Identification and validation of mouse sperm proteins correlated with epididymal maturation

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two‐dimensional gels. Of eight caput epididymal sperm‐differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm‐differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ‐actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S ‐transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post‐translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm‐differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm‐differential proteins are involved in ATP production that promotes sperm functions such as motility.