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.

RNA profiling of human testicular cells identifies syntenic lncRNAs associated with spermatogenesis.

STUDY QUESTION: Is the noncoding transcriptional landscape during spermatogenesis conserved between human and rodents? SUMMARY ANSWER: We identified a core group of 113 long noncoding RNAs (lncRNAs) and 20 novel genes dynamically and syntenically transcribed during spermatogenesis. WHAT IS KNOWN ALREADY: Spermatogenesis is a complex differentiation process driven by a tightly regulated and highly specific gene expression program. Recently, several studies in various species have established that a large proportion of known lncRNAs are preferentially expressed during meiosis and spermiogenesis in a testis-specific manner. STUDY DESIGN, SIZE, DURATION: To further investigate lncRNA expression in human spermatogenesis, we carried out a cross-species RNA profiling study using isolated testicular cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testes were obtained from post-mortem donors (N = 8, 51 years old on average) or from prostate cancer patients with no hormonal treatment (N = 9, 80 years old on average) and only patients with full spermatogenesis were used to prepare enriched populations of spermatocytes, spermatids, Leydig cells, peritubular cells and Sertoli cells. To minimize potential biases linked to inter-patient variations, RNAs from two or three donors were pooled prior to RNA-sequencing (paired-end, strand-specific). Resulting reads were mapped to the human genome, allowing for assembly and quantification of corresponding transcripts. MAIN RESULTS AND THE ROLE OF CHANCE: Our RNA-sequencing analysis of pools of isolated human testicular cells enabled us to reconstruct over 25 000 transcripts. Among them we identified thousands of lncRNAs, as well as many previously unidentified genes (novel unannotated transcripts) that share many properties of lncRNAs. Of note is that although noncoding genes showed much lower synteny than protein-coding ones, a significant fraction of syntenic lncRNAs displayed conserved expression during spermatogenesis. LARGE SCALE DATA: Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI Gene Expression Omnibus under accession number GSE74896. LIMITATIONS, REASONS FOR CAUTION: Isolation procedures may alter the physiological state of testicular cells, especially for somatic cells, leading to substantial changes at the transcriptome level. We therefore cross-validated our findings with three previously published transcriptomic analyses of human spermatogenesis. Despite the use of stringent filtration criteria, i.e. expression cut-off of at least three fragments per kilobase of exon model per million reads mapped, fold-change of at least three and false discovery rate adjusted P-values of less than <1%, the possibility of assembly artifacts and false-positive transcripts cannot be fully ruled out. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study has led to the identification of a large number of conserved germline-associated lncRNAs that are potentially important for spermatogenesis and sexual reproduction. In addition to further substantiating the basis of the human testicular physiology, our study provides new candidate genes for male infertility of genetic origin. This is likely to be relevant for identifying interesting diagnostic and prognostic biomarkers and also potential novel therapeutic targets for male contraception. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by l'Institut national de la santé et de la recherche médicale (Inserm); l'Université de Rennes 1; l'Ecole des hautes études en santé publique (EHESP); INERIS-STORM to B.J. [N 10028NN]; Rennes Métropole 'Défis scientifiques émergents' to F.C (2011) and A.D.R (2013). The authors have no competing financial interests.

Meiotic Genes Are Enriched in Regions of Reduced Archaic Ancestry.

About 1-6% of the genetic ancestry of modern humans today originates from admixture with archaic humans. It has recently been shown that autosomal genomic regions with a reduced proportion of Neanderthal and Denisovan ancestries (NA and DA) are significantly enriched in genes that are more expressed in testis than in other tissues. To determine whether a cellular segregation pattern would exist, we combined maps of archaic introgression with a cross-analysis of three transcriptomic datasets deciphering the transcriptional landscape of human gonadal cell types. We reveal that the regions deficient in both NA and DA contain a significant enrichment of genes transcribed in meiotic germ cells. The interbreeding of anatomically modern humans with archaic humans may have introduced archaic-derived alleles that contributed to genetic incompatibilities affecting meiosis that were subsequently purged by natural selection.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

Androgen-regulated microRNA-135a decreases prostate cancer cell migration and invasion through downregulating ROCK1 and ROCK2.

Androgen signaling, via the androgen receptor (AR), is crucial in mediating prostate cancer (PCa) initiation and progression. Identifying new downstream effectors of the androgens/AR pathway will allow a better understanding of these mechanisms and could reveal novel biomarkers and/or therapeutic agents to improve the rate of patient survival. We compared the microRNA expression profiles in androgen-sensitive LNCaP cells stimulated or not with 1 nM R1881 by performing a high-throughput reverse transcriptase-quantitative PCR and found that miR-135a was upregulated. After androgen stimulation, we showed that AR directly activates the transcription of miR-135a2 gene by binding to an androgen response element in the promoter region. Our findings identify miR-135a as a novel effector in androgens/AR signaling. Using xenograft experiments in chick embryos and adult male mice, we showed that miR-135a overexpression decreases in vivo invasion abilities of prostate PC-3 cells. Through in vitro wound-healing migration and invasion assays, we demonstrated that this effect is mediated through downregulating ROCK1 and ROCK2 expression, two genes that we characterized as miR-135a direct target genes. In human surgical samples from prostatectomy, we observed that miR-135a expression was lower in tumoral compared with paired adjacent normal tissues, mainly in tumors classified with a high Gleason score (⩾8). Moreover, miR-135a expression is lower in invasive tumors, showing extraprostatic extension, as compared with intraprostatic localized tumors. In tumor relative to normal glands, we also showed a more frequently higher ROCK1 protein expression determined using a semi-quantitative immunohistochemistry analysis. Therefore, in tumor cells, the lower miR-135a expression could lead to a higher ROCK1 protein expression, which could explain their invasion abilities. The highlighted relationship between miR-135a expression level and the degree of disease aggressiveness suggests that miR-135a may be considered as a prognostic marker in human PCa.

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.

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.