EPPIN interaction proteins in mouse sperm: identification, expression and potential physiological roles

Author(s)

Noemia Aparecida Partelli Mariani

• Document type: Master's Dissertation
• Press: Botucatu. 2019-09-16.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
• Defense date: 2019-08-30
• Advisor: Erick José Ramo Silva

Abstract

EPPIN (Epididymal peptidase inhibitor), a cysteine-rich antimicrobial protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for the development of new male contraceptive drugs due to its critical role in sperm motility. The development of contraceptive drugs based on EPPIN functions requires a deeper understanding on its physiological roles and further evaluation of efficacy and safety of the potential drug with preclinical models. Herein, we investigated the expression profile of EPPIN in tissues and in spermatozoa from testis and epididymis and identified potential proteins that interact with EPPIN in mature sperm. To characterize the expression profile of EPPIN, we processed reproductive and non-reproductive organs, as well as spermatozoa for RT-PCR, qPCR, Western blot and immunohistochemistry/immunofluorescence assays. For the identification of potential EPPIN’s partners, we collected spermatozoa from the corpus/cauda epididymis and incubated them with the phosphate-soluble fraction of seminal vesicle fluid from the same animals. Then, we performed co-immunoprecipitation assays using anti-EPPIN Q20E antibody or rabbit IgG (negative control) to immunoprecipitate EPPIN and co-immunoprecipitate its partner proteins, processed samples for trypsin protein digestion and identified tryptic peptides by mass spectrometry (LC-MS/MS). Our results showed that the Eppin is an androgendependent gene, abundantly expressed in the testis and epididymis, but also present in the vas deferens, seminal vesicle and adrenal gland of mice. We observed EPPIN-positive immunostaining in the acrosomal region and flagellum of epididymal spermatozoa, suggesting potential actions of EPPIN in the acrosome reaction and sperm motility. Co-immunoprecipitation with anti-EPPIN antibody and analysis by LC-MS/MS and Western blot using mature epididymal spermatozoa pre-incubated with seminal vesicle fluid revealed that the SVS2, SVS3a, SVS5 and SVS6 are potential interaction proteins of EPPIN in the sperm surface. We performed Western blot assays of the samples obtained from co-immunoprecipitation assays using anti-EPPIN, anti- SVS2 and anti-SVS3 antibodies to confirm the co-immunoprecipitation of SVS2 and SVS3, and Far-Western blot assays with recombinant EPPIN and SVS2 to further investigate their interaction. Our data suggest that EPPIN is a protein that acts as a hub for the interaction of SVS proteins in mouse spermatozoa. In addition, our results provide new insights into the physiological roles of EPPIN in males and highlight its importance for protein-protein interactions in the spermatozoon, which can protect and prepare the male gamete during its journey through the female reproductive system and facilitate the fertilization process. Thus, our data provide support for the use of the mouse as an experimental model to study physiological functions and mechanisms of EPPIN, as well as its further development as a contraceptive drug target. (AU)

• FAPESP's process: 17/11363-8 - EPPIN interacting proteins on mouse sperm surface: identification, expression and potential physiological roles
• Grantee: Noemia Aparecida Partelli Mariani
• Support Opportunities: Scholarships in Brazil - Master