Gamete Biology

Localization of the Chaperone Proteins GRP78 and HSP60 on the Luminal Surface of Bovine Oviduct Epithelial Cells and Their Association with Spermatozoa

Abstract
Upon their transit through the female genital tract, bovine spermatozoa bind to oviduct epithelial cells where they are maintained alive for long periods of time until fertilization. Although carbohydrate components of the oviduct epithelial cell membrane are involved in these sperm/oviduct interactions, no protein candidate has been identified to play this role. In order to identify the oviduct factors involved in their survival, sperm cells were pre incubated for 30 min with apical membranes isolated from oviduct epithelial cells, washed extensively and further incubated for up to 12 h in the absence of apical membranes. During this incubation, sperm viability, motility and acrosomal integrity were improved compared to cells pre-incubated in the absence of apical membranes. This suggests that, during the 30 min pre-incubation with apical membrane extracts, either an oviductal factor triggered intracellular events resulting in positive effects on spermatozoa or that such a factor strongly attached to sperm cells to promote a positive action. Similarly, spermatozoa were incubated with apical membranes isolated from oviduct epithelial cells labelled with [³⁵S]-methionine and, upon extensive washes, proteins were separated by 2-D gel electrophoresis in order to identify the factors suspected to have beneficial effects on spermatozoa. The 6 major proteins, according to their signal intensity on the autoradiographic film were extracted from a 2-D gel of oviduct epithelial cell proteins ran in parallel and processed for N-terminal sequencing of the first 15 amino acids. Of these, one was identical to heat shock protein 60 (HSP60) and one to the glucose regulated protein 78 (GRP78). Their identity and association with spermatozoa was confirmed using an antibody directed against these proteins. This paper reports the localization of both GRP78 and HSP60 on the luminal/apical surface of oviduct epithelial cells, their binding to spermatozoa and the presence of endogenous HSP60 in the sperm midpiece.

Key words: Female Reproductive Tract • Gamete Biology • Fertilization • Sperm • Sperm motility and transport

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