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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

ABSTRACT: Purpose: Azoles are widely used antifungal agents. However, many azoles have been shown to have endocrine disrupting properties including triticonazole and flusilazole that we previously showed have adverse effects in vivo. The purpose of this study was to elaborate on how flusilazole and triticonazole can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes. Methods: Testicular mRNA profiles of fetal rats at gestational day 17 or 21 exposed to vehicle, flusilazole, and triticonazole during gestation (from day 7-21) were generated by 3'digital gene expression sequencing, qRT–PCR validation was performed using TaqMan assays and testis histology was assesed by H&E staining and bright field microscopy, protein expression was assesed by immunofluorescence staining and fluorescence microcopy. Results: We identified 17,350 transcripts in the fetal rat testes. 9 transcripts showed differential expression between control and flusilazole or triticonazole exposed rats, with a fold change ≥1.5 and p value <0.05. Altered expression of 2 genes was confirmed with qRT–PCR. Testis histology was normal. Conclusions: Sequencing revealed few transcriptional changes in the fetal rat testes exposed to flusilazole or triticonazole. This suggests that flusilazole may be directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas triticonazole may primarily disturbe androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE154012 | GEO | 2023/07/08

REPOSITORIES: GEO

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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.

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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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