[Frontiers in Bioscience E2, 566-581, January 1, 2010]

[Frontiers in Bioscience E2, 566-581, January 1, 2010]

A novel testis-specific Na⁺/H⁺ exchanger is involved in sperm motility and fertility
Tao Liu^1,3, Jun-Cheng Huang¹, Wu-Lin Zuo², Cui-Ling Lu¹, Min Chen¹, Xue-Sen Zhang¹, Yi-Chuan Li¹, Huan Cai¹, Wen-Liang Zhou², Zhao-Yuan Hu¹, Fei Gao¹, Yi-Xun Liu¹

1State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China, ²School of Life Science, SunYat-Sen University, Guangzhou 510275, China², ³National Center for Molecular Genetics and Breeding of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods: 3.1. Materials; 3.2. cDNA amplification; 3.3. Northern blotting and In situ hybridization analysis; 3.4. Recombinant protein purification in E.coli and polyclonal antisera production; 3.5. Immunohistochemical and Western blot analysis; 3.6. Construction of pCR-NHE; 3.7. Immunization with DNA vaccine; 3.8. Confocal analysis; 3.9. Antibody detection by ELISA; 3.10. Assessment of sperm acrosome reaction and fertilization; 3.11. Sperm motility analysis; 3.12. Immunocontraceptive test; 3.13. Measurement of pHi and (Ca2+)i in spermatozoa; 3.14. Statistic analysis
4. Results: 4.1. Cloning and sequence analysis of mtsNHE cDNA and its protein; 4.2. Expression pattern of mtsNHE and its protein in testis and sperm; 4.3. Construction of DNA vaccine and confirmation of its specificity.; 4.4. ELISA; 4.5. Immunocontraceptive effects of pCR-NHE; 4.6. Inhibitory effect of antisera on in vitro fertilization; 4.7. Inhibitory effect of antisera on spermatozoa acrosome reaction; 4.8. Alterant spermatozoa motility by antisera; 4.9. Effect of antisera on sperm intracellular pH and calcium concentration
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

Sodium-hydrogen exchanger as a channel for regulation of intracellular pH might be a crucial modulator of sperm capacitation and motility. Three members of this family have been identified in spermatozoa. A novel protein testis-specific sodium-hydrogen exchanger named mtsNHE was cloned in the present study. The mtsNHE localizing on principle piece of sperm flagellum contained 12 predicted transmembrane regions without cytoplasmic fragment at carboxyl terminus. Hydrophilic region was common in the sodium-hydrogen exchanger family members. Polyclonal antibodies to trans-membrane region significantly reduced sperm motility, acrosome reaction and ratio of in vitro fertilization. By in-pouring the antibodies in sperm solution, intracellular pH and calcium concentration were decreased. Muscle injection of female mice with the specific gene vaccine of mtsNHE, significantly stepped down fertility rate. Considering its specific expression and involvement in the regulation of fertility, the mtsNHE might be a potential target molecule for developing a new male contraceptive.