Localization of the Chaperone Proteins GRP78 and HSP60 on the Luminal Surface of Bovine Oviduct Epithelial Cells and Their Association with Spermatozoa1

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. To identify the oviduct factors involved in their survival, sperm cells were preincubated 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 with cells preincubated in the absence of apical membranes. This suggests that, during the 30-min preincubation 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 labeled with [35S]-methionine and, upon extensive washes, proteins were separated by two-dimensional (2-D) gel electrophoresis to identify the factors suspected to have beneficial effects on spermatozoa. The six major proteins, according to their signal intensity on the autoradiographic film, were extracted from a 2-D gel of oviduct epithelial cell proteins run 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 identities and association with spermatozoa were 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.

Effects of HSPA8, an evolutionarily conserved oviductal protein, on boar and bull spermatozoa

“…Like the study reported here, experiments by Boilard and colleagues (Boilard et al 2001) identified three chaperone proteins from cow oviducts that bound to bull spermatozoa; two of these were identified as Heat shock 60 kDa protein 1 (chaperonin; HSPD1 previously known as HSP60) and HSPA5 (Boilard et al 2004). Interestingly, HSPA5 is also present in our sperm-binding 70 kDa sAPM band.…”

“…They are known to be synthesized during spermatogenesis (Allen et al 1988, Maekawa et al 1989 and they adopt specific localizations within sperm e.g. within their plasma membrane (Boilard et al 2004, Mitchell et al 2007. While endogenous heat shock proteins clearly have roles in sperm function, data in the present study clearly show that exogenous heat shock proteins, such as HSPA8 can also have an important effect on sperm function.…”

Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity

“…HSPA8 probably reverses sperm membrane damage and restores cell membrane integrity via interactions with, and alterations in, membrane properties. A number of studies have reported on the ability of different HSPs to enhance viability via interactions with cellular membranes in different cell types (Johnson et al 1990, Johnson & Tytell 1993, Boilard et al 2004. It is known that HSPs regulate membrane stability and fluidity (Chen et al 2005, Horvath et al 2008 and that HSPs are involved in stabilizing cellular and organellar membranes in stressful conditions (Carratu et al 1996, Vigh et al 1998.…”

Extracellular cell stress (heat shock) proteins—immune responses and disease: an overview

“…Proteomic analysis of porcine oviductal fluid has revealed that epithelial cells in the oviductal lumen secrete several molecules in response to the presence of spermatozoa, most notable of which are heat shock (stress) proteins (HSPs) (116). Heat shock proteins have also been identified in soluble fractions of pig and cow oviductal apical plasma membranes (sAPM) and in the human apical epithelium (117)(118)(119). These are potentially important findings, as the oviduct and oviductal sperm storage play key roles in reproduction by providing a secure reservoir in which spermatozoa can attain full fertilising properties.…”