Oolemmal proteomics--identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane

Abstract

Background: The mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development.

Methods: In order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin.

Results: The putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface localization of HSP70 or calnexin by this method.

Conclusions: We report here the identification of nine highly abundant molecular chaperones in the mouse egg proteome. In addition, we present preliminary data suggesting that these molecules localize to the oolemma of the mature mouse egg.

Figure 1

2D Elecrophoresis of whole egg extracts identifies abundant egg proteins. 2D electrophoresis of 2850 zona-free mouse egg proteins, extracted with Celis buffer and stained with coomassie. Black arrows indicate protein spots that were cored for TMS. Each spot is labelled with the resulting protein identification. Horizontal axis shows isoelectric point and vertical axis shows molecular weight (kDa).

Figure 2

2D Electrophoresis, biotinylation and avidin blotting identifies surface-labeled oolemmal proteins. 2D electrophoresis of 800 zona-free mouse egg proteins, surface labeled with 2 mg/ml Sulfo-NHS biotin, and separated by 2D electrophoresis. Blotted with streptavidin-HRP and visualized by ECL (A). Corresponding 2D electrophoretic gel of 800 unlabeled zona-free mouse eggs visualized by silver staining. Labelled black arrows indicate previously identified protein spots (B). Horizontal axis of each shows isoelectric point and vertical axis shows molecular weight (kDa).

Figure 3

Immunofluorescent localization of GRP94. Immunofluorescence using rat monoclonal antibodies to GRP94 on live, zona-free mouse eggs (A). Compare to control egg population incubated with rat IgG. Secondary antibody used for both is cy3-conjugated anti-rat antibody (B). Red asterisks mark activated eggs.

Figure 4

Immunofluorescent localization of HSP90. Immunofluorescence using mouse monoclonal antibodies to HSP90 on mature, zona-free mouse eggs (A). Compare to control egg population incubated with mouse IgG. Secondary antibody used for both is cy3-conjugated anti-rat antibody. n = 10 (B). Red asterisks mark activated eggs.

Figure 5

Immunofluorescent localization of Calreticulin and GRP78. Immunofluorescence using rabbit polyclonal antibodies to (A) calreticulin, and (B) GRP78, on mature, zona-free mouse eggs. Compare to control egg population incubated with normal rabbit sera. (C). Secondary antibody used for both is cy3-conjugated anti-rabbit antibody. Red asterisks mark activated eggs.