The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa.
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. 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 in a dose-dependent manner. Finally, fluo-3 analysis demonstrated that the A23187-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.


CONCLUSIONS
These results demonstrate that eppin can modulate intracellular calcium concentrations and subsequently affect the calcium ionophore A23187-induced acrosome reaction.
Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China.
Jie
J
Zhang
Xinliang
X
Ding
Zenghui
Z
Bian
Yankai
Y
Xia
Chuncheng
C
Lu
Shoulin
S
Wang
Ling
L
Song
Xinru
X
Wang
D020101:Acrosome Reaction / Q000187:drug effects / Q000502:physiology*; D000906:Antibodies / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D005298:Fertility / Q000502:physiology*; D006801:Humans; D008297:Male; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory / Q000037:antagonists & inhibitors / Q000276:immunology / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D000906:Antibodies; C431553:Eppin protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D019000:Phosphotyrosine; D000001:Calcimycin; D002118:Calcium