4701040_monash_120418.pdf (4.36 MB)
The identification of molecular chaperone associated proteins potentially involved in the regulation of Sperm-Oocyte interactions
thesisposted on 2017-02-28, 03:48 authored by Whelan, Dean
The union of the mammalian spermatozoon and oocyte represents the culmination of a series of complex interactions between male and female gametes. Yet despite the importance of this highly regulated and species specific event, relatively little is known about the molecular basis of the interaction. Mammalian testicular spermatozoa, are incapable of fertilisation. Before fertilisation can occur, spermatozoa must undergo both epididymal maturation in the male reproductive tract and capacitation in the female tract. Only sperm that have traversed the epididymis attain the functional endpoints of capacitation, the ability to acrosome react and fertilise an egg. Capacitation is correlated with an increase in the level of tyrosine phosphorylation of a number of proteins, several of which become exposed on the cell surface. Previous analysis of the surface phosphoproteome of capacitated sperm demonstrated that the molecular chaperone HSPD1 is exposed on the plasma membrane overlying the acrosome, an ideal position for interaction with the zona pellucida. Although HSPD1 is not directly involved in zona binding, it has been proposed that during capacitation, intracellular tyrosine phosphorylation activates HSPD1 inside the cell, orchestrating the assembly of a zona binding complex and its subsequent exposure on the outside of sperm. To investigate the role of HSPD1 in fertilization, a proteomics-based approach was employed to identify chaperone associated proteins in capacitated sperm. HSPD1 was immunoprecipitated from capacitated sperm and associated proteins identified by liquid chromatography tandem mass spectrometry LC-MS/MS. analysis. Protein interactions were confirmed by reciprocal co-immunoprecipitation followed by Western blotting. The expression and localisation of the identified proteins during sperm maturation and fertilisation related events were investigated using indirect immunofluorescence and flow cytometry. To this end we have identified a number of proteins including an aldose reductase, another chaperone HSPE1, citron kinase and the proacrosin binding protein. These proteins co-localise with HSPD1 to the acrosomal region. However, this expression pattern is lost once sperm have undergone calcium ionophore A23187 induced acrosome reaction, as would be expected of molecules potentially involved in sperm-egg interactions. Based on these data we hypothesize that during epididymal transit, proteins important for fertilisation are deposited on sperm. During capacitation these proteins are assembled into functional protein complexes by chaperones including HSPD1, and chaperoned to sites including the cell surface where they affect the functional competence of sperm. The characterization of these HSPD1-interacting proteins is the subject of several chapters in this thesis. In addition, a proteomic analysis of the surface proteome of functionally mature mouse sperm was performed. This work was designed to complement and reinforce the work identifying chaperone-associated proteins in sperm, and to establish a reference proteome for ongoing and future comparative proteome studies. Protocols were developed and optimized to label sperm surface proteins with a number of different biotinylation reagents. Isolated proteins were subsequently separated by SDS-PAGE and proteins identified by LC-MS/MS. The broad goals of the work were to investigate the molecular basis of sperm-egg recognition with a view to identifying the key proteins that orchestrate the process.