Differential Susceptibility to Heavy-Metal-Induced Injury in a Human Testis Tissue Equivalent (hTTE): Implications...

Oxidative stress driven by environmental and occupational exposure to heavy metals is increasingly recognized as a central, modifiable contributor to male infertility. Yet, the lack of physiologically relevant human models has limited mechanistic insight. We have previously described a three-dimensional Human Testis Tissue Equivalent (hTTE) that recapitulates the cellular composition and stem cell niche of the seminiferous tubule and is more resistant than two-dimensional (2D) culture to gonadotoxic chemotherapeutics. Here, we used the hTTE platform to compare heavy-metal cytotoxicity, cell-type-associated transcript responses, and exploratory microRNA (miRNA) profiles elicited by three prototypical pro-oxidant heavy metals: mercury (Hg²⁺), thallium (Tl⁺), and lead (Pb²⁺).

To determine whether the hTTE model exhibits greater resistance to heavy-metal cytotoxicity than matched 2D cultures and whether cell-type-associated responses differ following heavy-metal exposure within the established redox biology of these toxicants.

Adult human testicular cells from three donors were expanded, characterized by RT-qPCR and flow cytometry, and assembled into hTTEs alongside matched 2D cultures. Cultures were exposed for 48 h to Hg²⁺, Tl⁺, or Pb²⁺ across graded concentrations; IC₅₀ values were derived from ATP-based viability assays and used for selected downstream exposure concentrations. The relative abundances of cell-type-associated transcripts were profiled by digital PCR for ZBTB16 (spermatogonia), STAR (Leydig), SOX9 (Sertoli), and CD34 (peritubular cells), and global miRNA expression was quantified using the NanoString nCounter Human v3b miRNA panel (827 miRNAs). Exploratory Ingenuity Pathway Analysis of miRNA data was used to identify predicted mRNA associations with a testis/reproductive disease filter.

hTTEs exhibited significantly greater IC₅₀ values than matched 2D cultures for all three metals (p < 0.05), demonstrating greater resistance to acute heavy-metal cytotoxicity in the hTTE culture system, consistent with enhanced antioxidant buffering in the 3D microenvironment. Metal-associated changes in cell-type marker transcripts were distinct: Hg²⁺ produced broad reductions in markers associated with all four populations; Pb²⁺ produced greater reductions in Sertoli and peritubular cell markers while relatively preserving spermatogonial and Leydig cell markers; and Tl⁺ reduced somatic support cell markers with relative enrichment of spermatogonial (~7-fold) and Leydig cell marker expression. Heavy-metal exposure was associated with exploratory miRNA changes previously linked to redox imbalance, BCL2-mediated apoptosis, MYC/CDKN1A-driven nucleolar stress, AHR/CYP1A1 activation, and EGFR/COX-2 signaling.

These findings demonstrate greater resistance of the hTTE culture system to acute heavy-metal cytotoxicity and suggest metal-specific differences in testicular cell-type responses. Within the established redox biology of Hg²⁺, Tl⁺, and Pb²⁺, the observed patterns provide a framework for investigating oxidative stress mechanisms directly in a human-relevant testicular model. This system may provide a platform for future studies incorporating direct redox measurements and antioxidant rescue strategies.