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Single cell RNA-seq reveals that granulosa cells are a target of phthalate toxicity in the ovary

ABSTRACT: Phthalates are known endocrine disrupting chemicals and ovarian toxicants that are used widely in consumer products. Phthalates have been shown to exert ovarian toxicity on multiple endpoints, altering transcription of genes responsible for normal ovarian function. However, the molecular mechanisms by which phthalates act on the ovary are not well understood. In this study, we hypothesized that phthalates specifically target granulosa cells within the ovarian follicle. To test our hypothesis, we cultured whole mouse antral follicles for 96 hours in the presence of vehicle or 10 ug/mL of a phthalate metabolite mixture. At the end of the culture period, follicles were dissociated into single cell suspensions and subjected to single cell RNA sequencing. We used markers from published studies to identify major cell type clusters, the largest of which were granulosa and theca/stroma cells. We further identified sub-populations of granulosa, theca, and stromal cells and analyzed differentially expressed genes between the phthalate treatment and control. Granulosa cells, specifically mural granulosa cells, had the most differentially expressed genes. Pathway analysis of differentially expressed genes from the overall granulosa cell cluster revealed disruption of cell cycle and mitosis, whereas pathway analysis of the mural granola cell subcluster identified terms related to translation and ribosome. Our findings suggest that phthalates have both broad impacts on major cell types and specific impacts on individual cell types, emphasizing the complexity of phthalate toxicity and highlighting how bulk sequencing can mask effects on vulnerable cell types.

ORGANISM(S): Mus musculus

PROVIDER: GSE277115 | GEO | 2025/01/06

REPOSITORIES: GEO

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Project description:Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are three phthalates commonly found in consumer products, including the plastic coating of pharmaceuticals and personal care products. Folliculogenesis, a tightly regulated process occurring in the ovary, is the maturation of an immature primordial follicle to a mature antral follicle. Follicles house the oocyte and antral follicles specifically play a crucial role in ovarian steroidogenesis and ovulation. DBP, BBP, and DEHP have been associated with inhibited antral follicle growth in vitro, decreased ovulation rates in vitro, and decreased antral follicle counts in women. However, little is known about the effects of a three-phthalate mixture on antral follicles in vivo. The objective of this study was to evaluate the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. CD-1 female mice (60 days old) were pipet fed tocopherol stripped corn oil (vehicle control) only or a phthalate mixture (52% DBP, 36% DEHP, and 12% BBP dissolved in vehicle) which modeled human follicular fluid concentrations. The mice were treated with 32µg/kg/day (PHT Mix 32; cumulative estimate in general population) and 500µg/kg/day (PHT Mix 500; cumulative estimate in occupationally exposed individuals) for 10 consecutive days. Proteome profiling of antral follicles (>250µm) was performed via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were identified in the three groups, of which 194 were differentially abundant between the vehicle and PHT Mix 32 group, and 136 between the vehicle and PHT Mix 500 group. Gene ontology analysis revealed that the two treatments of the phthalate mixture upregulate and downregulate distinctive processes, supporting non-monotonic effects of phthalates on the antral follicle proteome. Taken together, these results reveal that a human relevant mixture of DBP, BBP, and DEHP alters the antral follicle proteome and merits further evaluation to elucidate the molecular mechanisms by which phthalates cause negative reproductive outcomes.

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Single cell RNA-seq reveals that granulosa cells are a target of phthalate toxicity in the ovary

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