveolar development using dexamethasone administered postnatally. We show that the effects of dexamethasone are concentration dependent, dose dependent, long lasting and result in a severe loss of alveolar surface area. When RA is administered to these animals as adults, lung architecture and the surface area per unit of body weight are completely restored to normal. This remarkable effect may be because RA is required during normal alveolar development and administering RA re-awakens gene cascades used during development. We provide evidence that RA is required during alveologenesis in the mouse by showing that the levels of the retinoid binding proteins, the RA receptors and two RA synthesizing enzymes peak postnatally. Furthermore, an inhibitor of RA synthesis, disulphiram, disrupts alveologenesis. We also show that RA is required throughout life for the maintenance of lung alveoli because when rats are deprived of dietary retinol they lose alveoli and show the features of emphysema. Alveolar regeneration with RA may therefore be an important novel therapeutic approach to the treatment of respiratory diseases characterized by a reduced gas-exchanging surface area such as bronchopulmonary dysplasia and emphysema for which there are currently no treatments.     

Dietary tocopherol and sexual reproduction in the rotifers Brachionus calyciflorus and Asplanchna sieboldi.

Brachionus calyciflorus contained no detectable tocopherol (vitamin E) when cultured for long periods on the yeast Rhodotorula glutinis. Using a spectrofluorometric assay and the Asplanchna body-wall-outgrowth response bioassay the limits of detection were 6.4 ng and 3.78 pg alpha-tocopherol per g dry weight Brachionus, respectively. These tocopherol-free Brachionus reproduced normally both parthenogenetically and sexually, producing active and potent males and fertilized resting eggs which developed into viable young rotifers. The apparent lack of a tocopherol requirement for male fertility in B. calyciflorus probably also applies to Asplanchna sieboldi and A. brightwell -- rotifers whose male-producing (mictic) females are induced by dietary tocopherol and whose males therefore are never tocopherol-deficient. The adaptive significance of the tocopherol requirement for the initiation of sexual reproduction in Asplanchna sieboldi--formerly hypothesized to be the result of a tocopherol requirement for male fertility--is now thought to be related to the role of tocopherol in controlling female polymorphism in this species.     

Endogenous retinoids in the hair follicle and sebaceous gland

Vitamin A and its derivatives (retinoids) are critically important in the development and maintenance of multiple epithelial tissues, including skin, hair, and sebaceous glands, as shown by the detrimental effects of either vitamin A deficiency or toxicity. Thus, precise levels of retinoic acid (RA, active metabolite) are needed. These precise levels of RA are achieved by regulating several steps in the conversion of dietary vitamin A (retinol) to RA and RA catabolism. This review discusses the localization of RA synthesis to specific sites within the hair follicle and sebaceous gland, including their stem cells, during both homeostasis and disease states. It also discusses what is known about the specific roles of RA within the hair follicle and sebaceous gland. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.     

Retinoic Acid Can Exacerbate T Cell Intrinsic TLR2 Activation to Promote Tolerance

The contribution of vitamin A to immune health has been well established. However, recent evidence indicates that its active metabolite, retinoic acid (RA), has the ability to promote both tolerogenic and inflammatory responses. While the outcome of RA-mediated immunity is dependent upon the immunological status of the tissue, the contribution of specific innate signals influencing this response have yet to be delineated. Here, we found that treatment with RA can dampen inflammation during intestinal injury. Importantly, we report a novel and unexpected requirement for TLR2 in RA-mediated suppression. Our data demonstrate that RA treatment enhances TLR2-dependent IL-10 production from T cells and this, in turn, potentiates T regulatory cell (T_REG) generation without the need for activation of antigen presenting cells. These data also suggest that combinatorial therapy using RA and TLR2 ligands may be advantageous in the design of therapies to treat autoimmune or inflammatory disease.     

Cellular retinoic acid-binding proteins are essential for hindbrain patterning and signal robustness in zebrafish

The vitamin A derivative retinoic acid (RA) is a morphogen that patterns the anterior-posterior axis of the vertebrate hindbrain. Cellular retinoic acid-binding proteins (Crabps) transport RA within cells to both its nuclear receptors (RARs) and degrading enzymes (Cyp26s). However, mice lacking Crabps are viable, suggesting that Crabp functions are redundant with those of other fatty acid-binding proteins. Here we show that Crabps in zebrafish are essential for posterior patterning of the hindbrain and that they provide a key feedback mechanism that makes signaling robust as they are able to compensate for changes in RA production. Of the four zebrafish Crabps, Crabp2a is uniquely RA inducible and depletion or overexpression of Crabp2a makes embryos hypersensitive to exogenous RA. Computational models confirm that Crabp2a improves robustness within a narrow concentration range that optimizes a 'robustness index', integrating spatial information along the RA morphogen gradient. Exploration of signaling parameters in our models suggests that the ability of Crabp2a to transport RA to Cyp26 enzymes for degradation is a major factor in promoting robustness. These results demonstrate a previously unrecognized requirement for Crabps in RA signaling and hindbrain development, as well as a novel mechanism for stabilizing morphogen gradients despite genetic or environmental fluctuations in morphogen availability.     

Androgens in rheumatoid arthritis: when are they effectors?

Neither hormone receptor genes nor plasma androgens seem significantly altered in female subjects before they became affected by rheumatoid arthritis (RA) and, therefore, do not seem to play a role as risk factors for its development. However, serum testosterone levels are inversely correlated with RA activity and dehydro-epiandrosterone sulfate (DHEAS) plasma levels are inversely correlated with both disease duration and clinical severity in patients already affected by active RA. In particular, gonadal and adrenal androgens (that is, testosterone and DHEAS) are significantly decreased in inflamed synovial tissue/fluids during active disease as a consequence of the inflammatory reaction, which supports a pro-inflammatory milieu in RA joints. Recently, male gender has been found to be a major predictor of remission in early RA.     

Rare sexual reproduction events in the clonal reproduction system of introduced populations of the little fire ant

A unique reproductive system has previously been described in Wasmannia auropunctata, a widespread invasive ant species, where males are produced clonally, female queens are parthenogens, and female workers are produced sexually. However, these findings were mostly based on samples originating from only a limited part of the native range of the species in South America. We used microsatellite markers to uncover the reproductive modes displayed by a large number of nests collected in various invasive W. auropunctata populations introduced 40 years ago into New Caledonia, where the species now forms a single 450-km-long supercolony. Although the main reproduction system in New Caledonia remained clonality for both male and female reproductives, we found evidence of rare sexual reproduction events that led to the production of both new queen and male clonal lineages. All clonal lineages observed in New Caledonia potentially derived from sexual reproduction, recombination, and mutation events from a single female and a single male genotype. Hence, the male and female gene pools are not strictly separated in New Caledonia and the two sexes do not follow independent evolutionary trajectories. Our results also suggest genetic determination for both parthenogenesis and caste. We discuss the evolutionary implications of the emergence of sex in the clonal reproduction system of introduced populations of W. auropunctata.     

Dicer调节生殖功能的研究进展

Dicer是微小非编码RNA生成的关键内切酶,介导微小RNA(micro RNA,miRNA)和小干扰RNA(small interfering RNA,siRNA)的产生,通过RNA干扰(RNA interference,RNAi)途径实现转录或转录后水平基因调控,在调节细胞增殖、分化、凋亡等方面起重要作用。近年来Dicer基因在生殖领域的研究越来越受关注,最近的研究表明Dicer与男性生精细胞发育、精子形成及成熟、精子活力和形态生成、卵泡发育、排卵及黄体形成、性激素合成、输卵管功能、子宫内膜容受性等方面都有密切关系。繁衍后代需要精子和卵子的共同参与,Dicer可能通过影响精子和卵子的数量或者质量进而导致胚胎发育异常,因此理解Dicer在雄性与雌性生殖的重要调节作用对于理解生殖调节异常相关的疾病如无精子症、复发性流产等的发病机制具有重要的作用。本文对Dicer在雄性生殖道与雌性生殖中的关键作用进行了综述,旨在进一步从分子层面深入理解Dicer与生殖相关疾病的关系。     

The control of morphogen signalling: regulation of the synthesis and catabolism of retinoic acid in the developing embryo

We consider here how morphogenetic signals involving retinoic acid (RA) are switched on and off in the light of positive and negative feedback controls which operate in other embryonic signalling systems. Switching on the RA signal involves the synthetic retinaldehyde dehydrogenase (RALDH) enzymes and it is currently thought that switching off the RA signal involves the CYP26 enzymes which catabolise RA. We have tested whether these enzymes are regulated by the presence or absence of all-trans-RA using the vitamin A-deficient quail model system and the application of excess retinoids on beads to various locations within the embryo. The Raldhs are unaffected either by the absence or presence of excess RA, whereas the Cyps are strongly affected. In the absence of RA some, but not all domains of Cyp26A1, Cyp26B1 and Cyp26C1 are down-regulated, in particular the spinal cord (Cyp26A1), the heart and developing vasculature (Cyp26B1) and the rhombomeres (Cyp26C1). In the presence of excess RA, the Cyps show a differential regulation-Cyp26A1 and Cyp26B1 are up-regulated whereas Cyp26C1 is down-regulated. We tested whether the Cyp products have a similar influence on these genes and indeed 4-oxo-RA, 4-OH-RA and 5,6-epoxy-RA do. Furthermore, these 3 metabolites are biologically active in that they fully rescue the vitamin A-deficient quail embryo. Finally, by using retinoic acid receptor selective agonists we show that these compounds regulate the Cyps through the RARalpha receptor. These results are discussed with regard to positive and negative feedback controls in developing systems.     

Sexual dimorphism in the nutritional requirement for adult lifespan in Drosophila melanogaster

The nutritional requirements of Drosophila have mostly been studied for development and reproduction, but the minimal requirements for adult male and female flies for lifespan have not been established. Following development on a complete diet, we find substantial sex difference in the basic nutritional requirement of adult flies for full length of life. Relative to females, males require less of each nutrient, and for some nutrients that are essential for development, adult males have no requirement at all for lifespan. The most extreme (and surprising) sex differences were that chronic cholesterol and vitamin deficiencies had no effect on the lifespan of adult males, but they greatly decreased lifespan in females. Female oogenesis rather than chromosomal karyotype and mating status is the key cause of this gender difference in life‐sustaining nutritional requirements. These data are important to the way we understand the mechanisms by which diet modifies lifespan.     

Cyp26 enzymes generate the retinoic acid response pattern necessary for hindbrain development

Retinoic acid (RA) is essential for normal vertebrate development, including the patterning of the central nervous system. During early embryogenesis, RA is produced in the trunk mesoderm through the metabolism of vitamin A derived from the maternal diet and behaves as a morphogen in the developing hindbrain where it specifies nested domains of Hox gene expression. The loss of endogenous sources of RA can be rescued by treatment with a uniform concentration of exogenous RA, indicating that domains of RA responsiveness can be shaped by mechanisms other than the simple diffusion of RA from a localized posterior source. Here, we show that the cytochrome p450 enzymes of the Cyp26 class, which metabolize RA into polar derivatives, function redundantly to shape RA-dependent gene-expression domains during hindbrain development. In zebrafish embryos depleted of the orthologs of the three mammalian CYP26 genes CYP26A1, CYP26B1 and CYP26C1, the entire hindbrain expresses RA-responsive genes that are normally restricted to nested domains in the posterior hindbrain. Furthermore, we show that Cyp26 enzymes are essential for exogenous RA to rescue hindbrain patterning in RA-depleted embryos. We present a ;gradient-free' model for hindbrain patterning in which differential RA responsiveness along the hindbrain anterior-posterior axis is shaped primarily by the dynamic expression of RA-degrading enzymes.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Retinoic acid is a negative physiological regulator of N‐cadherin during early avian heart morphogenesis

The vitamin A‐deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A‐active form, retinoic acid (RA). Here we examine the role of N‐cadherin (N‐cad) in RA‐regulated early cardiovascular morphogenesis. N‐cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N‐cad overproducing VAD embryos reveal N‐cad involvement in the RA‐regulated cardiovascular development and suggest that N‐cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N‐cad. We hypothesize that a critical endogenous level of N‐cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage‐specific, developmentally regulated RA signaling.     

Role of retinoid signaling in the regulation of spermatogenesis

While the need for vitamin A for the normal progression of male germ cell differentiation has been known for many years, the molecular mechanisms underlying this requirement are poorly understood. This review will explore the aspects of the effects on spermatogenesis of dietary deprivation of vitamin A, in particular as to how they compare to the male sterility that results from the genetic ablation of function of the retinoid receptor RARalpha. The effects of other genes involved with retinoid synthesis, transport, and degradation are also considered. The possible cellular mechanisms that may be affected by the lack of retinoid signaling are discussed, in particular, cell cycle regulation and cell-cell interaction, both of which are critical for normal spermatogenesis.