Expression of the endocannabinoid system and response to cannabinoid components by the human fetal testis.

BACKGROUND: Cannabis consumption by pregnant women continues to increase worldwide, raising concerns about adverse effects on fetal growth and deleterious impacts on the newborn, in connection with evidence of placental transfer of cannabis compound. Cannabis action is mediated by the endocannabinoid system (ECS), which expression is well established in the brain but unknown in the developing testis. The fetal testis, whose endocrine function orchestrates the masculinization of many distant organs, is particularly sensitive to disruption by xenobiotics. In this context, we aimed to determine whether cannabis exposure has the potential to directly impact the human fetal testis. METHODS: We determined the expression of components of the ECS in the human fetal testis from 6 to 17 developmental weeks and assessed the direct effects of phytocannabinoids Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) on the testis morphology and cell functions ex vivo. RESULTS: We demonstrate the presence in the human fetal testis of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and to a lower level anandamide (AEA), as well as a range of enzymes and receptors for the ECS. Ex vivo exposure of first trimester testes to CBD, THC, or CBD/THC [ratio 1:1] at 10-7 to 10-5 M altered testosterone secretion by Leydig cells, AMH secretion by Sertoli cells, and impacted testicular cell proliferation and viability as early as 72 h post-exposure. Transcriptomic analysis on 72 h-exposed fetal testis explants revealed 187 differentially expressed genes (DEGs), including genes involved in steroid synthesis and toxic substance response. Depending on the molecules and testis age, highly deleterious effects of phytocannabinoid exposure were observed on testis tissue after 14 days, including Sertoli and germ cell death. CONCLUSIONS: Our study is the first to evidence the presence of the ECS in the human fetal testis and to highlight the potential adverse effect of cannabis consumption by pregnant women onto the development of the male gonad.

Proteome analysis and genome-wide regulatory motif prediction identify novel potentially sex-hormone regulated proteins in rat efferent ducts.

The efferent ducts are a series of tubules that conduct sperm from the rete testis to the epididymis. They absorb most fluid and proteins originating from the rete testis during concentration of spermatozoa prior to their entry into the epididymis. Proteome analysis of micro-dissected efferent duct samples from adult rats was combined with genome-wide computational prediction of conserved hormone response elements to identify factors likely regulated by oestrogens and androgens. We identified 165 proteins and found subsets of the promoters controlling their corresponding genes to contain androgen- and oestrogen response elements (ARE/EREs) at similar frequencies. Moreover, EREs were significantly enriched among the loci identified compared with their genome-wide occurrence. The expression and localization of Anxa6, Ckb, Krt19, Park7, Pdzk1 and Tpt1 in the efferent ducts and other related hormone controlled tissues was further validated at the RNA or protein level. This study identifies many novel proteins predicted to play roles in sperm maturation and male fertility and provides significant computational evidence that the efferent ducts express genes transcriptionally controlled by sex hormones.

Mu-opioid receptor activation induces transcriptional plasticity in the central extended amygdala.

Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.

Transcriptome analysis identifies genes with enriched expression in the mouse central extended amygdala.

The central extended amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the mouse EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than twofold higher expression level in the EAc compared with whole brain. Among these, 43 genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to toward genetic manipulations within the EAc.

Gene expression is altered in the lateral hypothalamus upon activation of the mu opioid receptor.

The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.

Genome-wide identification of Sox8-, and Sox9-dependent genes during early post-natal testis development in the mouse.

The SOX8 and SOX9 transcription factors are involved in, among others, sex differentiation, male gonad development and adult maintenance of spermatogenesis. Sox8(-/-) mice lacking Sox9 in Sertoli cells fail to form testis cords and cannot establish spermatogenesis. Although genetic and histological data show an important role for these transcription factors in regulating spermatogenesis, it is not clear which genes depend upon them at a genome-wide level. To identify transcripts that respond to the absence of Sox8 in all cells and Sox9 in Sertoli cells we measured mRNA concentrations in testicular samples from mice at 0, 6 and 18 days post-partum. In total, 621 and 629 transcripts were found at decreased or increased levels, respectively, at different time points in the mutant as compared to the control samples. These mRNAs were categorized as preferentially expressed in Sertoli cells or germ cells using data obtained with male and female gonad samples and enriched testicular cell populations. Five candidate genes were validated at the protein level. Furthermore, we identified putative direct SOX8 and SOX9 target genes by integrating predicted SOX-binding sites present in potential regulatory regions upstream of the transcription start site. Finally, we used protein network data to gain insight into the effects on regulatory interactions that occur when Sox8 and Sox9 are absent in developing Sertoli cells. The integration of testicular samples with enriched Sertoli cells, germ cells and female gonads enabled us to broadly distinguish transcripts directly affected in Sertoli cells from others that respond to secondary events in testicular cell types. Thus, combined RNA profiling signals, motif predictions and network data identified putative SOX8/SOX9 target genes in Sertoli cells and yielded insight into regulatory interactions that depend upon these transcription factors. In addition, our results will facilitate the interpretation of genome-wide in vivo SOX8 and SOX9 DNA binding data.

Retinoic acid induces TGFbeta-dependent autocrine fibroblast growth.

To evaluate the role of murine TFIID subunit TAF4 in activation of cellular genes by all-trans retinoic acid (T-RA), we have characterized the T-RA response of taf4(lox/-) and taf4(-/-) embryonic fibroblasts. T-RA regulates almost 1000 genes in taf4(lox/-) cells, but less than 300 in taf4(-/-) cells showing that TAF4 is required for T-RA regulation of most, but not all cellular genes. We further show that T-RA-treated taf4(lox/-) cells exhibit transforming growth factor (TGF)beta-dependent autocrine growth and identify a set of genes regulated by loss of TAF4 and by T-RA corresponding to key mediators of the TGFbeta signalling pathway. T-RA rapidly and potently induces expression of connective tissue growth factor (CTGF) via a conserved DR2 type response element in its proximal promoter leading to serum-free autocrine growth. These results highlight the role of TAF4 as a cofactor in the cellular response to T-RA and identify the genetic programme of a novel cross talk between the T-RA and TGFbeta pathways that leads to deregulated cell growth.

GOAnno: GO annotation based on multiple alignment.

UNLABELLED: GOAnno is a web tool that automatically annotates proteins according to the Gene Ontology (GO) using evolutionary information available in hierarchized multiple alignments. GO terms present in the aligned functional subfamily can be cross-validated and propagated to obtain highly reliable predicted GO annotation based on the GOAnno algorithm. AVAILABILITY: The web tool and a reduced version for local installation are freely available at http://igbmc.u-strasbg.fr/GOAnno/GOAnno.html SUPPLEMENTARY INFORMATION: The website supplies a detailed explanation and illustration of the algorithm at http://igbmc.u-strasbg.fr/GOAnno/GOAnnoHelp.html.