Male infertility affects approximately 1 in 20 men in the Western world and is the sole cause in one-third of couples undergoing assisted reproductive technologies. Approximately 75% of men present with low sperm count, poor motility and morphology, together with high levels of sperm DNA damage—factors closely linked to embryo loss. Testicular hyperthermia is a key contributor, as clinical studies show scrotal cooling improves semen quality in 66% of infertile men, with 44% achieving natural conception within a year despite five years of prior unsuccessful attempts.
Although the requirement for cooler testicular temperature has been known for over a century, the molecular basis for this sensitivity remains unresolved. Our research investigates thermally sensitive CDC-like kinases (CLKs), maximally active at 33°C but sharply inhibited by a 1°C rise—mirroring spermatogenesis sensitivity. In mammals, four CLK paralogues exist (CLK1–4). Mice lacking CLK1 or CLK2 remain fertile. As such, we developed transgenic models, including a Drosophila DOA knockdown and mouse Clk3⁻/⁻ and Clk4⁻/⁻ lines. While Clk4 deletion had no impact, loss of DOA or CLK3 caused male-specific infertility with hallmark features of heat stress including low sperm count, impaired motility, and elevated DNA damage. Females remain fertile and otherwise the animals are healthy.
We propose testicular temperature directly regulates CLK3 activity. Supporting this, hyperthermia causes a loss of piRNA by ~50%. piRNA are essential for spermatogenesis and transposable element (TE) repression. Notably, Heat stress, DOA knockdown, and CLK3 deletion all led to TE activation. As unregulated TE activity causes DNA damage, instability, and epigenetic disruption, we suggest CLK3 acts as a thermal sensor safeguarding germ cell genome integrity via piRNA-mediated TE suppression. These findings provide a mechanistic explanation for heat-induced male infertility and identifies CLK3 as a novel regulator of testicular temperature sensitivity.