Identification of calcium-binding proteins associated with the human sperm plasma membrane

Abstract

Background: 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. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.

Methods: Surface 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.

Results: Nine 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.

Conclusion: The 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.

Figure 1

2D autoradiogram of radioiodinated acidic and neutral human sperm surface proteins. The positions of 80K-H and SAP are indicated by arrows, as well as those of previously reported surface proteins; oxygen regulated protein 150 (HYOU1), angiotensin converting enzyme (ACE), HSP86 (HSPC1), Bip (HSPA5), sperm-specific surface hyaluronidase (PH-20) and HSP70-1 (HSPA2). The positions of four intracellular proteins; valosine containing protein (VCP), calreticulin (CRT), tubulin and actin are indicated by rectangular boxes. Additional non-labelled tubulin isoforms were detected by mass spectrometry to the right of the boxed area. See also Figure 3A and Additional file 1 - Supplementary Figure 1.

Figure 2

2D autoradiogram demonstrating ⁴⁵Ca-binding human sperm proteins. The nine identified calcium binding proteins are indicated by oblique arrows.

Figure 3

SAP is a ⁴⁵ Ca-binding protein exposed on the surface of human sperm. Immuno-staining of PVDF membranes used to detect radioiodinated surface proteins (A) and ⁴⁵Ca-binding proteins (C) by autoradiography confirmed that the 26.5 kDa calcium binding surface protein with a pI of 5.2 (oblique downward arrows) is SAP (B & D).

Figure 4

The majority of SAP molecules bind to the human sperm surface in a calcium-independent fashion. SAP is slowly released from the surface of washed, swim-up harvested human sperm when incubated in a Ca²⁺-free DMEM medium (A, left panel). The addition of 5 mM EDTA to the medium induced a similar strong discharge of surface bound SAP within minutes (A, right panel). However, most SAP antigens remained attached to the sperm, despite the removal of calcium from the medium (B).

Figure 5

Demonstration of SAP on the surface of non-permeabilized, motile sperm. A: IF image demonstrating patches of SAP over the neck and tail regions of intact, motile human sperm (arrows). The nuclear DNA is stained blue with the DNA intercalating dye DAPI II. B: Secondary antibody alone control. C: Sperm agglutination floccules visualized by differential interface contrast microscopy. Antiserum against SAP agglutinated human sperm in a loose, tangled binding pattern, i.e. tail-to-tail, head-to-tail, and head to head. Round cells did not appear to be incorporated into the agglutination floccules. D: No antibody control without agglutination. Scale bar = 100 μm. E: A positive control with monoclonal antibody against CD52 induced strong agglutination.