Journal
MOLECULAR HUMAN REPRODUCTION
Volume 7, Issue 10, Pages 923-933Publisher
OXFORD UNIV PRESS
DOI: 10.1093/molehr/7.10.923
Keywords
calcium regulation; calcium storage; calreticulin; human spermatozoa; inositol trisphosphate receptor
Funding
- NICHD NIH HHS [U54 HD 29099, F32-HD08002, T32-HD07382] Funding Source: Medline
- NIDDK NIH HHS [P32-DK07642] Funding Source: Medline
- PHS HHS [P30-28934] Funding Source: Medline
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Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several prefertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a Ca-45 overlay technique was employed on 2-D blots of human sperm extracts. Microsequencing by Edman degradation and CAD mass spectrometry identified a relatively abundant 60.5 kDa CBP with a pI of 4.2 as calreticulin (CRT). Immunofluorescent labelling with anti-CRT antibodies localized CRT to the acrosome, with highest fluorescence in the equatorial segment, and in the cytoplasmic droplets of 94 and 48% of human spermatozoa respectively. Double immunolabelling experiments demonstrated co-localization of CRT and the inositol 1,4,5-trisphosphate receptor (IP3R) in the acrosome, in the equatorial segment, and vesicular structures in the cytoplasmic droplets of the neck region. Electron microscopic immunogold labelling localized CRT to the equatorial segment of acrosome-reacted spermatozoa and to membrane-enclosed vesicles within the cytoplasmic droplet of both acrosome-intact and acrosome-reacted spermatozoa. Localization of the IP3 receptor to the CRT-containing vesicles, in the sperm neck and to the acrosome, suggests that capacitative calcium entry in human spermatozoa may be regulated from these putative calcium storage sites.
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