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During ejaculation, cauda epididymal spermatozoa are suspended in a protein-rich solution of seminal plasma, which is composed of proteins mostly secreted from the seminal vesicle. These seminal proteins interact with the sperm cells and bring about changes in their physiology, so that they can become capacitated in order for the fertilization to take place. Sulfhydryl oxidase (SOX) is a member of the QSOX family and its expression is found to be high in the seminal vesicle secretion (SVS) of mouse. Previously, it has been reported to cross-link thiol-containing amino acids among major SVS proteins. However, its role in male reproduction is unclear. In this study, we determined the role of SOX on epididymal sperm maturation and also disclosed the binding effect of SOX on the sperm fertilizing ability in vitro. In order to achieve the above two objectives, we constructed a Sox clone (1.7 kb) using a pET-30a vector. His-tagged recombinant Sox was overexpressed in Shuffle Escherichia coli cells and purified using His-Trap column affinity chromatography along with hydrophobic interaction chromatography. The purified SOX was confirmed by western blot analysis and by its activity with DTT as a substrate. Results obtained from immunocytochemical staining clearly indicated that SOX possesses a binding site on the sperm acrosome. The influence of SOX on oxidation of sperm sulfhydryl to disulfides during epididymal sperm maturation was evaluated by a thiol-labeling agent, mBBr. The SOX protein binds onto the sperm cells and increases their progressive motility. The effect of SOX binding on reducing the [Ca2+]i concentration in the sperm head was determined using a calcium probe, Fluo-3 AM. The inhibitory influence of SOX on the sperm acrosome reaction was shown by using calcium ionophore A32187 to induce the acrosome reaction. The acrosome-reacted sperm were examined by staining with FITC-conjugated Arachis hypogaea (peanut) lectin. Furthermore, immunocytochemical analysis revealed that SOX remains bound to the sperm cells in the uterus but disappears in the oviduct during their transit in the female reproductive tract. The results from the above experiment revealed that SOX binding onto the sperm acrosome prevents sperm capacitation by affecting the [Ca2+]i concentration in the sperm head and the ionophore-induced acrosome reaction. Thus, the binding of SOX onto the sperm acrosome may possibly serve as a decapacitation factor in the uterus to prevent premature capacitation and acrosome reaction, thus preserving their fertilizing ability.

Proteins are versatile biological macromolecules that are involved in many essential processes and basic functions of a cell, including catalytic activity, storage, transport, cell structure, metabolism, cell signaling, and immunity. The functions of proteins are dictated by their structures. For instance, the shape, catalytic activity, and specificity of enzymes depend on both the sequence of amino acids in their active site to which the substrate or drug binds and the nature of protein folding. The stability of protein will determine if a protein is in native folded conformation or the unfolded or denatured state. The key role of drug designing is to enhance protein stability since the marginal stability of a protein could cause loss of protein function, increased degradation, and difficulty in synthesizing protein-based drugs. The folded structure of a protein is stabilized by several atomic interactions such as electrostatic, hydrophobic, van der Waals, disulfide, and hydrogen bonds, while the entropic or non-entropic interactions dominate the unfolded protein conformations. This chapter provides an overview of the techniques to determine the structure and stability of proteins addressing the principles involved in structure prediction with specific highlights on widely used experimental methods and computational techniques, namely protein purification techniques, biophysical/biochemical characterization of proteins, protein structure determining methods, factors contributing to protein stability, and conformational analysis of protein folding. This combination of advanced experimental and computational approaches in predicting the protein structure and measuring its stability serves to an exciting future in drug designing and stability engineering.