Project description:Background: The regular use of cannabis by young men has been associated with an increased incidence of testicular germ cell tumors (TGCT). TGCT, the most common cancer in young adults, is believed to arise from an alteration of testicular fetal germ cells differentiation during development, with a possible subsequent environmental trigger (eg drugs or other chemicals) during puberty or adulthood leading to cancer. Cannabis consumption by pregnant women is currently increasing worldwide, and legalization for its recreational and therapeutic purposes is debated in numerous countries. In this context, we aimed to determine whether cannabis exposure can affect development of the fetal testis. Methods: Since phytocannabinoids act on an endogenous system called the endocannabinoid system (ECS), we first investigated these signaling pathways in the human fetal testis, from 6 to 17 developmental weeks. We next investigated the effects of the two main components of cannabis, (−)-Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the human fetal testis ex vivo. Results: We show the presence of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), albeit with lower levels. The human fetal testis also expressed several enzymes and receptors of this signaling pathway. Human fetal testicular explants collected from first trimester were exposed to CBD, THC or CBD/THC [ratio 1:1] at concentrations ranging from 10-7 to 10-5M during 72h to 14 days. Phytocannabinoids treatments affected fetal testicular cell proliferation and viability, as well as testosterone secretion by Leydig cells and AMH secretion by Sertoli cells. Transcriptomic analysis performed by BRB-seq on exposed versus unexposed fetal testis explants showed 187 differentially expressed genes (DEGs), some of which involved in toxic substances response and steroid synthesis. Conclusions: Our study provides the first evidence of the presence of ECS in human fetal testis and support a potential adverse effect of cannabis consumption in pregnant women on the male reproductive function development.

Project description:ITo derive a scaffold from human testis for the purpose of tissue engineering and regenerative medicine, we developed a method to produce a cytocompatible decellularized testicular matrix (DTM) while maintaining the native tissue-specific characteristics and components. The potential benefits of tissue-specific scaffolds consisting of naturally-derived extracellular matrix (ECM) have been demonstrated using a wide variety of animal and human tissue sources. However, so far, testis scaffolds have never been considered for constructive remodelling purposes. We have therefore developed a protocol for the preparation of cell-free extracellular matrix and characterized the material extensively using a combination of proteomics, immunohistochemsitry, and cell population assays. To prepare cell-free matrix, human cadaveric testicular tissue was exposed for 24 h or 48 h to 1% Triton X-100 and/or 1% sodium dodecyl sulfate (SDS). The extent of decellularization was evaluated by histology. Confirmation of cell removal in DTM was done by a DNA quantification technique. The protein composition was analysed by LC-MS/MS. The retention of testicular tissue-specific characteristics was evaluated by immunohistochemistry, Alcian blue staining and scanning electron microscopy. Soluble toxicity and testicular cell attachment was assessed to check the cytocompatibility of DTM scaffolds. Histological analysis showed that DTM could be obtained by mechanical agitation in 1% SDS for 24 h. The resulting DTM was found to be clear of cells while retaining the typical three-dimensional structure. Proteomics analysis revelaed the presence of the major components of the native tissue scaffold, including collagen type I and IV, fibronectin, laminin and glycosaminoglycans. In addition, numerous additional ECM proteins in DTM were detected, indicating its complex nature. Importantly, we demonstrated that DTM scaffolds are not cytotoxic, as evidenced by MTT assay showing a normal fibroblast proliferation activity after indirect exposure, and support testicular cell attachment and infiltration.

Project description:The purpose of this study is to assess the potential of TOTM to induce testicular maldevelopment in the rat. We studied the effects of TOTM on the expression of genes in pathways involved in steriodogenesis and testes development. Certain phthalate esters have previously been shown to be involved in the induction of rat testicular mal-development (TMD) through effects on the expression of genes in pathways involved in steroidogenesis and testes development. In order to assess the effects of a potential alternate plasticizer, tris(2-ethylhexyl)trimellitate (TOTM), rats were exposed daily, in utero, to TOTM in order to assess the potential of this compound to induce developmental effects on the fetal testes. Pregnant rats were exposed between gestational day 12 and 19 and fetal testes RNA was analysed using whole genome microarrays. The effects of TOTM on the expression of genes in pathways involved in steroidogenesis and testes development were examined. The effects of TOTM were also compared with di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)phthalate (MEHP), an active metabolite of DEHP, and 2-ethylhexanol (2-EH), which were used as positive and negative controls, respectively. MEHP & DEHP (500mg/kg) caused a repression of genes in TMD pathways involved in cholesterol synthesis and transport (HMGCS, HMGCR, StAR, SCARB1, FDFT1, FDPS), steroidogenesis (Cyp11a, HSD3B1, SC4MOL) and testes development (INSL3, INHA). 2-EH caused minor repression of some of the genes in the TMD pathway. This was rationalised on the basis that 2-EH, a DEHP metabolite, is also a weak PPARα agonist. It has been shown that in utero treatment with DBP will repress the genes from fetal testes involved in steroidogenesis and that this effect is associated with direct DBP-mediated binding of PPARα to the promoters of these genes (Plummer et. al. 2010). TOTM did not cause a significant repression of genes in the TMD pathway. Based on these data, it is highly unlikely that TOTM will cause testicular dysgenesis in rats. Rats were exposed to TOTM in utero by the administration of daily oral gavage doses to pregnant dams between gestational day 12 and 19. The foetal testes were obtained by micro-dissection and prepared to facilitate the isolation of RNA, which was subsequently analysed using whole rat genome microarrays.

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3'DGE-seq analysis of fetal rat testis reveals limited transcriptional changes after exposure to azole fungicides triticonazole and flusilazole despite adverse in vivo effects

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