Abstract

Epidemiological studies continually reveal associations between the organochlorine pesticide, dichlorodiphenyltrichloroethane (DDT) and reproductive disease across aquatic species including urogenital carcinoma in sea lions, sex reversal in fish, and eggshell thinning in condors.^1-3 However, the exact mechanisms by which DDT harms the female reproductive tract remain unclear. The Japanese medaka (Oryzias latipes) is a valuable fish model for sex-specific toxicology studies due to its mapped genome, short generation time, and chromosomal sex determination. Medaka exposed to DDT-contaminated water experience ovarian abnormalities and reduced egg production and fertilization success.^4-6 However, no molecular analyses have been performed to elucidate potential mechanisms. Single-cell mRNA sequencing (scRNA-seq) analyzes transcriptomes of individual cells, revealing heterogeneous cell responses, rare cell types, and transcripts with low expression that may play roles in disease but are masked by traditional bulk RNAseq. The ovary is a highly organized composite of diverse cell types. Synchronized crosstalk between germ and surrounding somatic cells produces critical molecular signals required for oogenesis. Therefore, pesticide-induced signaling perturbations in one cell type can have profound effects on overall ovarian function. Furthermore, intercellular signaling networks required for healthy oogenesis have not been fully resolved in medaka.

This is the first published report of a medaka ovary single-cell atlas despite widespread use as endocrine disruption models. The study objectives are to use single-cell sequencing to 1) define ovarian cell types and marker genes in the medaka ovary and 2) identify the contribution of various cell types and gene regulatory networks to DDT-induced decreases in fertility. Due to the heterogeneous functional roles of ovarian cells, we hypothesized that the diverse cell populations respond to DDT exposure with markedly different expression profiles.

Female medaka hatchlings were exposed to 2 µg/L o,p’-DDT or control solutions over a key 30-day window of ovarian differentiation then grown out to 80 days (sub-adult stage) in clean water. Fish were euthanized and ovaries pooled by treatment (n=15 ovaries/pool), then enzymatically dissociated into single-cell suspensions (∼900 cells/µl; 91% viability) for scRNA-seq analysis on a 10X Genomics Chromium System. Resulting ovary libraries (2 DDT-treated replicates, 2 control replicates) contained an average of ∼13.6 K cells (∼35 K median reads/cell; 77.4% of reads mapped to the genome) expressing ∼990 median genes/cell. Libraries were combined for cell type identification, clustering, and differential expression analyses of a final library containing 26,961 DDT-treated and 27,365 control cells. Using cell visualization and K-means clustering, we identified 15 germ and somatic cell clusters and their signature molecular markers. Somatic cell types included steroidogenic (follicle, theca, ovarian stroma), blood (erythrocytes, lymphocytes, neutrophils, macrophages), and endothelial cells. Interestingly, one cluster shared transcriptomic profiles of both follicle (estrogen-producing) and stroma cells. Germ cell populations included early-stage germ, meiotic cells, and oocytes. Oocytes appeared to be most affected from early-life DDT exposure, as DDT-treated oocytes clustered separately from controls. Differential expression and gene ontology analyses are underway to identify genes and corresponding biological processes driving this pattern. By combining medaka with cutting-edge scRNA-seq, we aim to promote a novel experimental and computational workflow for predicting adverse cellular effects of chemical exposures.

Acknowledgements

Research reported in this project was supported by NIEHS of the National Institutes of Health under award numbers T32 ES007059 and 1F30ES033550-01A1, the Dorothea and Vivian Hagaman Graduate Fellowship, and the UC Davis Aquatic Health Program (AHP). We are especially grateful to Aishwarya Bhusal, Jessica Munson, and AHP members for their vital assistance with experimental set-up and fish husbandry, as well as support from the LaSalle Lab for single-cell dissociation optimization and Christiaan Henkel (Norwegian University of Life Sciences) for sharing modified medaka genome files. All experiments were performed under approved IACUC Protocol #21475.

*Presenting author
+Student presenter

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