Maternal Glyphosate Exposure Induces Transgenerational Testicular Fibrosis and Dysfunction in Male Offspring Mediated by Sperm-Borne piRNAs

Abstract Glyphosate, the most widely used herbicide worldwide, has experienced a more than 12-fold increase in application over the past decade, raising substantial public health concerns regarding its potential reproductive toxicity. Previous toxicological studies have primarily focused on the effects of high-dose exposures, while the impacts of long-term, low-dose, and transgenerational exposure, which more closely reflect real-world environmental exposure, remain poorly understood. A three-generation rat model of continuous maternal glyphosate exposure was established. Pregnant F0 dams were administered glyphosate (5 μg/kg bw/day) in drinking water, and the exposure was maintained through their female offspring (F1 and F2) during gestation and lactation and continued in all offspring postweaning. Male offspring from each generation (F1–F3) were assessed for testicular histology, serum testosterone levels, and sperm quality. Transcriptomic profiling was performed by RNA sequencing, and sncRNA-seq was conducted to characterize sperm miRNA and piRNA expression profiles. Key molecules and signaling pathways were further examined by RT-qPCR and molecular docking analyses. Significant reproductive alterations were observed in F3 males at postnatal day 90 (PND 90), including reduced body weight, decreased sperm count, lower serum testosterone levels, and disrupted seminiferous tubule morphology. Transcriptomic analysis showed progressive dysregulation of extracellular matrix (ECM) and collagen formation-related pathways across generations, with the most pronounced changes in F3 males. This dysregulation was related to the activation of the TGF-β signaling pathway, as protein–protein interaction network analysis identified the TGF-β receptor 2(Tgfbr2) and its downstream mediator (Ccn2) as central hubs, together with the major collagen gene Col1a1. Moreover, sperm sncRNA-seq identified differentially expressed miRNAs and piRNAs, among which piR-rno-2217383_L2 was bioinformatically predicted to target Tgfbr2 and Ccn2, and molecular docking indicated stable binding of glyphosate to TGFBR2 and CCN2 proteins. These findings suggest the possibility that the piR-rno-2217383_L2/TGFBR2 and CCN2/Col1a1 axis may contribute to testicular fibrosis, although functional validation is required to confirm this regulatory cascade.