Study of EPPIN as sperm target for male contraception: structural characterization, protein-protein interaction and potential mechanisms of action

• Grant number: 20/04841-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: January 01, 2022
• End date: November 30, 2024
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Erick José Ramo da Silva
• Grantee: Noemia Aparecida Partelli Mariani
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated scholarship(s): 22/10159-6 - Functional studies on mouse SEMG1 as a modulator of CatSper in sperm flagellum, BE.EP.DR

Abstract

EPPIN (Epididymal protease inhibitor) is a sperm surface protein recognized as a target for the development of male contraceptive drugs, due to its critical role on sperm function. EPPIN is expressed in the testis and epididymis, and is found on the human sperm surface, where it inhibits sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). Little is known, however, on the mechanisms by which EPPIN modulates sperm motility, as well as its other potential roles in the male reproductive tract. In the last years, our research group has been dedicated to the study of the regulation, function and mechanisms of action of EPPIN using the mouse as an experimental model. Our previous studies demonstrated that the seminal plasma protein SVS2 (Seminal vesicle-secretory protein 2), which is orthologous to human SEMG1, emerged as EPPIN an interacting partner on mouse spermatozoa. Here, we propose to investigate the roles of EPPIN on mouse sperm function, focusing on its binding to SVS2 and the potential mechanisms involved on sperm motility inhibition. First, we will characterize the expression profile of EPPIN, as well as its co-localization with SVS2, in mouse ejaculate spermatozoa collected from different regions of the female reproductive tract (oviduct, uterus and vagina) after copulation by Western blot and immunofluorescence assays. In parallel, we will evaluate the effects of recombinant SVS2 and anti-EPPIN antibodies on mouse sperm intracellular Ca2+ levels and pH. Furthermore, we will identify EPPIN and SVS2 binding surfaces using Alpha Screen technology. Based on these results, we will perform docking studies using EPPIN and SVS2 build 3D structural models and analyze their stability by molecular dynamics simulations. The results of this project will support to improve our knowledge about evolutionary aspects of EPPIN, its role in male fertility, as well as its development as pharmacological target for male fertility control.