Compartmentalization of proteins in epididymosomes coordinates the association of epididymal proteins with the different functional structures of bovine spermatozoa

Abstract

Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis. In bovine, many proteins are associated with epididymosomes, and some of them, such as the glycosylphosphatidylinositol (GPI)-anchored protein P25b, macrophage migration inhibitory factor (MIF), and aldose reductase (AKR1B1), are transferred to spermatozoa during the epididymal maturation process. P25b is associated with detergent-resistant membrane (DRM) domains of epididymal spermatozoa, whereas MIF and AKR1B1 are cytosolic proteins associated with detergent-soluble fractions. In this study, we tested the hypothesis that DRM domains are also present in the epididymosomes and that P25b DRM-associated proteins in these vesicles are transferred to the DRMs of spermatozoa. The presence of DRMs in epididymosomes was confirmed by their insolubility in cold Triton X-100 and their low buoyant density in sucrose gradient. Furthermore, DRMs isolated from epididymosomes are characterized by the exclusive presence of ganglioside GM1 and by high levels of cholesterol and sphingomyelin. Biochemical analysis indicated that P25b is linked to DRM in epididymosomes, whereas MIF and AKR1B1 are completely excluded from these membrane domains. Proteolytic treatment of epididymosomes and immunoblotting studies showed that P25b is affected by trypsin or pronase proteolysis. In contrast, MIF and AKR1B1 are not degraded by proteases, suggesting that they are localized within epididymosomes. Interaction studies between epididymosomes and epididymal spermatozoa demonstrated that P25b is transferred from the DRM of epididymosomes to the DRM of the caput epididymal spermatozoa as a GPI-anchored protein. Together, these data suggest that specific localization and compartmentalization of proteins in the epididymosomes coordinate the association of epididymal proteins with the different functional structures of spermatozoa.

FIG. 1.

P25b protein is associated with the plasma membrane of caput epididymal spermatozoa, whereas MIF and AKR1B1 are exclusively found as soluble proteins. Sperm proteins were isolated from different subcellular compartments following nitrogen cavitation, ultrasonic treatment, and centrifugations. Different fractions enriched from specific subcellular compartments, such as the sperm heads and flagella, membranes, and cytosolic fractions were obtained from caput epididymal spermatozoa and analyzed by Western blotting. Western blot (WB) detection of P25b molecules (28 kDa) as well as MIF (12 kDa) and AKR1B1 (36 kDa) was performed in reducing conditions. The equivalent of 25 × 10⁶ spermatozoa per sample for each fraction was loaded onto gel. Results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on the left. Flag, flagella; PM, plasma membrane; OM, other membranes; Cyt, cytosolic fraction.

FIG. 2.

Ganglioside GM1 and high light scattering intensity were found mainly in the low-density fractions of sucrose gradient isolated from bovine cauda epididymosomes. Epididymosomes were treated with 1% cold Triton X-100 and subjected to fractionation by the sucrose gradient method. The fractions collected along the sucrose gradient were analyzed. A) The collected fractions were dotted on membranes, and GM1 molecules were detected in each fraction with HRP-conjugated CTB. B) Light scattering intensity was assessed in each fraction by measuring the absorbance at 400 nm (A400). Results are expressed as mean ± SEM for three independent experiments.

FIG. 3.

P25b is associated with DRM domains of epididymosomes, whereas MIF and AKR1B1 are found in T-S fractions. Epididymosomes were treated with 1% cold Triton X-100 and subjected to fractionation in discontinuous density sucrose gradient. Western blot detection of P25b molecules (28 kDa) as well as MIF (12 kDa) and AKR1B1 (36 kDa) was performed in reducing conditions. A total of 25 × 10⁶ spermatozoa per sample for each fraction were loaded. Results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on the left.

FIG. 4.

P25b is affected by proteolytic treatment, whereas MIF and AKR1B1 proteins remain intact in cauda epididymosomes. Epididymosomes, purified MIF from epididymal fluid, or recombinant AKR1B1 was treated in the absence (control) or presence of 25 μg/ml trypsin or 15 μg/ml pronase for 30 min. P25b protein (28 kDa), MIF (12 kDa), and AKR1B1 (36 kDa) were detected by Western blot analysis. A total of 25 μg of proteins per sample was loaded onto gel. Results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on left. Ctrl, Control.

FIG. 5.

P25b is specifically transferred from the DRM domain of epididymosomes to the DRM domain of caput epididymal spermatozoa. Biotinylated epididymosomes were treated with 1% Triton X-100 and subjected to fractionation in discontinuous density sucrose gradient. A) Collected fractions were analyzed by Western blot (WB) analysis to detect biotinylated proteins. B) The DRM (fractions 3–5) and T-S (fractions 9–11) fractions collected from the sucrose gradient were pooled. The DRM proteins were solubilized by Triton X-100. Biotinylated proteins from DRM and T-S fractions were affinity precipitated (AP) using streptavidin agarose (SA-Agarose), were analyzed, and were immunoblotted to detect P25b protein as well as biotinylated proteins. C) Biotinylated epididymosomes were coincubated with caput epididymal spermatozoa. Spermatozoa were then extensively washed and treated with 1% Triton X-100. Treated spermatozoa were subject to fractionation in discontinuous density sucrose gradient. Different fractions were analyzed to detect biotinylated proteins in collected fractions. D) Biotinylated protein in DRM and T-S fractions isolated from spermatozoa were affinity precipitated with streptavidin-agarose beads. Western blot detection of P25b molecules (28 kDa) (WB: P25b) and biotinylated proteins (WB: NA-HRP [neutravidin-conjugated HRP]) were performed in reducing conditions. Results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on the left.

FIG. 6.

P25b is transferred from epididymosomes to membrane spermatozoa as a GPI-anchored protein. Biotinylated epididymosomes were coincubated with caput epididymal spermatozoa. Membrane-enriched fraction was isolated from these spermatozoa by nitrogen cavitation and ultrasonic treatment. Spermatozoa as well as membrane-enriched fractions were treated in the absence or presence of phospholipase C (PLC) and centrifuged in order to separate the pellet from the supernatant. The pellet and supernatant were then analyzed to detect biotinylated proteins (30 kDa). Molecular weight standards (kDa) are indicated on the left. PM+OM, plasma membrane and other membrane-enriched fraction.