A semi-synthetic methylenedioxyphenyl compound piperonyl butoxide (PBO) has been used as a ubiquitous synergist to increase the insecticidal effect of pesticides for agricultural and household use. Despite previously demonstrated effects of PBO, the detailed mechanism of PBO in spermatozoa and reproductive toxic effects on male germ cells have not been fully elucidated. Therefore, this study evaluated the effects of PBO on various sperm functions during capacitation and clarified the mechanisms of reproductive toxic effects on male fertility at different concentrations of PBO (0.1, 1, 10, and 100 μM). Sperm motility and kinematics were assessed using computer-assisted sperm analysis and the status of capacitation was evaluated using combined H33258/chlortetracycline (CTC) staining. Intracellular adenosine triphosphate (ATP) and cell viability levels were also measured. In addition, protein kinase A (PKA) activity and protein tyrosine phosphorylation were evaluated. In addition, in vitro fertilization was performed to determine the effects of PBO on cleavage and blastocyst formation rates. We found that PBO significantly decreased sperm motility, kinematics, and acrosome-reacted and capacitated spermatozoa. In addition, PBO suppressed the intracellular ATP levels and directly affected cell viability. Moreover, PBO detrimentally decreased the activation of PKA and altered the levels of tyrosine-phosphorylated proteins. Consequently, cleavage and blastocyst formation rates were significantly reduced in a dose-dependent manner. In line with our observations, the synergist of pesticides PBO may directly and/or indirectly cause disorder in male fertility. Hence, we suggest that careful attention is made to consider reproductive toxicity when using PBO as a synergist.

半合成的亚甲基二氧苯基化合物胡椒基丁醚 (PBO) 已被用作无处不在的增效剂，以增加农用和家用杀虫剂的杀虫效果。尽管先前已证明 PBO 的作用，但 PBO 在精子中的详细机制和对男性生殖细胞的生殖毒性作用尚未完全阐明。因此，本研究评估了 PBO 在获能期间对各种精子功能的影响，并阐明了不同浓度 PBO（0.1、1、10 和 100 μM）对男性生育能力的生殖毒性作用机制。使用计算机辅助精子分析评估精子活力和运动学，并使用联合 H33258/金霉素 (CTC) 染色评估获能状态。还测量了细胞内三磷酸腺苷 (ATP) 和细胞活力水平。此外，还评估了蛋白激酶 A (PKA) 活性和蛋白酪氨酸磷酸化。此外，进行体外受精以确定 PBO 对卵裂和囊胚形成率的影响。我们发现 PBO 显着降低了精子活力、运动学以及顶体反应和获能精子。此外，PBO 抑制细胞内 ATP 水平并直接影响细胞活力。此外，PBO 有害地降低了 PKA 的激活并改变了酪氨酸磷酸化蛋白的水平。因此，卵裂和囊胚形成率以剂量依赖性方式显着降低。根据我们的观察，杀虫剂 PBO 的增效剂可能直接和/或间接导致男性生育能力紊乱。因此，我们建议在使用 PBO 作为增效剂时要仔细考虑生殖毒性。