Glucose-regulated protein precursor (GRP78) and tumor rejection antigen (GP96) are unique to hamster caput epididymal spermatozoa

Abstract

The immotile testicular mammalian spermatozoon gets transformed into a motile spermatozoon during 'epididymal maturation'. During this process, the spermatozoa transit from the caput to the cauda epididymis and undergo a number of distinct morphological, biophysical and biochemical changes, including changes in protein composition and protein modifications, which may be relevant to the acquisition of motility potential. The present proteome-based study of the hamster epididymal spermatozoa of caput and cauda led to the identification of 113 proteins spots using Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis. Comparison of these 113 protein spots indicated that 30 protein spots (corresponding to 20 proteins) were significantly changed in intensity. Five proteins were increased and eleven were decreased in intensity in the cauda epididymal spermatozoa. In addition, two proteins, glucose-regulated protein precursor (GRP78) and tumor rejection antigen (GP96), were unique to the caput epididymal spermatozoa, while one protein, fibrinogen-like protein 1, was unique to cauda epididymal spermatozoa. A few of the five proteins, which increased in intensity, were related to sperm metabolism and ATP production during epididymal maturation. The changes in intensity of a few proteins such as ERp57, GRP78, GP96, Hsp60, Hsp70, and dihydrolipoamide S-acetyltransferase were validated by immunoblotting. The present study provides a global picture of the changes in protein composition occurring during hamster sperm epididymal maturation, besides being the first ever report on the proteome of hamster spermatozoa.

Figure 1

Representative 2D maps of the proteins of hamster caput (A) and cauda (B) epididymal spermatozoa resolved in the pI range of pH 5–8. The mol. wt. of the standard proteins is indicated. The protein spots encircled and numbered in (A) and (B) are listed in Supplementary Table 1. Encircled proteins within a rectangle in A and B represent spots unique to caput and the encircled protein within a triangle represents a protein unique to cauda epididymal spermatozoa. The encircled proteins in (B) are those proteins that exhibit increase or decrease in intensity in cauda epididymal spermatozoa compared with caput epididymal spermatozoa.

Figure 2

Immunofluorescent localization of ERp57 in the spermatozoa of hamster (A, B), mouse (C, D), rat (E, F) and humans (G). A', B', C', D' and E', F′ and G′ are the corresponding brightfield images of A, B, C, D and E, F and G. Caput epididymal spermatozoa of hamster (A) show intense staining in the mid-piece [M], with faint staining in the principal piece [PP]. Cauda epididymal spermatozoa of hamster (B) did not show any positive staining in the mid-piece [M], but showed faint staining in the principal piece [PP]. In mouse, both caput and cauda epididymal spermatozoa (C, D) staining are observed in the acrosome [Ac] and principal piece [PP]. In rat, the caput epididymal spermatozoa (E) showed staining both in the head [H] and in the entire flagellum, whereas the cauda epididymal spermatozoa (F) showed staining limited only to the acrosome [Ac]. Ejaculated human spermatozoa (G) show staining in the acrosome [Ac] and entire flagellum. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Scale bars = 20 μm.

Figure 3

Immunofluorescent localization of GRP78 and GP96 in hamster caput and cauda epididymal spermatozoa. Immunofluorescence localization of GRP78 showed an intense staining in the acrosome (Ac) and principal piece (PP) of caput spermatozoa of hamster (A), while GP96 showed intense staining only in the acrosome (Ac) of caput epididymal spermatozoa (C). Cauda epididymal spermatozoa did not show any positive staining for either GRP78 (B) or GP96 (D) proteins. A', B', C', D' are the corresponding brightfield images of A, B, C, D. 'H' denotes the head and 'M' denotes the mid-piece of the spermatozoa. Scale bars = 20 μm.

Figure 4

Immunoblot analysis of GRP78, ERp57, GP96 (A), Hsp60 (B), Hsp70 (C) and dihydrolipoamide S-acetyltransferase (D) using hamster caput [Cp] and cauda [Cd] epididymal sperm lysates. Densitometry analysis of Hsp60 (B′) and Hsp70 (C′) showed a decrease in intensity in cauda epididymal spermatozoa, while dihydrolipoamide S-acetyltransferase (D′) showed an increase in intensity in cauda epididymal spermatozoa. ^*P < 0.05, Mann-Whitney test, compared with Cp. Tubulin was used as an internal control.

Figure 5

Immunoblot analysis of GRP78, ERp57 and GP96 using hamster caput [Cp] and cauda [Cd] epidydimal sperm lysates resolved in the pI range of 5–8 (A) and 3–10 (B).