Abstract
BackgroundThe precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.MethodsSurface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.ResultsNine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.ConclusionThe presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.
Highlights
The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood
The precise composition of the sperm surface, the molecular interactions that define domain specific functions, and the changes induced during the capacitation process, still remain to be fully elucidated
Activation of the putative zona pellucida (ZP)-receptor leads to a transient influx of calcium through T-type voltage-dependent calcium channels in the plasma membrane that are thought to be released from inactivation by the capacitation-induced hyperpolarization of the membrane potential [5]
Summary
The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Activation of the putative ZP-receptor leads to a transient influx of calcium through T-type voltage-dependent calcium channels in the plasma membrane that are thought to be released from inactivation by the capacitation-induced hyperpolarization of the membrane potential [5]. This brief (< 500 ms) initial elevation of [Ca2+]i to micromolar levels activates the Ca2+-sensitive phospholipase PLCδ, causing the generation of diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), and consumption of the plasma membrane positioned substrate phosphatidylinositol biphosphate (PIP2) [4,6]. CAMP and small G protein activities act together to set in motion the SNARE machinery (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), which is required for the fusion between the outer acrosomal membrane and the overlying plasma membrane [4]
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