Numerous studies have confirmed that nicotine decreased sperm quality through unbalancing the spermatogenesis and damaged the testicular tissue, such as distorted convoluted tubules, thinning of spermatogenic epithelium, disordered germ cells and damage testicular blood-testis barrier (BTB) [2,17,27]. In the testis, the BTB plays a pivotal role on spermatogenesis through preventing the autoimmune reaction of sperm and avoiding harmful substances entering the seminiferous tubules [5]. At the same time, the BTB is easily damaged by various physical and chemical factors [25]. However, the potential mechanisms of nicotine exposure on BTB damage and sperm development are not fully understood and requires further clarification.

Ferroptosis is one of the programmed cell death types which was first detected in tumorigenesis cells and distinct from apoptosis and necroptosis, the other two types of cell death, in morphology, biochemical and genetics. Ferroptosis is recognized to be an iron-dependent regulated cell death (RCD) characterized via phospholipid peroxidation of plasma membranes caused by reactive oxygen species (ROS) produced during iron-mediated Fenton reactions [11]. Studies have shown that ferroptosis can reduce the damaged cells and maintain the balance of homeostasis [4]. On the other hand, ferroptosis is largely associated with the development and progression of cancer, neurodegenerative diseases, diabetes, and cardiomyopathy [40,46] and even on reproductive organs [44,47]. Therefore, we aimed to define the role of ferroptosis in testicular toxicity.

The increasing evidences indicated that the Bmal1, a key protein involved in circadian rhythm, plays an integral role in the mammalian circadian clock through the regulation of the clock-related genes expression by E-BOX, such as those genes of coding the PER (period circadian regulator) and CRY (cryptochrome circadian regulator) families [31]. Similarly, Study also found that Bmal1 also is involved in the direct regulation of Nrf2 by E-BOX [12,37]. Nrf2 plays a key role in regulating various physiological and pathological cellular events including oxidative homeostasis, inflammatory response and energy metabolism [14,50]. Intriguingly, Nrf2 activation protect against ferroptosis. Increasing evidence suggests that Nrf2 not only increases the REDOX imbalance in the testis but also aggravates dysgenesis via the Fenton reaction [39,51]. Of note, some of the key regulators of ferroptosis are downstream targets of Nrf2, such as GSH, which directly affect the function of GPX4, which plays keying action in the reduce the toxicity of lipid peroxides and alleviating the Fenton reaction [36,46,52]. However, the underlying mechanism of the Bmal1/Nrf2 axis in mediating ferroptosis in nicotine-induced BTB damage has not been explored to date.

In present study, we verified the crucial role of ferroptosis in nicotine-induced BTB damage and impaired sperm development. Nrf2 inactivation by nicotine leaded to oxidative stress and subsequent ferroptosis, and Bmal1 was indispensable for nicotine-induced ferroptosisviadirect regulation of Nrf2 transcription. We believe that this study provides important clues for developing novel male reproductive injury by causing smoking/tobacco smoke targeting ferroptosis through mediating iron hemostasis and lipid peroxidation.