Effect of heat stress on bovine testicular spheroids

Downregulated genes were mainly associated with spermatogenesis, spermatid development, and flagellar motility, reflecting an underrepresentation of the germ cell compartment, which was confirmed by reduced DDX4 expression. In contrast, spheroids exhibited increased expression of the somatic cell markers WT1 and ACTA2, together with enrichment of biological processes related to extracellular matrix organization, collagen synthesis, vascularization, and seminiferous tubule morphogenesis.

These findings indicate that the spheroids retain the major somatic cell populations while undergoing structural remodelling characteristic of tissue organization. To investigate heat stress responses, spheroids were cultured for seven days under physiological testicular temperature (33.5°C) and then maintained at either 33.5°C (control) or exposed to 38.5°C for 48 h. Heat stress triggered a marked transcriptional response, with 871 upregulated and 688 downregulated genes.

Downregulated genes were enriched for spermatogenesis-related pathways, whereas upregulated genes were predominantly involved in inflammatory and immune signaling, including NF-κB, TNF, and IL-17 pathways, as well as cytokine production and signaling. Expression of DDX4, WT1, and ACTA2 remained unchanged after heat stress, whereas STAR, a Leydig cell marker, was significantly upregulated. Overall, these results demonstrate that bovine testicular spheroids faithfully preserve key somatic features of the testis and recapitulate molecular responses to elevated temperature.

This model provides a novel platform for investigating the mechanisms of heat-induced testicular dysfunction and developing strategies to mitigate heat stress-associated male infertility in cattle.