Molecular Human Reproduction, Vol. 7, No. 10, 923-933,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Co-localization of the inositol 1,4,5-trisphosphate receptor and calreticulin in the equatorial segment and in membrane bounded vesicles in the cytoplasmic droplet of human spermatozoa
1 Department of Cell Biology and 2 Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA and 3 Ludwig Institute for Cancer Research, Royal Free and University College Medical School, University College London, London W1P 8BT, UK
Abstract
Modulation of the intracellular calcium concentration within mammalian spermatozoa is important in several pre-fertilization events including hyperactivated motility and the acrosome reaction. To identify calcium binding proteins (CBP) potentially regulating these processes, a 45Ca 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.
calcium regulation/calcium storage/calreticulin/human spermatozoa/inositol trisphosphate receptor
Notes
4 To whom correspondence should be addressed at: University of Virginia, Health Sciences Center, Box # 439, Charlottesville, VA 22908, USA. E-mail: jch7k{at}virginia.edu
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