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

BackgroundThe 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.MethodsIn 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.ResultsThe 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.ConclusionsWe 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.

The function of chaperone proteins in the assemblage of protein complexes involved in gamete adhesion and fusion processes

“…Despite this, the individual chaperone proteins, calreticulin, calnexin, HSPA1A, HSPA5, HSP90AA1, and HSP90B1, have been confirmed on the surface of mature mouse oocytes (Calvert et al 2003) and may act in a coordinated manner to activate fusion machinery on the surface of the oocyte. In an independent study, calreticulin has also been found to localize to the cortical granules of hamster oocytes and appears to be released into the perivitelline space following oocyte activation (MunozGotera et al 2001), raising the possibility that it is involved in the priming of oocytes in preparation for fusion with a spermatozoon.…”

“…Of the chaperone proteins identified on the oolemma, HSP70 is constitutively synthesized in the preovulatory mouse oocyte (Calvert et al 2003) as well as the mature bovine oocyte (Kawarsky & King 2001). Following zygotic gene activation in the two-cell mouse embryo, HSP70 is one of the primary genes to be expressed.…”

“…For a sperm cell to interact with the oocyte, the activation of nascent, receptor-like proteins on the sperm surface is required, a process that has frequently been linked to the coordinated action of a subset of molecular chaperones (Ikawa et al 2001, Asquith et al 2004. Members of the HSP families, in addition to several other germ cell-specific chaperones, have been identified in both the mature spermatozoon of a number of species, including mouse (Asquith et al 2004), human (Mitchell et al 2007, and pig (Spinaci et al 2005), and in mature murine (Calvert et al 2003) and bovine (Kawarsky & King 2001) oocytes and have been extensively implicated in gamete adhesion and fusion processes , Asquith et al 2004, Nixon et al 2005, Huszar et al 2007, Ikawa et al 2011.…”

Expression and function of HSP110 family in mouse testis after vasectomy

“…HSPA4l is highly expressed in spermatogenic cells of testis from late pachytene spermatocytes to postmeiotic spermatids3 while HSPA4 is predominant in the testis and ovary 45. ORP150 is expressed in the plasma membrane of mature mouse oocytes 6. This expression pattern suggests a critical role for the HSP110 family proteins in reproduction.…”

Identification and characterization of an oocyte factor required for sperm decondensation in pig

“…Several investigators have explored the role of maternal proteins using proteomics, including from bovine, porcine, and mouse oocyte proteomes (Calvert et al 2003, Ellederova et al 2004, Memili et al 2007, Susor et al 2007, Zhang et al 2009, Wang et al 2010. Wang et al (2010) successfully identified 2781 proteins in the germinal vesicle (GV) of mouse oocytes, 2973 proteins in meta-phase II (MII) oocytes, and 2082 proteins in zygote.…”