Comparative transcriptomics reveals a core molecular signature of hypothyroid testis development across three TPO inhibitors

Thyroid hormone (TH) signaling is critical for normal testis development; however, the molecular mechanisms linking developmental hypothyroidism to adverse testis outcomes remain poorly understood. Improved mechanistic insight is needed to support predictive approaches in chemical risk assessment. Here, we build on previous work to define a molecular signature in testes from hypothyroid rats induced by three thyroperoxidase (TPO)-inhibiting chemicals: the pharmaceuticals propylthiouracil (PTU), and methimazole (MMI), and the pesticide amitrole (AMI). Transcriptomic data for MMI and AMI were obtained from a previously published study. For the PTU study, pregnant Sprague-Dawley rat dams were exposed by oral gavage to PTU (1.25 or 2.5 mg/kg bw/day) from gestational day (GD) 7 to pup day (PD) 28. Reproductive toxicity endpoints were assessed, and testes were collected from offspring on GD21, PD16, and PD78 for bulk RNA barcoding and sequencing (BRBseq). Comparative transcriptomic analysis across PTU, MMI, and AMI revealed substantial transcriptional disruption, with effects that were both age- and chemical-dependent. Notably, at PD16, there was an overlap of 278 differentially expressed genes between the three chemicals, a finding that was preserved when applying a previous bioinformatic pipeline. The shared transcriptional changes were associated with biological processes related to cell cycle regulation, developmental growth, and pathways involved in Sertoli cell proliferation, germ cell development, and testis morphogenesis. Collectively, these findings support the existence of a TH-mediated molecular signature that aligns with the classical morphologic phenotype of hypothyroid testis and underscores its potential value for the development of alternative, mechanism-based test methods for regulatory purposes.