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Soybean trypsin inhibitor as a probe for the acrosome reaction in motile cynomolgus macaque sperm

Published online by Cambridge University Press:  01 May 2000

Theodore L. Tollner ,
Ashley I. Yudin ,
Gary N. Cherr  and
James W. Overstreet
Theodore L. Tollner
Affiliation:
Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of California, Davis, California, USA.
Ashley I. Yudin
Affiliation:
Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of California, Davis, California, USA.
Gary N. Cherr
Affiliation:
Bodega Marine Laboratory, University of California, Davis, California, USA.
James W. Overstreet
Affiliation:
Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of California, Davis, California, USA.
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Abstract

Soybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases, and has been shown to bind to acrosin, an acrosomal hydrolase which is not exposed on the surface of macaque sperm until after the acrosome reaction. Following activation with caffeine and dibutyryl cAMP, cynomolgus macaque sperm were induced to acrosome react with calcium ionophore A23187 in the presence of SBTI and were fixed for ultrastructural observation. Transmission electron microscopy (TEM) revealed secondary labelling of anti-SBTI-IgG with colloidal gold in association with the acrosomal matrix and fused membranes of sperm undergoing the acrosome reaction, but gold labelling was not observed on acrosome-intact sperm. When SBTI was conjugated with the fluorochrome Alexa 488, labelled (acrosome-reacted) sperm showed bright fluorescence that ranged from a patchy or punctate appearance to solid labelling over the region of the acrosomal cap. Following treatment with ionophore, the percentages of total acrosome-reacted sperm (motile and non-motile) as assessed with Alexa-SBTI, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and TEM were 54.6%, 51.6% and 61.5%, respectively. Measures of acrosomal status with FITC-PSA and Alexa-SBTI were highly correlated (r = 0.94; n = 3). Macaque zonae pellucidae were co-incubated with activated sperm for 1 min and then rinsed in medium containing Alexa-SBTI and immediately observed with epifluorescence microscopy. The mean percentage of Alexa-SBTI-labelled (acrosome-reacted) motile sperm bound to the zona was 45.7 ± 14 (range: 22–80.4%; n = 4). Fewer than 1% of the motile sperm in suspension surrounding the zonae were acrosome-reacted. Alexa-SBTI had no effect on sperm motility, survival, or zona binding capability.

Keywords

AcrosinAcrosome reactionMacaqueSBTISperm
Type
Research Article
Information
Zygote , Volume 8 , Issue 2 , May 2000 , pp. 127 - 137
Copyright
2000 Cambridge University Press

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