Department of Pharmacology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu-SP, Brazil.
Biotechnology Institute (IBTEC), São Paulo State University (UNESP), Botucatu-SP, Brazil; Department of Genetics, Institute of Biosciences, São Paulo State University (UNESP), Botucatu-SP, Brazil.
Mol Cell Endocrinol. 2020 Apr 15;506:110754. doi: 10.1016/j.mce.2020.110754. Epub 2020 Feb 7.
EPPIN is a sperm-surface drug target for male contraception. Here we investigated EPPIN-interacting proteins in mouse spermatozoa. We showed that EPPIN is an androgen-dependent gene, expressed in the testis and epididymis, but also present in the vas deferens, seminal vesicle and adrenal gland. Mature spermatozoa presented EPPIN staining on the head and flagellum. Immunoprecipitation of EPPIN from spermatozoa pre-incubated with seminal vesicle fluid (SVF) followed by LC-MS/MS or Western blot revealed the co-immunoprecipitation of SVS2, SVS3A, SVS5 and SVS6. In silico and Far-Western blot approaches demonstrated that EPPIN binds SVS2 in a protein network with other SVS proteins. Immunofluorescence using spermatozoa pre-incubated with SVF or recombinant SVS2 demonstrated the co-localization of EPPIN and SVS2 both on sperm head and flagellum. Our data show that EPPIN's roles in sperm function are conserved between mouse and human, demonstrating that the mouse is a suitable experimental model for translational studies on EPPIN.
EPPIN 是精子表面的一种药物靶点，可用于男性避孕。本研究旨在探索小鼠精子中的 EPPIN 相互作用蛋白。结果表明，EPPIN 是一种雄激素依赖性基因，在睾丸和附睾中表达，也存在于输精管、精囊和肾上腺中。成熟精子的头部和鞭毛上有 EPPIN 染色。将精子与精囊液（SVF）预孵育后进行 EPPIN 免疫沉淀，然后进行 LC-MS/MS 或 Western blot 分析，发现 SVS2、SVS3A、SVS5 和 SVS6 共同免疫沉淀。通过计算机模拟和远 Western blot 分析表明，EPPIN 在与其他 SVS 蛋白的蛋白质网络中结合 SVS2。使用与 SVF 或重组 SVS2 预孵育的精子进行免疫荧光染色，结果表明 EPPIN 和 SVS2 在精子头部和鞭毛上共定位。本研究数据表明，EPPIN 在精子功能中的作用在小鼠和人类之间是保守的，这表明小鼠是研究 EPPIN 的转化研究的合适实验模型。
