Impairment of spermatogenesis and enhancement of testicular germ cell apoptosis induced by exogenous all-trans-retinoic acid in adult lizard Podarcis sicula

In mammals, retinoic acid is involved in the regulation of testicular function by interaction with two families of nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Among RAR isoforms, the testicular cells of the lizard were found to express only RARalpha (3.7 kb) and RARbeta (3.4 kb) mRNAs, as reported here. In this study, the effects of exogenous all-trans-retinoic acid (atRA) on spermatogenesis of a non-mammalian seasonal reproducer were investigated. Daily intraperitoneal injections of atRA or atRA plus testosterone (atRA+T) were given for 2 weeks to adult males of the lizard Podarcis sicula. In animals treated with atRA, the seminiferous tubules were markedly reduced in cross-area. The seminiferous epithelium collapse was responsible for a sensible reduction in the number of germ cells and disruption in normal epithelial organization. In comparison, in atRA+T-treated lizards the loss of germinal cells was significantly less. The loss of germ cells observed in both experimental groups results from an induction of apoptotic process, as revealed by TUNEL analysis. Although low in number, apoptotic germ cells were also observed in the control groups (saline- and T-treated lizard), where the main germ cells undergoing apoptosis are primary spermatocytes (most frequently) and some spermatogonia.In conclusion, it is shown here that retinoic acid has deleterious effects on lizard spermatogenesis, causing a severe depletion of seminiferous epithelium, probably via induction of apoptotic processes. These effects are not completely inhibited by simultaneous administration of testosterone, although this hormone, once injected, is able to stimulate spermatogenesis and protect germinal cells from apoptotic cell death.     

Requirement of retinoic acid receptor isotypes alpha, beta, and gamma during the initial steps of neural differentiation of PCC7 cells

Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes alpha, beta, and gamma and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXRalpha and RARalpha, lower levels of RXRbeta, and barely detectable amounts of RARbeta, RARgamma, and RXRgamma. At receptor-selective concentrations, only a RARalpha or RARgamma agonist induced the typical tissue-like differentiation pattern consisting of neuronal and nonneuronal cells. Differentiation-associated processes, such as the down-regulation of Oct4, up-regulation of certain nuclear receptors and proneuronal genes, and the induction of neuronal markers could be triggered by receptor-selective concentrations of a RARalpha-, beta-, or gamma-selective agonist, although with distinct efficacy. The differences are only partially explained by the distinct RARalpha, beta, and gamma expression levels and the dissociation constants for the bound retinoids, suggesting differential requirement of RAR isotypes during the initial stages of neural differentiation of PCC7 cells.     

Chimeric analysis of retinoic acid receptor function during cardiac looping

Retinoids (vitamin A and its derivatives) play essential roles during vertebrate development. Vitamin A deprivation leads to severe congenital malformations affecting many tissues, including diverse neural crest cell populations and the heart. The vitamin A signal is transduced by the retinoic acid receptors (RARalpha, RARbeta, and RARgamma). However, these receptors exhibit considerable functional redundancy, as judged by the mild phenotype of RAR single null mutants relative to the defects evoked by loss of multiple RARs. To circumvent this redundancy, the endogenous RARgamma2 allele was replaced with a ligand-binding RARgamma mutant (RARgammaE(305)) by gene targeting in mouse embryonic stem (ES) cells. Chimeric embryos derived from hemizygous RARgammaE(305) ES cells displayed several defects similar to those observed in certain RAR double null mutants, including hypoplasia or absence of the caudal pharyngeal arches and myocardial deficiencies. The latter defects were not due to abnormal cardiac specification as affected hearts still expressed chamber-specific markers in an appropriate manner. Chimeras also displayed cardiac looping anomalies, which were associated with a reduction of Pitx2. This work suggests a role for RAR signaling in late looping morphogenesis and illustrates the utility of using a dominant-negative gene substitution approach to circumvent the functional redundancy inherent to the RAR family.     

A new class of RAR subtype selective retinoids: correlation of pharmacological effects with receptor activity

The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARbeta or RARbeta,gamma selectivity with weak RARalpha activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARgamma transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARbeta selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis.     

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.