Environmentally-relevant doses of bisphenol A and S exposure in utero disrupt germ cell programming across generations resolved by single nucleus multi-omics

Abstract

Background: Exposure to endocrine-disrupting chemicals (EDCs), such as bisphenol (BP) A, disrupts reproduction across generations. Germ cell epigenetic alterations are proposed to mediate these transgenerational defects. Previously, we have shown that prenatal exposure to environmentally relevant doses of BPA or its substitute, BPS, caused transgenerationally maintained reproductive impairments associated with neonatal spermatogonial epigenetic changes in male mice. However, the mechanisms sustaining these changes across generations remain unclear.

Objectives: This study aimed to systematically elucidate the mechanism of transgenerational inherence by prenatal BPA and BPS exposure in the murine germline from F1 to F3 generations at both transcriptomic and epigenetic levels.

Methods: Pregnant CD-1 females (F0) were orally administered BPA or BPS at doses of 0 (vehicle control), 0.5, 50, or 1000 µg/kg/b.w./day from gestational day 7 to birth. Sperm counts and motility were examined in F1, F2, and F3 adult males. THY1⁺ germ cells on postnatal day 6 from F1, F2, and F3 males at a dose of 50 µg/kg/b.w./day were used for analysis by single-nucleus (sn) multi-omics (paired snRNA-seq and snATAC-seq on the same nucleus).

Results: Prenatal exposure to BPA and BPS with 0.5, 50, and 1000 µg/kg/b.w./day reduced sperm counts in mice across F1 to F3 generations. In the F1 generation, BPA or BPS exposure with 50 µg/kg/b.w./day disrupted the balance between maintaining the undifferentiated and differentiating spermatogonial populations. Differentially accessible peaks (DAPs) by snATAC-seq were primarily located in the promoter regions, with elevated activity of key transcription factors, including SP1, SP4, and DMRT1. Notably, similar gene expression and chromatin changes were observed in directly exposed F1 and F2 generations but differed in the indirectly exposed F3 generation. Approximately 80% of DAPs in F1 and F2 spermatogonia overlapped with histone post-translational modifications linked to transcription activation (e.g., H3K4me1/2/3 and H3K27ac). Across F1 to F3 generations, although BPA exerted more potent effects on gene expression in F1 spermatogonia, BPS induced longer-lasting effects. Interestingly, DMRT1 motif activity was persistently elevated in all three generations following ancestral BPA or BPS exposure.

Discussion: Our work provides the first systematic analyses of transgenerational gene and chromatin dynamics following prenatal exposure to BPA or BPS. These results suggest that prenatal exposure to environmentally relevant doses of BPA or BPS alters chromatin accessibility and transcription factor motif activities, consequently contributing to disrupted transcriptional levels in neonatal spermatogonia, and some are sustained to F3 generations, ultimately leading to the reduction of sperm counts in adults. https://doi.org/10.1289/EHP16981.