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International audience; 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.

International audience

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Although mammalian development is directly affected by exposition of the mother during pregnancy, there is this last decade increasing evidence for the preconceptional impact of the father on their offspring. This article provides a summary of the major factors able to impact male gametes and considers epigenetics changes as supporting the existence of inheritance of paternal background.

International audience; 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 (dpp) 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, affect 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 suggests that the current ADI for glyphosate could be overestimated.

Depuis le debut des annees 90, la sante reproductive des hommes et le role qu’une sous-classe de produits chimiques appelee « perturbateurs endocriniens » (PE) pourrait jouer dans la diminution de la qualite du sperme et dans l’augmentation de l’incidence des cancers des testicules et de malformations congenitales ont suscite de vives inquietudes. Les hommes sont exposes simultanement a un grand nombre de produits chimiques pouvant jouer un role dans ces troubles. Ils y seront exposes a l’âge adulte ou durant des phases particulaires de vulnerabilite que sont le developpement, l’enfance ou la puberte. Les testicules assurent deux grandes fonctions: la production des cellules sexuelles mâles et la production des steroides sexuels. Ces deux fonctions s’etablissent durant la vie fœtale et cette ontogenese est fondamentale, puisqu’elle conditionne la fertilite de l’individu adulte et la masculinisation des organes genitaux. Le lien entre reproduction masculine et perturbation endocrinienne a fait l’objet ces dernieres decennies d’un grand nombre d’etudes toxicologiques et epidemiologiques. Beaucoup de composes exogenes incluant des plastifiants, des pesticides, des fongicides, des medicaments ou encore des composes recreatifs ont ete teste au travers de modeles animaux ou humain, ex-vivo ou in-vivo, afin de demontrer le potentiel anti-androgenique de certaines molecules ou leur capacite a venir alterer la fonction des cellules de Sertoli, cellules somatiques du testicule, ou de la lignee germinale. Certains travaux ont egalement mis en lumiere la capacite de certain PE a avoir des effets transgenenerationnels par leur impact sur l’epigenome. Les methodes utilisees dans le cadre de ce domaine de recherche vont de methodes classiques (histologie, immunohistochimie…) a l’utilisation de methodes innovantes avec l’arrivee de donnees de sequencage a haut debit. Alors que le potentiel perturbateur endocrinien de certain compose est aujourd’hui clairement avere, apporter la preuve que le mecanisme interferant avec le systeme endocrinien est bien responsable de l’effet observe represente un veritable challenge. Ainsi l’etude des PE represente un enjeu majeur pour la recherche et les pouvoirs publics car les sources d’exposition sont nombreuses et difficiles a maitriser, tandis que les consequences biologiques de ces expositions sont encore mal apprehendees et complexes a etudier.

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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.

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International audience; 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.

International audience; 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), RARβ, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (Aal) spermatogonia, as well as during the transition from Aal to A1 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 Aal to A1 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.

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. Stem Cells 2015;33:515–525

Supplementary material. (PDF 2459 kb)

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., Author Summary Differentiation of spermatozoa from immature germ cells, called spermatogonia, critically depends on retinoic acid (ATRA), the active metabolite of vitamin A that acts though binding to nuclear receptors called RXR and RAR. To understand the mechanism by which ATRA control germ cell differentiation, we generated mice simultaneously lacking all RXR or all RAR specifically in spermatogonia. From their phenotypic analysis, we demonstrate that meiosis does not require a RAR/RXR-dependent pathway in germ cells and propose that this process is either ATRA-independent or requires an ATRA signal originating from somatic cells. We also show that RXR, in the form of dimers with RAR, can drive spermatogonia differentiation through binding to a regulatory region located in the Sall4 gene. This finding is significant, as the transcription factor encoded by Sall4 is known to regulate the expression of KIT, a key tyrosine kinase receptor which is frequently deregulated in testicular cancer.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2095-y) contains supplementary material, which is available to authorized users.

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.

Additional file 1: Fig. S1. Gestational CD exposure increases H2Aub in embryonic ovaries. Fig. S2. Increased levels of H3K27me3 in meiotic cells following embryonic CD exposure. Fig. S3. Gestational exposure to CD leads to decrease in expression of developmental genes. Fig. S4. Gestational exposure to CD leads to decrease in expression of genes associated with signaling, transcription regulation and DNA repair in three-month-old ovaries. Fig. S5. Decrease in the levels of AMH in adult ovaries after gestational CD exposure. Fig. S6. Gestational exposure to CD leads to decrease in body and ovarian weights in 5-month-old animals. Fig. S7. CD induces increased H3K4me3 occupancy at ZBTB17 binding site. Table S1. H3K4me3 differential peaks identified in CD exposed ovaries. Table S2. Targets of ZFP57 identified by ChEA. Table S3. Targets of TRIM28 identified by ChEA. Table S4. Oligonucleotides used for RT-qPCR.

Additional file 1: Fig. S1. Gestational CD exposure increases H2Aub in embryonic ovaries. Fig. S2. Increased levels of H3K27me3 in meiotic cells following embryonic CD exposure. Fig. S3. Gestational exposure to CD leads to decrease in expression of developmental genes. Fig. S4. Gestational exposure to CD leads to decrease in expression of genes associated with signaling, transcription regulation and DNA repair in three-month-old ovaries. Fig. S5. Decrease in the levels of AMH in adult ovaries after gestational CD exposure. Fig. S6. Gestational exposure to CD leads to decrease in body and ovarian weights in 5-month-old animals. Fig. S7. CD induces increased H3K4me3 occupancy at ZBTB17 binding site. Table S1. H3K4me3 differential peaks identified in CD exposed ovaries. Table S2. Targets of ZFP57 identified by ChEA. Table S3. Targets of TRIM28 identified by ChEA. Table S4. Oligonucleotides used for RT-qPCR.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Uma tendência significativa para um declínio mundial na fertilidade masculina humana tem sido relatada nos últimos anos. Enquanto isso, a exposição a químicos tem sido proposta para potencialmente contribuir para os distúrbios reprodutivos masculinos, em que químicos ambientais e farmacológicos/recreacionais podem induzir efeitos prejudiciais à saúde reprodutiva masculina. Entre uma variedade complexa de produtos químicos ambientais, o cádmio (Cd) é um dos metais pesados que se classifica entre os metais prioritários com importância para a saúde pública, uma vez que o Cd é considerado como um desregulador endócrino. Os seres humanos estão expostos diariamente ao Cd por meio de fontes ocupacionais (e.g. produção de baterias) e não ocupacionais (e.g. alimentos/água contaminados e fumaça de cigarro). Além disso, o consumo de cannabis para fins terapêuticos e recreativos está em ascensão. Neste contexto de produtos químicos e fertilidade masculina, a exposição aos compostos da cannabis, chamados fitocanabinoides (canabinoides), pode resultar em resultados adversos para a saúde reprodutiva masculina. Portanto, a presente tese teve como objetivo avaliar o impacto dessas duas tendências globais, cádmio e canabinoides presentes na cannabis, no processo espermatogênico. Já se sabe que o cádmio induz danos ao testículo. No entanto, os mecanismos pelos quais o cádmio promove o aparecimento de patologias testiculares e a relação entre dose, via e tempo de exposição e lesões são pouco conhecidos. Nesse sentido, realizamos uma revisão sistemática (Capítulo 1) para responder a essas questões. Depois de recuperar estudos originais em bancos de dados, organizamos os resultados em uma estrutura de Adverse Outcome Pathway (AOP). Além disso, uma análise de viés foi realizada. Nos 37 estudos selecionados, ficou claro que o cádmio induz histopatologias significativas em testículos de modelos murinos independente de via, de maneira dose e tempo-dependente. Os danos podem ser observados nas primeiras horas de exposição, principalmente danos vasculares, sugerindo que a falha na vascularização é o mecanismo primário envolvido. A AOP mostrou que potenciais eventos moleculares iniciais podem ser o mimetismo e a interferência com elementos essenciais desregulando proteínas (estruturais e antioxidantes), alteração na atividade da enzima de fosforilação oxidativa e alteração da expressão gênica, que levam à falha reprodutiva. No entanto, a análise de viés mostrou que as evidências atuais apresentam qualidade metodológica pobre. Em relação ao impacto dos canabinoides na saúde reprodutiva masculina, realizamos dois ensaios biológicos. Em um manuscrito (Capítulo 2), buscamos responder se os dois principais fitocanabinoides presentes na cannabis poderiam representar um perigo para o testículo humano adulto. Para isso, expusemos testículos humanos adultos ao Δ 9- tetrahidrocanabinol (THC), canabidiol (CBD), ou CBD/THC (proporção 1:1) nas concentrações de 10 -9 M a 10 -5 M por 48 horas, utilizando nosso modelo ex vivo único de cultura organotípica. Nossos achados não mostraram alteração na histologia testicular, produção de testosterona, número de células apoptóticas e proliferativas e expressão de genes que codificam importantes proteínas testiculares. Uma vez que alguns problemas reprodutivos masculinos foram hipotetizados como sendo de origem fetal, no último estudo (Capítulo 3) avaliamos se a exposição pré-natal ao CBD/THC poderia levar a distúrbios reprodutivos masculinos na vida adulta. Para isso, expomos camundongos fêmeas prenhas do dia de gestação 6,5 (E6,5) ao E15,5 a 5mg/kg/dia de CBD/THC (proporção 1:1), por gavagem. Observamos diminuição do peso testicular e alterações morfológicas testiculares, estas identificadas por análises morfométricas, na prole adulta. No entanto, essas alterações não foram acompanhadas por mudanças nos números de espermatogônias indiferenciadas e células de Sertoli. A análise da expressão gênica revelou um aumento na expressão de genes pró-apoptótico (Bax), de diferenciação de células germinativas (Sohlh1) e de regulação meiótica (Stra8). Não foram observadas alterações nos níveis séricos de testosterona. Em geral, nossos resultados demonstram que o cádmio, um poluente ambiental, pode afetar a saúde reprodutiva masculina de modelos murinos e conduzir à infertilidade masculina. Entretanto, os dois fitocanabinoides presentes na cannabis, THC e CBD, os quais são também utilizados como químicos farmacológicos, não causam efeitos agudos diretos na histofisiologia testicular usando nossa cultura organotípica bem caracterizada de testículo humano adulto. Porém, exposição pré-natal a uma mistura desses dois canabinoides derivados da canabis pode conduzir ao peso testicular diminuído de camundongos na vida adulta. Ainda, a exposição pré-natal pode acelerar a meiose e a diferenciação celular e induzir apoptose, baseado nos achados moleculares, provavelmente, contribuindo de forma compensatória para a diminuição do peso do testículo. Assim, em camundongos, exposição pré-natal a uma mistura de THC/CBD pode afetar a biologia testicular de proles adultas. No geral, os resultados apresentados por esta tese fornecem novos conhecimentos sobre a toxicologia reprodutiva e a saúde reprodutiva masculina, que podem ser considerados para preservar a fertilidade em toda a população. Palavras-chave: Toxicologia reprodutiva. Contaminante ambiental. Adverse Outcome Pathway. Δ 9 -tetrahidrocanabinol. Canabidiol. Cultura organotípica. A significant trend towards a worldwide decline in human male fertility has been reported over the last years. In the meantime, exposure to chemicals has been proposed to potentially contribute to male reproductive disorders, in which environmental and pharmacological/recreational chemicals might induce detrimental effects on male reproductive health. Among a complex variety of environmental chemicals, cadmium (Cd) is one of the heavy metals that rank among the priority metals that are of public health significance, since Cd is considered as an endocrine disruptor. Human beings are daily exposure to Cd through occupational (e.g. production of batteries) and non-occupational (e.g. contaminated food/water, cigarette smoke) sources. Furthermore, cannabis consumption for medical and recreational purposes is on the rise. In this context of chemicals and male fertility, exposure to cannabis compounds, called phytocannabinoids (cannabinoids), could result in adverse outcomes to male reproductive health. So, this present thesis aimed to assess the impact of these two global trends, cadmium and cannabinoids, in the spermatogenic process. It is already known that cadmium induces damage to the testis. However, the mechanisms that cadmium leads to testicular histopathologies and the relationship between dose, route, and time of exposure and injuries are poorly understood. In this sense, we conducted a systematic review (Chapter 1) to answer these questions. After retrieving original studies on databases, we organized the results into an Adverse Outcome Pathway (AOP) framework. Also, a bias analysis was performed. In the 37 studies selected, it was clear that cadmium induces significant histopathologies in the murine model’s testis regarding routes, in a dose- and time-dependent manner. The damages can be observed in the first hours of exposure, mainly vascular damages suggesting that vasculature failure is the primary mechanism involved. The AOP showed that potential molecular initiating events may mimic and interfere with essential elements disrupting proteins (structural and antioxidants), change in the oxidative phosphorylation enzyme activities, and gene expression alteration, which lead to reproductive failure. However, analysis of bias showed that the current evidence presents poor methodological quality. Regarding the cannabinoid impact on male reproductive health, we performed two biological essays. In one manuscript (Chapter 2), we aimed to answer whether the two main phytocannabinoids present in cannabis could represent a hazard for adult human testis. For that, we exposed adult human testis to Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), or CBD/THC (ratio 1:1) at the concentrations from 10 -9 M to 10 -5 M for 48 hours, by using our unique ex vivo model of organotypic culture. Our findings show no alteration in testis histology, testosterone production, number of apoptotic and proliferative cells, and expression of some genes encoding important testicular proteins. Since some male reproductive problems have been hypothesized to have a fetal origin, in the last study (Chapter 3), we assessed whether prenatal exposure to CBD and THC could lead to male reproductive disorders in adult life. For that, we exposed pregnant mice from embryogenic day 6.5 (E6.5) to E15.5 to 5mg/kg/day of CBD/THC (ratio 1:1), by gavage. Then, we observed decreased testicular weight and testicular morphological alterations identified by morphometric analyses in adult offspring. However, these alterations were not followed by changes in the undifferentiated spermatogonia and Sertoli cell numbers. The gene expression analysis revealed an increase in the expression of pro-apoptotic (Bax), germ cell differentiation (Sohlh1), and regulating meiotic (Stra8) genes. Alterations in the serum testosterone levels were not observed. In general, our findings demonstrate that cadmium, an environmental pollutant, can affect the male reproductive health of murine models and lead to male infertility. On the other hand, the two main cannabinoids present in cannabis, THC and CBD, which are also used as pharmacological chemicals, do not cause acute direct effects in the testicular histophysiology using our well-characterized organotypic culture of adult human testis. However, prenatal exposure to a mixture of these cannabis-derived cannabinoids can lead to decreased testis weight of mice in adult life. Also, prenatal exposure can accelerate meiosis and cellular differentiation and induce apoptosis, based on the molecular findings, probably, contributing compensatory to the decreased testis weight. So, in mice, prenatal exposure to a mixture of THC/CBD might affect the testis biology of adult offspring. Overall, the results presented by this thesis provide new insights into male repro-toxicology and reproductive health, which might be considered to preserve fertility across the population. Keywords: Repro-toxicology. Environmental contaminant. Adverse Outcome Pathway. Δ 9 - tetrahydrocannabinol. Cannabidiol. Organotypic culture.