The Acute Exposure of Human Adult Testis Tissue to Cannabinoids THC and CBD Does Not Impact Testosterone Production Nor Germ Cell Lineage.

PURPOSE: While an increased risk of developing germ cell tumors has been established in regular cannabis consumers, there is conflicting evidence about an association between cannabis use and testosterone levels in those regular consumers. In this context, we aimed to determine whether Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the two major and best-studied cannabinoids present in cannabis, also the most used for therapeutic applications, can directly impact the steroidogenic function and germ cell lineage of the human adult testis. MATERIALS AND METHODS: We used our well-characterized organotypic culture of human testis, in which adult testis explants were exposed to CBD, THC, or CBD/THC [ratio 1:1] from 10-9 to 10-5 M for up to either 48 hours or 9 days of culture. The testes were obtained from multi-organ donors (n=13; mean age: 55.15±5.62 y). RESULTS: The testosterone production and the spatial distribution of Leydig cells did not change upon CBD and/or THC exposure versus controls after 48 hours or 9 days. The overall tissue morphology of the cannabinoids-exposed testis explants was similar to their control upon 24 hours or 9 days of exposure, a finding confirmed by morphometric analyses on short-term cultures. In line, the number of apoptotic cells was not affected by either 48 hours or 9 days of cannabinoids treatment versus mock. Cannabinoids had no impact on the number of proliferating cells nor on mRNA expression of genes encoding proteins involved in germ cell differentiation, meiosis, or Sertoli and Leydig functions after 24 hours exposure. CONCLUSIONS: Altogether, these findings show an absence of acute direct effects of exposure to cannabis-derived cannabinoids THC and CBD on testicular testosterone production and germ cells ex vivo. Further studies are warranted to explore an indirect impact of cannabinoids on testis functions through the hypothalamic-pituitary-testis axis, as well as the potential effects of long-term exposures.

Developmental exposure to chlordecone induces transgenerational effects in somatic prostate tissue which are associated with epigenetic histone trimethylation changes.

BACKGROUND: Chlordecone (CD), also known as Kepone, is an organochlorine insecticide that has been used in banana crops in the French West Indies. Due to long-term contamination of soils and water, the population is still exposed to CD. Exposure to CD in adulthood is associated with an increased risk of prostate cancer (PCa). OBJECTIVES: We examined the transgenerational effects of CD on murine prostate tissue. METHODS: We exposed pregnant Swiss mice to CD. The prostates from directly exposed (F1) and non-exposed (F3) male progeny were analyzed. We used immunofluorescence, RNA-seq and ChIP-seq techniques for the comprehensive analyses of chromatin states in prostate. RESULTS: We observed an increased prostatic intraepithelial neoplasia phenotype (PIN) in both F1 and F3 generations. Transcriptomic analysis in CD-derived F1 and F3 prostate using RNA-seq revealed that 970 genes in F1 and 218 in F3 genes were differentially expressed. The differentially expressed genes in both datasets could be clustered accordingly to common biological processes, "cell differentiation", "developmental process", "regulating of signaling", suggesting that in both generations similar processes were perturbed. We detected that in both datasets several Hox genes were upregulated; in F1, the expression was detected mainly in Hoxb and Hoxd, and in F3, in Hoxa family genes. Using a larger number of biological replicates and RT-qPCR we showed that genes implicated in testosterone synthesis (Akr1b3, Cyp11a1, Cyp17a1, Srd5a1) were dramatically upregulated in PIN samples; Cyp19a1, converting testosterone to estradiol was elevated as well. We found a dramatic increase in Esr2 expression both in F1 and F3 prostates containing PIN. The PIN-containing samples have a strong increase in expression of self-renewal-related genes (Nanog, Tbx3, Sox2, Sox3, Rb1). We observed changes in liver, F1 CD-exposed males have an increased expression of genes related to DNA repair, matrix collagen and inflammation related pathways in F1 but not in F3 adult CD-derived liver. The changes in RNA transcription were associated with epigenetic changes. Specifically, we found a global increase in H3K4 trimethylation (H3K4me3) and a decrease in H3K27 trimethylation (H3K27me3) in prostate of F1 mice. ChIP-seq analysis showed that 129 regions in F1 and 240 in F3 acquired altered H3K4me3 occupancy in CD-derived prostate, including highest increase at several promoters of Hoxa family genes in both datasets. The alteration in H3K4me3 in both generations overlap 73 genes including genes involved in proliferation regulation, Tbx2, Stat3, Stat5a, Pou2f3 and homeobox genes Hoxa13, Hoxa9. CONCLUSIONS: Our data suggest that developmental exposure to CD leads to epigenetic changes in prostate tissue. The PIN containing samples showed evidence of implication in hormonal pathway and self-renewal gene expression that have the capacity to promote neoplasia in CD-exposed mice.

Ovarian dysfunction following prenatal exposure to an insecticide, chlordecone, associates with altered epigenetic features.

Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features.

Perinatal Exposure to Glyphosate and a Glyphosate-Based Herbicide Affect Spermatogenesis in Mice.

Glyphosate is the most widely used herbicide in the world. Several studies have investigated the effects of glyphosate and glyphosate-based herbicides (GBHs) on male reproduction, but there is still little and conflicting evidence for its toxicity. In this study, we analyzed the effects of glyphosate, alone or in formula, on the male reproductive system. Pregnant mice were treated from E10.5 to 20 days postpartum by adding glyphosate or a GBH (Roundup 3 Plus) to their drinking water at 0.5 (the acceptable daily intake, ADI dose), 5 and 50 mg/kg/day. Male offspring derived from treated mice were sacrificed at 5, 20, and 35 days old (d.o.) and 8 months old (m.o.) for analysis. Our result showed that exposure to glyphosate, but not GBH, affects testis morphology in 20 d.o. and decrease serum testosterone concentrations in 35 d.o. males. We identified that the spermatozoa number decreased by 89% and 84% in 0.5 and 5 mg/kg/day of GBH and glyphosate groups, respectively. Moreover, the undifferentiated spermatogonia numbers were decreased by 60% in 5 mg/kg/day glyphosate group, which could be due to the alterations in the expression of genes involved in germ cell differentiation such as Sall4 and Nano3 and apoptosis as Bax and Bcl2. In 8 m.o. animals, a decreased testosterone level was observed in GBH groups. Our data demonstrate that glyphosate and GBHs could cause endocrine-disrupting effects on male reproduction at low doses. As glyphosate has effects at the ADI level, our data suggest that the current ADI for glyphosate could be overestimated.

Gestational exposure to chlordecone promotes transgenerational changes in the murine reproductive system of males.

Environmental factors can affect epigenetic events during germline reprogramming and impose distinctive transgenerational consequences onto the offspring. In this study, we examined the transgenerational effects of chlordecone (CD), an organochlorine insecticide with well-known estrogenic properties. We exposed pregnant mice to CD from embryonic day 6.5 to 15.5 and observed a reduction in spermatogonia (SG) numbers in F3, meiotic defects in spermatocytes and decrease in spermatozoa number in the first and third generation of male progeny. The RNA qRT-PCR expression analysis in F1 and transcriptomics analysis in F3 males using the whole testes revealed changes in the expression of genes associated with chromosome segregation, cell division and DNA repair. The expression of the master regulator of pluripotency, Pou5f1, decreased in foetal and increased in adult F1, but not in F3 adult testes. Analysis of histone H3K4me3 distribution revealed widespread changes in its occupancy in the genome of F1 and F3 generations. We established that 7.1% of altered epigenetic marks were conserved between F1 and F3 generations. The overlapping changes common to F1 and F3 include genes implicated in cell adhesion and transcription factor activities functions. Differential peaks observed in F1 males are significantly enriched in predicted ESR1 binding sites, some of which we confirmed to be functional. Our data demonstrate that CD-mediated impairment of reproductive functions could be transmitted to subsequent generations.

Embryonic exposure to the widely-used herbicide atrazine disrupts meiosis and normal follicle formation in female mice.

The widely-used herbicide atrazine (ATZ) is detected in ground and surface water in many countries. Several studies in animals have demonstrated that ATZ has endocrine-disrupting effects on male and female reproduction in many vertebrate species. In this study, we investigated the effects of ATZ exposure on meiosis, a key step in gametogenesis in mammals. The treatment was initiated before oocyte entry into meiosis, which occurs during the embryonic period in females. We found that embryonic exposure to ATZ increases the level of 8-oxo-guanine in the nucleus of meiotic cells, reflecting oxidative stress and affecting meiotic double-strand break repair, chromosome synapsis and crossover numbers. Finally, embryonic exposure to ATZ reduces the number of primordial follicles and increases the incidence of multi-oocyte follicles in adult mice. Our data demonstrate that embryonic exposure to ATZ disrupts prophase I of meiosis and affects normal follicle formation in female mice.

Exposure to the widely used herbicide atrazine results in deregulation of global tissue-specific RNA transcription in the third generation and is associated with a global decrease of histone trimethylation in mice.

The epigenetic events imposed during germline reprogramming and affected by harmful exposure can be inherited and transferred to subsequent generations via gametes inheritance. In this study, we examine the transgenerational effects promoted by widely used herbicide atrazine (ATZ). We exposed pregnant outbred CD1 female mice and the male progeny was crossed for three generations with untreated females. We demonstrate here that exposure to ATZ affects meiosis, spermiogenesis and reduces the spermatozoa number in the third generation (F3) male mice. We suggest that changes in testis cell types originate from modified transcriptional network in undifferentiated spermatogonia. Importantly, exposure to ATZ dramatically increases the number of transcripts with novel transcription initiation sites, spliced variants and alternative polyadenylation sites. We found the global decrease in H3K4me3 occupancy in the third generation males. The regions with altered H3K4me3 occupancy in F3 ATZ-derived males correspond to altered H3K4me3 occupancy of F1 generation and 74% of changed peaks in F3 generation are associated with enhancers. The regions with altered H3K4me3 occupancy are enriched in SP family and WT1 transcription factor binding sites. Our data suggest that the embryonic exposure to ATZ affects the development and the changes induced by ATZ are transferred up to three generations.

The epigenetic processes of meiosis in male mice are broadly affected by the widely used herbicide atrazine.

BACKGROUND: Environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. We studied the effects of the widely used herbicide atrazine (ATZ) on meiosis, a key step of gametogenesis, in male mice. METHODS: Gene expression pattern was analysed by Gene-Chip array. Genome-wide mapping of H3K4me3 marks distribution was done by ChIP-sequencing of testis tissue using Illumina technologies. RT-qPCR was used to validate differentially expressed genes or differential peaks. RESULTS: We demonstrate that exposure to ATZ reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. Using Gene-Chip and ChIP-Seq analysis of H3K4me3 marks, we found that a broad range of cellular functions, including GTPase activity, mitochondrial function and steroid-hormone metabolism, are affected by ATZ. Furthermore, treated mice display enriched histone H3K4me3 marks in regions of strong recombination (double-strand break sites), within very large genes and reduced marks in the pseudoautosomal region of X chromosome. CONCLUSIONS: Our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.

Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

Evidence for a subventricular zone neural stem cell phagocytic activity stimulated by the vitamin K-dependent factor protein S.

Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

Retinoic acid induces Sertoli cell paracrine signals for spermatogonia differentiation but cell autonomously drives spermatocyte meiosis.

Direct evidence for a role of endogenous retinoic acid (RA), the active metabolite of vitamin A in the initial differentiation and meiotic entry of spermatogonia, and thus in the initiation of spermatogenesis is still lacking. RA is synthesized by dedicated enzymes, the retinaldehyde dehydrogenases (RALDH), and binds to and activates nuclear RA receptors (RARA, RARB, and RARG) either within the RA-synthesizing cells or in the neighboring cells. In the present study, we have used a combination of somatic genetic ablations and pharmacological approaches in vivo to show that during the first, prepubertal, spermatogenic cycle (i) RALDH-dependent synthesis of RA by Sertoli cells (SC), the supporting cells of the germ cell (GC) lineage, is indispensable to initiate differentiation of A aligned into A1 spermatogonia; (ii) RARA in SC mediates the effects of RA, possibly through activating Mafb expression, a gene whose Drosophila homolog is mandatory to GC differentiation; (iii) RA synthesized by premeiotic spermatocytes cell autonomously induces meiotic initiation through controlling the RAR-dependent expression of Stra8. Furthermore, we show that RA of SC origin is no longer necessary for the subsequent spermatogenic cycles but essential to spermiation. Altogether, our data establish that the effects of RA in vivo on spermatogonia differentiation are indirect, via SC, but direct on meiotic initiation in spermatocytes, supporting thereby the notion that, contrary to the situation in the female, RA is necessary to induce meiosis in the male.

An endogenous vitamin K-dependent mechanism regulates cell proliferation in the brain subventricular stem cell niche.

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(-)-3-acetonylbenzyl)-4-hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease.

Spermatogonia differentiation requires retinoic acid receptor γ.

Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARß, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (A(al)) spermatogonia, as well as during the transition from A(al) to A(1) spermatogonia, which is known to require RA; and 2) ablation of Rarg, either in the whole mouse or specifically in spermatogonia, does not affect meiosis and spermiogenesis but impairs the A(al) to A(1) transition in the course of some of the seminiferous epithelium cycles. Upon ageing, this phenomenon yields seminiferous tubules containing only spermatogonia and Sertoli cells. Altogether, our findings indicate that RARG cell-autonomously transduces, in undifferentiated spermatogonia of adult testes, a RA signal critical for spermatogenesis. During the prepubertal spermatogenic wave, the loss of RARG function can however be compensated by RARA, as indicated by the normal timing of appearance of meiotic cells in Rarg-null testes. Accordingly, RARG- and RARA-selective agonists are both able to stimulate Stra8 expression in wild-type prepubertal testes. Interestingly, inactivation of Rarg does not impair expression of the spermatogonia differentiation markers Kit and Stra8, contrary to vitamin A deficiency. This latter observation supports the notion that the RA-signaling pathway previously shown to operate in Sertoli cells also participates in spermatogonia differentiation.