Specific autoantigens identified by sera obtained from mice that are immunized with testicular germ cells alone.

Hayato Terayama,Masahiro Itoh,Masaki Hiramoto,Munekazu Naito,Kou Sakabe,Zhong-Lian Li,Shogo Hayashi,Chiaki Katagiri,Naoyuki Hatayama,Hiraku Sasaki,Keisuke Miyazawa,Shota Moriya,Shuichi Hirai,Kenta Nagahori,Masayuki Matsushita,Ning Qu

doi:10.1038/srep35599

Abstract

There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body’s immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography–tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.

Highlights

Immunoregulatory molecules that have important roles in the regulation of immune responses in the testes
Experimental autoimmune orchitis (EAO) is classically induced by immunization with testicular homogenate (TH) plus complete Freund’s adjuvant (CFA) and Bordetella pertussis (BP); it is thought that treatment with the two adjuvants is required to enhance immune responses, resulting in the breakdown of testicular immune privilege[27,29,30]
The recombinant proteins of identified 10 TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction

Summary

Introduction

Immunoregulatory molecules that have important roles in the regulation of immune responses in the testes. Our EAO model is unique because serum autoantibodies are only against acrosomal regions of sperm and spermatids, but not Sertoli cells, Leydig cells and seminiferous tubular basement membrane[3,25,26,32] This model showed that the immunologically privileged state of the testis is overcome using only two TGC injections. Previous studies using TH + CFA +BP-induced EAO rat and vasectomized mouse models have demonstrated that the proteins endoplasmic reticulum 60, heat shock protein 70, a partial region of D3p domain of Zan with B cell epitope, and others are AIs that are involved in testicular autoimmune response[36,37]. The aim of this study is to identify TGC-specific AIs using sera obtained from mice with EAO induced by TGC alone

Objectives

Methods

Results

Discussion

Conclusion