Bisphenol A is an endocrine disruptor associated with hormone synthesis and reproduction alterations. However, the initiating events underpinning these dysfunctions are still unclear. Here, we address the hypothesis that BPA interferes with the highly evolutionary conserved process of mitochondrial cholesterol transport, a crucial step in steroid hormone biosynthesis, by using the model organism C. elegans. We observed that embryonic lethality and germline apoptosis, hallmarks of BPA's reproductive toxicity in C. elegans, are fully rescued by low exogenous cholesterol supplementation. We also observed that increasing BPA concentrations proportionally reduced mitochondrial cholesterol levels. Mutants for strl-1 (ortholog of StAR), but not C41G7.9 (ortholog of TSPO), show reproductive defects similar to BPA's while BPA exposure in a strl-1 background did not worsen these effects. Finally, cholesterol supplementation rescued these defects for all strl-1 genotype/BPA combinations assessed. Together, these results uncover a novel mechanism underlying BPA's germline toxicity through the alteration of cholesterol transport.

Caenorhabditis elegans is a valuable model to study organ, tissue, and cell-type responses to external cues. However, the nematode comprises multiple syncytial tissues with spatial coordinates corresponding to distinct nuclear transcriptomes. Here, we present a single-nucleus RNA sequencing (snRNA-seq) protocol that aims to overcome difficulties encountered with single-cell RNA sequencing in C. elegans. We describe steps for isolating C. elegans nuclei for downstream applications including snRNA-seq applied to the context of alcohol exposure. For complete details on the use and execution of this protocol, please refer to Truong et al. (2023).¹.

Single-cell transcriptomic platforms provide an opportunity to map an organism's response to environmental cues with high resolution. Here, we applied single-nucleus RNA sequencing (snRNA-seq) to establish the tissue and cell type-resolved transcriptome of the adult C. elegans and characterize the inter- and trans-generational transcriptional impact of ethanol. We profiled the transcriptome of 41,749 nuclei resolving into 31 clusters, representing a diverse array of adult cell types including syncytial tissues. Following exposure to human-relevant doses of alcohol, several germline, striated muscle, and neuronal clusters were identified as being the most transcriptionally impacted at the F1 and F3 generations. The effect on germline clusters was confirmed by phenotypic enrichment analysis as well as by functional validation, which revealed a remarkable inter- and trans-generational increase in germline apoptosis, aneuploidy, and embryonic lethality. Together, snRNA-seq represents a valuable approach for the detailed examination of an adult organism's response to environmental exposures.