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Hum. Reprod. Advance Access published online on October 3, 2009
Human Reproduction, doi:10.1093/humrep/dep356
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa

Jie Zhang, Xinliang Ding, Zenghui Bian, Yankai Xia, Chuncheng Lu, Shoulin Wang, Ling Song and Xinru Wang1

Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China

1 Correspondence address. Tel: +86-25-86862863; Fax: +86-25-86662863; E-mail: xrwang{at}njmu.edu.cn

BACKGROUND: Before a spermatozoon can fertilize an oocyte it must undergo a cascade of biochemical and physiological changes that facilitate its binding and penetration into the oocyte. Epididymal protease inhibitor (eppin) has been found to play a critical role in male fertility through an immunological approach.

METHODS: In this study, we used an anti-eppin antibody to clarify the effect of eppin on human sperm functions during fertilization. Immunofluorescence studies were performed on ejaculated human spermatozoa in uncapacitated, capacitated and ionophore-treated states. Human spermatozoa were incubated in the presence or absence of anti-eppin antibody under capacitating conditions and with A23187. [GenBank] The effects of the antibody were evaluated on sperm motility, protein phosphotyrosine content and free intracellular calcium.

RESULTS: Immunofluorescence results demonstrated that eppin is located on the acrosome and tail. After the acrosome reaction eppin is found on the equatorial segment and tail. We found that blocking eppin with antibodies significantly inhibited the human sperm acrosome reaction induced by A23187 [GenBank] in a dose-dependent manner. Finally, fluo-3 analysis demonstrated that the A23187 [GenBank] -induced elevation of sperm intracellular calcium concentration was markedly reduced after incubation with anti-eppin antibody. However, the tyrosine phosphorylation of sperm proteins did not change.

CONCLUSION: These results demonstrate that eppin can modulate intracellular calcium concentrations and subsequently affect the calcium ionophore A23187 [GenBank] -induced acrosome reaction.

Key words: eppin/spermatozoa/acrosome reaction/intracellular calcium/phosphorylation

Submitted on March 8, 2009; resubmitted on September 1, 2009; accepted on September 10, 2009.


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