Single-cell Transcriptomics reveals atrazine-induced Reprogramming of Porcine Leydig Cells

The development of fertile boars is critical for swine production. However, the prepubertal boar may be exposed to agrichemicals that impair testis development. Atrazine is a heavily used herbicide that contaminates groundwater in agricultural states. Of concern, atrazine is also a potent endocrine-disrupting chemical linked to reproductive dysfunction across species, including swine. Recent data from our group demonstrated that early-life atrazine exposure (eATZ) during critical windows of early testicular development (e.g., fetal and neonatal) decimates boar testis function. For example, eATZ ablated testicular steroidogenesis, including seven androgens (up to 67% decrease) and two estrogens (up to 29% decrease), consistent with impaired Leydig cell function. The objective of this study was to identify molecular mechanisms underlying Leydig cell dysfunction in neonatal eATZ boars. Pregnant littermate gilts drank water containing either an environmentally relevant dose of atrazine (20 µg/L) or vehicle control [0.002% (v/v) ethanol] ad libitum from 28 days of gestation through parturition (99.0 ± 0.9 days). Control and eATZ boars (n = 5/treatment) nursed their respective dams ad libitum prior to testis tissue collection on post-natal day 10. Samples were snap frozen for single-nuclei RNA-sequencing. Briefly, nuclei were isolated, processed with 10x Genomics technology, sequenced on an Illumina NovaSeq, and analyzed using the Sscrofa11.1 genome. Classical Leydig cell markers (e.g., STAR, CYP17A1, CYP11A1) were utilized for cluster analysis. A total of 79 differentially expressed genes (DEGs) were identified in Leydig cells from eATZ boars compared with controls (FDR adjusted P < 0.05; 33 transcripts down-regulated, 46 transcripts up-regulated). Fourteen DEGs associated with Leydig cell proliferation and differentiation were identified, including CDK6, LGR4, and MAP3K4. Notably, MAP3K4 is indispensable for normal testis differentiation in the mouse—thus, downregulation of MAP3K4 may contribute to atrazine-induced defects in porcine testis development. In addition, thirteen DEGs were associated with lipid availability, steroidogenesis, and steroid signaling, including LIPG (lipid hydrolysis), OSBPL1A (intracellular cholesterol transport), TSPOAP1 [supports StAR-mediated cholesterol transport (the rate limiting-step in steroidogenesis)], and ADCY3 (generates cAMP for steroidogenic signaling). KEGG analysis revealed enrichment of lipid metabolism, hormone synthesis, and hormone signaling pathways, including arachidonic acid metabolism, steroid synthesis, and estrogen signaling (q < 0.05; pathway-level FDR adjustment). Furthermore, 10 DEGs were related to cellular stress and toxicant response pathways including ATF3, DUOX2, and EPHX2. Notably, ATF3 is a stress-activated gene that was recently implicated in testicular ferroptosis and hypospadias. Its 4-fold upregulation in Leydig cells from eATZ boars suggests substantial cellular stress. Ultimately, these results provide novel insights into the mechanisms by which eATZ drives Leydig cell dysfunction in neonatal boars. These perturbations may alter the trajectory of testis development and compromise future fertility. Therefore, eATZ is an emerging threat to the development of fertile boars for the breeding herd.