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. 2012 Mar 21;86(6):181. doi: 10.1095/biolreprod.111.097477

ATP-Binding Cassette Transporter G2 Activity in the Bovine Spermatozoa Is Modulated Along the Epididymal Duct and at Ejaculation1

Julieta Caballero 3,4, Gilles Frenette 3,4, Olivier D'Amours 3,4, Maurice Dufour 3,4, Richard Oko 5, Robert Sullivan 3,4,2
PMCID: PMC4480151  PMID: 22441796

Abstract

During their epididymal maturation, stabilizing factors such as cholesterol sulfate are associated with the sperm plasma membrane. Cholesterol is sulfated in epididymal spermatozoa by the enzyme estrogen sulfotransferase. Because of its role in the efflux of sulfate conjugates formed intracellularly by sulfotransferases, the ATP-binding cassette membrane transporter G2 (ABCG2) might have a role in the translocation of this compound across the plasma membrane. In the present study we showed that ABCG2 is present in the plasma membrane overlaying the acrosomal region of spermatozoa recovered from testis, epididymis, and after ejaculation. Although ABCG2 is also present in epididymosomes, the transporter is not transferred to spermatozoa via this mechanism. Furthermore, although epididymal sperm ABCG2 was shown to be functional, as determined by its ability to extrude Hoechst 33342 in the presence of the specific inhibitor Fumitremorgin C, ABCG2 present in ejaculated sperm was found to be nonfunctional. Additional experiments demonstrated that phosphorylation of ABCG2 tyrosyl residues, but not its localization in lipid rafts, is the mechanism responsible for its functionality. Dephosphorylation of ABCG2 in ejaculated spermatozoa is proposed to cause a partial protein relocalization to other intracellular compartments. Prostasomes are proposed to have a role in this process because incubation with this fraction of seminal plasma induces a decrease in the amount of ABCG2 in the associated sperm membrane fraction. These results demonstrate that ABCG2 plays a role in epididymal sperm maturation, but not after ejaculation. The loss of ABCG2 function after ejaculation is proposed to be regulated by prostasomes.

Keywords: ABCG2, ATP-binding cassette transporters, bull spermatozoa, epididymis

ABCG2 is involved in cholesterol and cholesterol sulfate translocation through the sperm membrane during epididymal sperm maturation, but is inactive after ejaculation.

INTRODUCTION

During epididymal transit, testicular spermatozoa undergo modifications to acquire their fertilization potential [1]. One of the biochemical events putatively involved in the epididymal maturation of spermatozoa is the association of stabilizing factors, such as cholesterol sulfate, with the sperm plasma membrane. Cholesterol sulfate accumulates in the plasma membrane that covers the acrosomal region during epididymal maturation [2]. Previous results from our laboratory in the bovine model demonstrated that in vitro, cholesterol is sulfated by the enzyme estrogen sulfotransferase (EST) in epididymal spermatozoa [3]. Estrogen sulfotransferase is also present in the acrosomal region of epididymal spermatozoa. The presence of this enzyme was also reported in epididymosomes (i.e., membranous vesicles purified from epididymal luminal fluid), suggesting that the latter structures might transfer EST to the maturating spermatozoa [3]. Once produced intracellularly, it is still unknown how cholesterol sulfate is translocated to the plasma membrane of epididymal spermatozoa.

ABCG2 is an efflux pump that belongs to the ABCG subfamily of the ATP-binding cassette (ABC) transporters and can extrude a wide variety of compounds from the cell using the energy produced from ATP hydrolysis [4]. When functionally expressed in Lactobacillus lactis, this transporter was found to interact with sterols [5] and to promote the efflux of sulfate conjugates formed intracellularly by sulfotransferases [6]. ABCG2 is localized in the plasma membrane of mammalian cells. Moreover, it has been shown to be associated with lipid raft microdomains [7, 8], suggesting that ABCG2 may be functionally regulated by the lipid environment [9] and cholesterol content of the plasma membrane [10, 11]. The plasma membrane localization of ABCG2 is modulated by the phosphorylation of Tyr-362 by Pim-1 kinase [12]. Posttranslational modification of this highly conserved tyrosine residue protects ABC transporters from proteolytic and proteasomal degradation [13]. ABCG2 is expressed in the blood barrier of several tissues of major regulatory importance [14–18] as well as in epithelial cells and ducts [19]. In the male reproductive tract, ABCG2 was immunodetected in the blood-testis barrier [6, 20] and in the abluminal membrane of the caput region of the epididymis [6]. More recently, the presence of ABCG2 was also reported in spermatogonias and the acrosomal region of mouse and rat epididymal spermatozoa and human and bull ejaculated spermatozoa [21].

Taken together, the available evidence allowed us to suggest that ABCG2 present in epididymal spermatozoa might play an important role in epididymal sperm maturation because of its ability to translocate sterols across the plasma membrane. In the model proposed, cholesterol sulfate accumulated in epididymal spermatozoa [2, 22] exerts its inhibitory effect on capacitation [23], but after ejaculation, capacitation-associated events would be initiated in sperm [24], resulting in the removal of cholesterol. At this time, the protective functions of cholesterol sulfate, and therefore ABCG2, are not required any longer, and in consequence, dephosphorylation and internalization of the transporter in ejaculated spermatozoa cause its loss of function.

MATERIALS AND METHODS

Processing of Biological Material

Testes and epididymides from mature bulls were obtained by local professionals from the Abattoir Colbex slaughterhouse and sent to the laboratory on ice. Upon arrival, tissues were dissected on ice, and small pieces of different testicular and epididymal sections were processed immediately for RNA and protein extraction as well as for immunohistochemistry. Testicular spermatozoa were obtained by cutting the testis and collecting the fluid. Epididymal fluids were obtained from the caput, corpus, and cauda sections as previously described by Frenette et al. [25]. Briefly, caput fluid was obtained by cutting tubules and applying pressure on their proximal portion, and the corpus middle section was cut into small pieces, which were then immersed in 150 mM NaCl to drain off epididymal fluid; cauda fluid was collected using retrograde flushing by applying air pressure into the vas deferens. Freshly ejaculated bull semen samples were generously provided by the Centre d'insémination artificielle du Québec (L'Alliance Bovitec Inc.) and processed immediately. After resuspension in 150 mM NaCl, epididymal fluids or semen samples were centrifuged at 700 × g for 10 min. Pelleted spermatozoa were washed several times and processed for protein extraction, immunocytochemistry, subcellular fractionation, coincubation with membranous vesicles, and fractionation of plasma membrane domains, as well as flow cytometry analysis. Supernatants from epididymal and seminal plasma fluids were centrifuged at 3000 × g for 20 min to remove cell debris and then ultracentrifuged at 120 000 × g for 2 h. The resulting pellets were resuspended and subjected to chromatography on Sephacryl S-500 HR (Pharmacia Canada Ltd.) to purify membranous vesicles. Vesicle-free supernatant fluids isolated from seminal plasma samples were stored at −80°C. Purified membranous vesicles were resuspended in 150 mM NaCl and ultracentrifuged again. The latter preparations were aliquoted and stored at −80°C until use. Protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories).

RNA Extraction, Reverse Transcription, and Real-Time PCR

For RNA extraction, small tissue sections were homogenized with TRIzol reagent (Invitrogen Life Technologies), precipitated with CHCl3/isopropanol, and purified with the RNeasy Mini Kit (Qiagen) according to the manufacturers' instructions. The integrity of RNA samples was evaluated by agarose gel electrophoresis. Four micrograms of RNA were reverse transcribed using Superscript II and hexameric random primers (Invitrogen). From these samples, an amount of cDNA equivalent to 100 ng of RNA was amplified by real-time PCR using a Light Cycler-Fast Start DNA Master SYBR Green I kit (Roche Diagnostics). The primers used for ABCG2 (NM_001037478) were: forward, 5′-GGTTTCCGCTGTGAGCCCTATA-3′, and reverse, 5′-GAAGGCTCTTCGGTCTCGTT-3′. TUBA1B (Tubulin-α1b; NM_001114856) and GAPDH (NM_001034034) were used as housekeeping (control) genes, and the primers used were: forward, 5′-TCCATCCACGTTGGCCAGGCT-3′, and reverse, 5′-AGCCCCTGTCTCACTGAAGAAG-3′; and forward, 5′-GCATCGTGGAGGGACTTATGA-3′, and reverse, 5′-GTTTGAGCTCAGGGATGACCTT-3′, respectively. Amplified PCR products were sequenced to confirm the specificity of the reactions.

Subcellular Fractionation

Ejaculated or cauda epididymal spermatozoa were subjected to subcellular fraction following nitrogen cavitation [26, 27]. The complete procedure was performed at 4°C. Spermatozoa (2 × 109) were washed with PBS supplemented with a protease inhibitor cocktail (Roche) and subjected to nitrogen cavitation at 0.11 Pa for 10 min. Demembranated spermatozoa were pelleted by centrifugation at 16 000 × g for 15 min. The supernatant was then ultracentrifuged at 100 000 × g for 1 h. Plasma membrane and cytosolic fractions were recovered from the pellet and the supernatant, respectively. Demembranated spermatozoa were sonicated and centrifuged at 10 000 × g for 10 min. The supernatant thus obtained was ultracentrifuged at 100 000 × g for 1 h. Other membrane fractions (acrosomal, nuclear, and remnants of plasma membranes) and other cytosolic components (not removed by cavitation) were recovered from the pellet and supernatant, respectively. Sonicated spermatozoa were subjected to fractionation on a discontinuous sucrose gradient (1.6, 1.8, and 2.2 M) and centrifuged at 15 000 × g for 15 min. A flagella-enriched fraction was recovered at the upper/intermediate-layer interface (i.e., 1.6 M/1.8 M), and sperm heads were mainly found in the pellet.

Immunoprecipitation of ABCG2 and Tyrosine-Phosphorylated Proteins

The plasma membrane and cytosol fractions obtained through epididymal and ejaculated sperm subcellular fractionation were subjected to immunoprecipitation according to Lachance et al. [28]. The whole procedure was carried out at 4°C in the presence of protease and phosphatase inhibitors (Roche). Each subcellular fraction recovered from 200 × 106 spermatozoa was lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 0.1% [w/v] SDS, 1% Triton X-100, 0.5% [w/v] sodium deoxycholate, and 1 mM ethylene diamine tetraacetic acid, plus protease and phosphatase inhibitors) for 30 min on ice. After centrifugation at 10 000 × g for 20 min at 4°C, the soluble fraction was recovered and incubated with 1 μg of anti-ABCG2 antibody (BXP-53; ab24115; Abcam), normal rat immunoglobulin G (IgG), anti-phosphotyrosine antibody (clone 4G10; Upstate Biotechnology Inc.), or normal mouse IgG overnight at 4°C with constant rotary shaking. Then, samples were incubated with recombinant protein G-Sepharose beads (Invitrogen) for 2 h at 4°C with constant rotary shaking. The beads were then washed three times with RIPA buffer, and the immunocomplexes were eluted with Laemmli sample buffer (2% [w/v] SDS, 2.5% [v/v] β-mercaptoethanol, and 50 mM Tris, pH 6.8) and subjected to analysis by 8% SDS-PAGE.

Isolation of Raft Membrane Domains

Detergent-resistant membrane domains (DRMs; including rafts) and Triton-soluble membrane domains (TSs) were isolated from caput and cauda epididymal sperm and membranous vesicles (epididymosomes and prostasomes) as described by Girouard et al. [24], with minor changes. Spermatozoa (5 × 108 to 7 × 108) or the amount of membranous vesicles corresponding to 1 mg of proteins were detergent extracted with 2% (v/v) Triton X-100 (in PBS) on ice for 1 h. Spermatozoa and/or other particles were removed by centrifugation at 2000 × g for 5 min. Supernatants were subjected to fractionation on a discontinuous sucrose gradient (42.5%, 35%, and 5%) and centrifuged at 100 000 × g for 16 h at 4°C. Ten fractions were then recovered from top to bottom, namely, the low-density fractions (fractions 2–4) containing DRMs, the intermediate-density fractions (fractions 5–7), and the high-density fractions (fractions 8–10), which contained the TS proteins. Proteins from each fraction were precipitated with MeOH/CHCl3 [29].

Immunolocalization of ABCG2

Small epididymal tissue sections were cut and fixed in 4% (v/v) paraformaldehyde in PBS for 24 h and embedded afterward in paraffin. Sections of 6 μm were mounted, paraffin was removed by immersion in xylene, and tissues were rehydrated by immersion in EtOH solutions of decreasing concentration. Endogenous peroxidases were neutralized with 3% H2O2 in MeOH for 30 min. Antigens were unmasked by boiling in 10 mM sodium citrate (pH 6.0). After blocking unspecific sites with 5% (v/v) goat serum in PBS, slides were incubated overnight at 4°C with 12.5 μg/ml anti-ABCG2 antibodies or normal rat IgG as control in 0.5% (v/v) goat serum in PBS. After removing excess antibodies, slides were incubated with biotinylated goat anti-rat IgG, and the reaction was developed by addition of ABC Vectastain kit (Vector Laboratories). Nuclei were counterstained with a hematoxylin solution.

Ejaculated or epididymal spermatozoa from different epididymal sections were washed and smeared onto slides. After fixation with 3.7% formaldehyde in PBS, unspecific sites were blocked with 5% goat serum in PBS and incubated overnight at 4°C with 12.5 μg/ml anti-ABCG2 antibodies or normal rat IgG as control in 0.5% of goat serum in PBS. Unbound antibodies were washed with PBS, and anti-rat IgG Alexa 568-conjugated antibodies were added as secondary antibodies for 1 h at room temperature in darkness. To permeabilize plasma membranes, fixed sperm smears were incubated with 1% Triton X-100 in PBS for 10 min. Samples were mounted with Vectashield supplemented with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and observed under a Zeiss Axioskop 2 epifluorescence microscope (Carl Zeiss Canada). Images were also obtained in parallel in brightfield.

Immunogold Labeling for Electron Microscopy

Testes were fixed by perfusion of testicular blood vessels with 4% formaldehyde and 0.8% glutaraldehyde. Small pieces of tissues (1–2 mm3) were dehydrated in graded EtOH up to 90% and embedded in LR White (Polysciences Inc.). Sperm samples were obtained and washed as described above. After a 1-h fixation in the same solution with rotary shaking at 4°C, sperm cells were dehydrated and embedded in LR White. These embedded samples were cut into ultrathin sections and mounted on Formvar-coated nickel grids (Polysciences Inc.). After blocking with 10% (v/v) normal goat serum, the sections were incubated overnight at 4°C with the anti-ABCG2 primary antibody or normal rat serum as control. After washing, sections were incubated with goat anti-rat IgG conjugated with 12-nm colloidal gold (Jackson ImmunoResearch Laboratories Inc.). The sections were counterstained with uranyl acetate and lead citrate and were examined by transmission electron microscopy (Hitachi 7000).

Incubation of Epididymal Spermatozoa with Epididymosomes or Seminal Plasma Components

Caput or cauda epididymal spermatozoa (1.5 × 107) were resuspended in 50 μl of 150 mM NaCl containing membranous vesicles (caput or cauda epididymosomes or prostasomes) or 50 μl of prostasome-free seminal plasma or total seminal plasma for 1 h at 37°C. After incubation, spermatozoa were extensively washed in saline solution (0.9% [w/v] NaCl), and total spermatozoan proteins were extracted with Triton X-100 [25]. Experiments were performed in duplicate.

Protein Extraction and Western Immunoblotting

Small pieces of the different testicular and epididymal sections were homogenized in 25 mM Tris buffer (pH 7.5) containing 50 mM NaCl, 1% (v/v) SDS, and protease inhibitors (Roche), and supernatants were obtained after a 30-min centrifugation at 20 000 × g. The various subcellular fractions as well as sperm or membranous vesicle proteins were extracted with 1% (v/v) SDS. Proteins from spermatozoa coincubated with membranous vesicles were extracted with 1% (v/v) Triton X-100. Proteins were next precipitated with MeOH/CHCl3, solubilized in Laemmli sample buffer, subjected to SDS-PAGE, and electrotransferred onto nitrocellulose membrane. Membranes were blocked with 5% dry skimmed milk in PBS supplemented with 0.1% (v/v) Tween-20 and incubated with the anti-ABCG2 or anti-tubulin antibodies for 2 h at room temperature. Following rinsing, membranes were incubated for 1 h with goat anti-rat IgG or goat anti-mouse coupled to horseradish peroxidase, respectively, and revealed using ECL substrate (Amersham). Films were scanned, and images were analyzed and quantified using the Adobe Photoshop CS2 version 9.0.2 software using a previously described method (http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html). Semiquantitative determination of ABCG2 content on tissues and spermatozoa was done using tubulin as loading control. In the case of epididymosomes or spermatozoa incubated with epididymosomes or seminal plasma components, the analysis was performed based on the total amount of proteins and the number of spermatozoa, respectively.

Evaluation of ABCG2 Activity

The activity of ABCG2 was assessed by its ability to expel Hoechst 33342 from sperm cells [21]. Epididymal and ejaculated spermatozoa were washed twice with sperm medium (114 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM NaH2PO4, 10 mM sodium lactate, 2 mM CaCl2, 0.5 mM MgCl2, 0.2 mM sodium pyruvate, 10 mM d-glucose, and 1 mg/ml polyvinyl alcohol) at pH 6.5 (10 mM 2-(N-morpholino)ethanesulfonic acid) and 7.4 (10 mM HEPES), respectively. After adjusting sperm concentration at 5 × 106 per milliliter in the same medium supplemented with 5 μg/ml Hoechst 33342, cells were incubated for 10 min at 37°C. Samples were split into two halves and incubated in the presence or absence of 10 μM Fumitremorgin C (Sigma), a specific inhibitor of ABCG2 activity. Propidium iodide (2 μg/ml) was then added to exclude the dead sperm population, and the sperm suspensions were analyzed by flow cytometry (BD FACSAria-II; BD Biosciences). Blue (λem = 450 nm; λexc = 375 nm) and red (λem = 610 nm; λexc = 375 nm) Hoechst fluorescence was detected by forward and side scatters. Propidium iodide was measured at a λem of 610 nm using a λexc of 488 nm.

Statistical Analysis

Statistical analyses were performed using the GraphPad Prism 4 software (GraphPad Software). Results from semiquantitative densitometry values from Western immunoblotting and quantitative PCR were transformed and analyzed by ANOVA and Bonferroni post hoc correction. Data obtained from experiments with spermatozoa coincubated with membranous vesicles or seminal plasma components were analyzed by paired Student t-tests where controls were compared with each treatment condition. A difference with P < 0.05 was considered as significant.

RESULTS

Expression of ABCG2 in Bull Epididymal Spermatozoa

Immunoblotting experiments showed the presence of ABCG2 in total protein extracts from epididymal spermatozoa recovered from all three different epididymal sections, namely, the caput, corpus, and cauda (Fig. 1A), as a band of ∼63 kDa. Even though the expected Mr is 72 kDa, the present figure is in agreement with that reported by Pulido et al. [30], who used the same antibody with bovine mammary gland protein extracts. The abundance of ABCG2 protein was semiquantitatively determined using tubulin as a loading control. As shown in Figure 1A, ABCG2 expression exhibited variations along the epididymal transit. Quite notably, ABCG2 content was significantly decreased in spermatozoa recovered from the corpus region (Fig. 1A).

FIG. 1. .

FIG. 1. 

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Expression of ABCG2 in bull epididymal spermatozoa. A) Immunoblotting and semiquantitative analysis of ABCG2 (63 kDa) protein expression in total protein extracts from 1 × 107 epididymal spermatozoa. Ca-Sptz, caput; Co-Sptz, corpus; Cd-Sptz, cauda. Tubulin (55 kDa) was used as a loading control to normalize ABCG2 expression. Results are expressed as ABCG2/tubulin and represent the mean ± SD of four independent experiments. Values with different superscripts are significantly different (P < 0.05). B) Indirect immunofluorescence localization of ABCG2 in nonpermeabilized epididymal spermatozoa. Control conditions: normal rat IgG. Images are contrasted with brightfield. Bar = 10 μm. Results are representative of four independent experiments. The image represents ABCG2 localization in epididymal sperm populations recovered from caput, corpus, and cauda regions. C) Immunoblotting detection of ABCG2 and tubulin in total protein extracts from different cauda epididymal sperm subcellular fractions: plasma membrane and cytosol obtained after nitrogen cavitation (PM1 and Cyt1, respectively), other membranes and cytosol obtained following sonication (PM2 and Cyt2, respectively), and heads and flagella (H&F). Results are representative of two independent experiments.

Indirect immunofluorescence studies on spermatozoa recovered from each epididymal region showed that ABCG2 is mostly localized to the acrosomal region (Fig. 1B). Proximal postacrosomal staining was also observed in spermatozoa recovered from some bulls. Furthermore, using nitrogen cavitation followed by subcellular fractionation, ABCG2 was specifically detected in membrane-containing fractions from cauda epididymal spermatozoa (Fig. 1C).

The above results support that ABCG2 is present in the plasma membrane overlaying the acrosomal region of epididymal spermatozoa during epididymal transit. The localization in this region was also observed by indirect immunofluorescence studies on nonpermeabilized spermatozoa recovered from testis (Fig. 2A). ABCG2 immunodetection by electron microscopy on testicular tissues showed that this signal was present on the surface of the acrosomal region of the testicular spermatozoa (Fig. 2B). The resolution of the micrograph is not sufficient to discriminate whether the presence of ABCG2 is on the glycocalyx or the plasma membrane. However, anti-ABCG2 antibodies recognize specifically an internal epitope of the transporter, so we can suggest that ABCG2 is associated with the testicular and epididymal sperm plasma membrane.

FIG. 2. .

FIG. 2. 

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Sperm ABCG2 has a testicular origin. A) Indirect immunofluorescence localization of ABCG2 in nonpermeabilized testicular spermatozoa. Bar = 10 μm. Results are representative of two independent experiments. B) Immunoelectron microscopy and localization of ABCG2 in testis. Sperm ABCG2 localization revealed with anti-rat 12-nm gold particle-labeled antibodies. Red asterisks indicate immunolabeling. Control conditions: normal rat IgG. Bar = 0.5 μm.

ABCG2 Is Active in Epididymal Spermatozoa

One of the standard approaches to demonstrate ABCG2 functionality is to measure the accumulation of specific fluorescent dyes [31] by flow cytometry following treatment with specific inhibitors of the transporter [32]. Exposition to Hoechst 33342 in the presence of the specific pump inhibitor Fumitremorgin C led to an increase in dye accumulation by either caput or cauda epididymal spermatozoa compared with control sperm suspensions (minus the inhibitor; Fig. 3). These results suggest that ABCG2 is active in epididymal spermatozoa.

FIG. 3. .

FIG. 3. 

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ABCG2 is active in epididymal spermatozoa. Epididymal sperm suspensions were incubated in sperm medium (pH 6.5) at a concentration of 5 × 106 per milliliter with 5 μg/ml Hoechst 33342 for 10 min at 37°C. After incubation in the presence or absence of the specific ABCG2 inhibitor, Fumitremorgin C (FTC; 10 μM), samples were analyzed by flow cytometry. Propidium iodide was used to exclude dead sperm. Hoechst 33342 blue fluorescence of sperm plus or minus inhibitor (red and blue, respectively) was integrated. Results are representative of three independent experiments done with caput and caudal epididymal sperm populations.

Coincubation of Spermatozoa with Epididymosomes

Variations in ABCG2 expression were observed on epididymal spermatozoa as shown in Figure 1A. Because our group had reported that certain proteins present in the epididymosomes can be selectively transferred to maturing sperm [25], ABCG2 expression was first assessed in epididymosomes. The protein was also detected as a 63-kDa species in epididymosomes recovered from the three different epididymal regions (Fig. 4A). ABCG2 was not detected in epididymal fluid freed from epididymosomes (data not shown). Because ABCG2 content was seen to follow the same pattern as the protein as detected in spermatozoa recovered from the epididymis (Fig. 1A), epididymosomes and epididymal spermatozoa were coincubated in order to monitor the possible transfer of ABCG2 between the two structures. No significant change in total ABCG2 protein content could be observed in caput spermatozoa after incubation with neither caput (Fig. 4B), corpus (data not shown), nor cauda epididymosomes (Fig. 4B). However, there was trend towards an increase of ABCG2 content in spermatozoa following incubation with cauda epididymosomes.

FIG. 4. .

FIG. 4. 

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Coincubation of spermatozoa with epididymosomes. A) Immunoblotting detection of ABCG2 in 15 μg of total protein extracts from membranous vesicles (epididymosomes) recovered from epididymal fluids from different epididymal regions. Ca, caput; Co, corpus; Cd, cauda epididymosomes. Results are representative of three independent experiments. B) Semiquantitative analysis of Western immunoblots of ABCG2 from 1 × 107 caput epididymal spermatozoa after a 60-min incubation without (Ca-Sptz) or with (Ca-Ep) caput or cauda epididymosomes (Cd-Ep). Data (arbitrary units) were analyzed by t-test using control conditions as 100%. No significant differences were observed for the various treatments versus control.

ABCG2 Is Expressed in the Epididymis

Because epididymosomes are the apocrine secretory system of the epididymal epithelial cells, ABCG2 expression was next studied in the various epididymal tissues. The relative abundance of ABCG2 mRNA in the different epididymal tissue sections was quantified by real-time PCR using TUBA1B (tubulin-α1b) and GAPDH as housekeeping control genes, and the amount of ABCG2 protein was semiquantitatively analyzed by Western immunoblotting using tubulin as a loading control for total protein extracts. Because analysis of whole caput, corpus, and cauda sections showed that the ABCG2 expression pattern was different in each section but with a high interindividual variability (data not shown), a second experiment was carried out comparing different subsections obtained from the same individual. The ABCG2 mRNA as well as ABCG2 protein contents tended to be higher in the proximal regions of the corpus and the cauda (Fig. 5, A and B).

FIG. 5. .

FIG. 5. 

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ABCG2 is expressed in epididymis. A) Real-time PCR and quantitative analysis of ABCG2 mRNA in different epididymal tissue sections. Ca1, caput proximal; Ca2, caput distal; Co1, corpus proximal; Co2, corpus distal; Cd1, cauda proximal; Ca2, cauda distal. TUBA1B (tubulin) and GAPDH were used as housekeeping genes to normalize ABCG2 expression. Data are expressed as relative units, using the tissue displaying highest expression as 100% for each bull. Results represent the mean ± SD of one experiment run in duplicates. B) Immunoblotting and semiquantitative analysis of ABCG2 protein expression in different epididymal tissue sections. Ca1, caput proximal; Ca2, caput distal; Co1, corpus proximal; Co2, corpus distal; Cd1, cauda proximal; Ca2, cauda distal. TUBA1B (tubulin) was used as loading control to normalize ABCG2 expression. Data are expressed as relative units, using the tissue displaying highest expression as 100% for each bull. Results represent the mean ± SD of one experiment run in duplicates. C) Immunohistochemical localization of ABCG2 in cauda epididymal histological sections. Cd, cauda. Immunocomplexes appear in brown (arrows). Control conditions: normal rat IgG (Cd′). Nuclei are counterstained with hematoxylin. Bar = 50 μm. Results are representative of three independent experiments.

Because the protein present in the various tissues might come from different cell types as well as from spermatozoa and epididymosomes, immunohistochemistry experiments were carried out in order to delineate ABCG2 localization more precisely in each tissue section. ABCG2 was only detectable in the cauda region (Fig. 5C). In this distal segment of the epididymis, ABCG2 was specifically located in the apical region of epithelial cells.

Together, these results show that ABCG2 is expressed in bull epididymal tissues. Its expression is mostly associated with the apical border of the epithelium, and especially in the distal region of the epididymis.

ABCG2 Is Present in Ejaculated Spermatozoa, but Is Nonfunctional

Sperm cholesterol sulfate is accumulated during epididymal transit and has been suggested to inhibit capacitation. Early events that immediately follow ejaculation associated with capacitation have been described. In order to study the fate of ABCG2 in ejaculated spermatozoa, its abundance was evaluated by Western immunoblotting. An immunoreactive ABCG2 species was present in ejaculated spermatozoa with the same molecular mass (i.e., ∼63 kDa) as in epididymal spermatozoa (Fig. 6A).

FIG. 6. .

FIG. 6. 

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ABCG2 is present in ejaculated spermatozoa but is nonfunctional. A) Immunoblotting detection of ABCG2 in total protein extracts from 1 × 107 ejaculated spermatozoa (Ej-Sptz). Tubulin was used as a loading control. Results are representative of two independent experiments. B) Ejaculated sperm suspensions were incubated in sperm medium (pH 7.4) at a concentration of 5 × 106 per milliliter with 5 μg/ml Hoechst 33342 for 10 min at 37°C. After incubation with or without Fumitremorgin C (FTC; 10 μM), samples were analyzed by flow cytometry. Propidium iodide was used to exclude dead sperm. Hoechst blue fluorescence of sperm plus or minus inhibitor (red and blue, respectively) was integrated. Results are representative of two independent experiments.

However, the dye extrusion test used to assess ABCG2 functionality in epididymal spermatozoa revealed a different behavior in ejaculated spermatozoa. Indeed, Fumitremorgin C did not enhance the total fluorescence measured in ejaculated spermatozoa upon incubation with Hoechst 33342 (Fig. 6B), unlike the behavior observed above in epididymal spermatozoa (Fig. 3). A minor extra peak was also observed in these samples that might be caused by the presence of debris or cells of epithelial origin in fresh ejaculates.

The latter results suggest that although ABCG2 is still present in ejaculated spermatozoa, it appears to be inactive compared with epididymal sperm populations.

ABCG2 Membrane Domain Localization and Phosphorylation in Epididymal and Ejaculated Sperm

In order to explain the loss of ABCG2 activity in ejaculated spermatozoa, we assessed the localization of ABCG2 in various membrane domains in epididymal and ejaculated sperm. For this purpose, membranes were extracted from caput and cauda epididymal spermatozoa and ejaculated sperm with cold Triton X-100 and were subjected to centrifugation on a discontinuous sucrose gradient. Immunoblotting of the various sucrose gradient fractions revealed that ABCG2 is located in detergent-resistant membrane domains, or rafts, in epididymal as well as ejaculated spermatozoa (Fig. 7A).

FIG. 7. .

FIG. 7. 

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Membrane domain localization and phosphorylation of epididymal and ejaculated sperm ABCG2. A) Immunoblotting detection of ABCG2 in sucrose gradient fractions of Triton X-100 protein extracts from epididymal (Ca-sptz, caput spermatozoa; Cd-sptz, cauda spermatozoa) and ejaculated (Ej-sptz) spermatozoa. Results are representative of at least four independent experiments for each condition. B) Immunoprecipitation (IP) of ABCG2 from cauda epididymal sperm plasma membrane (PM) and cytosol (Cyt) protein extracts. Immunoblotting was carried out using anti-ABCG2 or anti-phosphotyrosine antibodies. Normal rat IgG (R IgG) and mouse IgG (M IgG) were used as negative controls. C) Immunoprecipitation of ABCG2 from ejaculated sperm PM and Cyt protein extracts. Immunoblotting was carried out using anti-ABCG2 or anti-phosphotyrosine antibodies. Normal rat IgG (R IgG) and mouse IgG (M IgG) were used as negative controls.

ABCG2 tyrosine phosphorylation status was also studied in sperm. In epididymal spermatozoa, ABCG2 was specifically immunoprecipitated in both plasma membrane and cytosol fractions (Fig. 7B). Under these extraction conditions, three different molecular forms with Mr values of 132, 73, and 63 kDa were detected. When protein blots were probed with anti-phosphotyrosine antibodies, only the 73-kDa isoform was detected in plasma membrane protein extracts. Conversely, when phosphotyrosine-containing proteins were precipitated, the 73-kDa ABCG2 isoform was associated with the plasma membrane fraction. On the other hand, only the 63-kDa ABCG2 isoform was detected in protein extracts from ejaculated spermatozoa (Fig. 7C). Importantly, no signal could be detected in immunoprecipitated ABCG2 probed with anti-phosphotyrosine antibodies and vice versa, indicating that this ABC transporter is not tyrosine phosphorylated in ejaculated sperm.

These results demonstrate that although ABCG2 is localized in the lipid raft plasma membrane microdomains of epididymal and ejaculated spermatozoa, tyrosine phosphorylation status of the pump appears to be lost at the time of or immediately following ejaculation.

ABCG2 Subcellular Localization in Ejaculated Sperm

In somatic cells, ABCG2 is a membrane transporter, and tyrosine phosphorylation is responsible for its localization at the plasma membrane. Because permeabilization protocols were required to expose the antigen in ejaculated spermatozoa (Fig. 8A), additional studies were carried out to assess the subcellular localization of ABCG2 in intact cells. Immunoelectron localization of ABCG2 in ejaculated spermatozoa (Fig. 8B) showed that the transporter is localized in the plasma membrane as well as in the cytosol and the acrosomal matrix. These results suggest that after ejaculation, a subpopulation of ABCG2 is exported from the plasma membrane to the cytosol and the acrosomal matrix.

FIG. 8. .

FIG. 8. 

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Ejaculated sperm ABCG2 subcellular localization. A) Indirect immunofluorescence localization of ABCG2 in permeabilized or nonpermeabilized ejaculated spermatozoa. Control conditions: normal rat IgG. Nuclei are counterstained with DAPI. Bar = 10 μm. Results are representative of two independent experiments. B) Immunoelectron microscopy and localization of ABCG2 in ejaculated spermatozoa revealed with anti-rat 12-nm gold particle-labeled antibodies. The lower inset shows a higher magnification of the acrosomal region. Arrows indicate immunolabeling. Bar = 0.5 μm.

Coincubation of Spermatozoa with Prostasomes

At the time of ejaculation, epididymal spermatozoa encounter various components found in seminal plasma. We thus hypothesized that prostasomes might regulate epididymal sperm ABCG2. Incubation of cauda epididymal spermatozoa with cauda epididymosomes induced no detectable change in sperm ABCG2 content, whereas prostasomes caused a significant decrease (P < 0.05) in Triton X-100-extractable ABCG2 content (Fig. 9A).

FIG. 9. .

FIG. 9. 

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Coincubation of spermatozoa with prostasomes. A) Semiquantitative analysis of ABCG2 immunoblotting from 1 × 107 cauda epididymal spermatozoa after a 60-min incubation without (Cd-Sptz) or with cauda epididymosomes (Cd-Ep), or with prostasomes (Prost). Data (arbitrary units) were analyzed by t-test using control conditions as 100%. Values with different superscripts are significantly different (P < 0.05). B) Semiquantitative analysis of ABCG2 immunoblotting from 1 × 107 cauda epididymal spermatozoa after a 60-min incubation without (Cd-Sptz) or with prostasomes (Prost), prostasome-free seminal plasma fluid (Fluid), or complete seminal plasma (SP). Data (arbitrary units) were analyzed by t-test using control conditions as 100%. Values with different superscripts are significantly different (P < 0.05).

To assess whether this effect was exclusive to the prostasome fraction, we compared the effect of incubating cauda epididymal spermatozoa with various seminal plasma fractions, namely, prostasomes, prostasome-free seminal plasma, and whole seminal plasma. A decrease (P < 0.05) in the amount of sperm ABCG2 was only observed upon coincubation with the prostasome fraction (Fig. 9B), in support of the notion that the reduction in ABCG2 content was caused by the prostasomes contained in seminal plasma.

DISCUSSION

When spermatozoa are expulsed from the testis, their transcriptional and translational machineries are silenced. However, during epididymal transit, sperm cells undergo several modifications that are responsible for acquisition of full fertilizing ability [1]. Several of these changes are under the control of the epididymal milieu that results from the active secretory activity of the epithelial cells that border the epididymis [33]. Among these epididymal sperm modifications, remodeling of the plasma membrane may be considered to be one of the most important events.

Cholesterol and cholesterol sulfate are plasma membrane components that are responsible for creating nucleating centers for the formation of lipid and protein clusters (or lipid rafts), and membrane stabilization, respectively [34, 35]. Spermatozoa have been shown to contain the enzymatic components (i.e., sulfotransferases) to produce cholesterol sulfate [3]. ABC transporters are key actors for the membrane transport of a wide variety of substrates, including various lipids types. Using the free energy released by ATP hydrolysis [36], ABC transporters can translocate phospholipids or cholesterol across the membrane [37]. ABCG2 displays a particularly high affinity for sterols [5] and sulfate conjugates formed intracellularly by sulfotransferases [6].

Several members of the ABC transporter family have been found in spermatozoa from different species [38–41]. Among them, ABCB1 has been recently reported to be acquired by mouse sperm during epididymal transit toward distal regions of the epididymis [42]. ABCG2 is present and active in mouse spermatogonia [43] and has been localized in the acrosomal region of cauda epididymal spermatozoa in mouse and rat as well as human and bull ejaculated spermatozoa [21]. In the male reproductive tract, ABCG2 has been detected in the endothelial cells and luminal membrane of testis and in the luminal and abluminal sides of caput epididymal ducts and in corpus endothelial cells [44]. Even the absence of the transporter in the knockout mouse model did not demonstrate to affect reproductive functions [45], redundant mechanisms for transporting these compounds through the plasma membrane could exist.

The present study is the first to report the expression of ABCG2 in bovine epididymis as well as in epididymal and testicular spermatozoa. ABCG2 is more specifically associated with the plasma membrane neighboring the acrosomal region of spermatozoa from the time of sperm formation in the testis, and it remains present in this region during epididymal transit. The presence of a functional ABCG2 in the acrosomal region suggests its possible involvement in the translocation of cholesterol sulfate. Indeed, the latter compound, like EST [3], has been found in the acrosomal region of spermatozoa [2].

Previous studies from our laboratory have demonstrated that in bovines, specific proteins can be selectively transferred from epididymosomes to maturating spermatozoa. Examples of such proteins are the glycosylphosphatidylinositol-anchored protein P25B, macrophage migration inhibitory factor, and aldose reductase (AKR1B1) [46]. The fact that ABCG2 was also found in epididymosomes, as well as the fact that the expression pattern of the transporter protein in epididymosomes from different epididymal sections followed the same distribution as that observed in spermatozoa recovered from the respective epididymal regions, led to the hypothesis that epididymosomes might control the differential expression of ABCG2. However, no detectable ABCG2 was transferred from the corpus or cauda epididymosomes to the caput spermatozoa as determined in vitro with coincubation experiments. Nevertheless, the possible transfer of ABCG2 from epididymosomes to spermatozoa cannot be ruled out completely because in vitro experiments may not faithfully mimic the maturation process of sperm in the epididymal duct, which extends over several days physiologically.

Because epididymosomes are formed through apocrine secretion of epididymal epithelial cells, ABCG2 expression was thus analyzed in epididymal tissues. We found that ABCG2 mRNA and protein contents both increased in the proximal regions of the corpus and cauda, with a corresponding decrease in distal regions. Because spermatozoa and epididymosomes were recovered from distal caput, intermediate corpus, and distal cauda, the level of ABCG2 expression in spermatozoa and epididymosomes provides a reliable measure of the tissue pattern of expression. Furthermore, ABCG2 expression is known to be regulated by sex hormones. Thus, ABCG2 is upregulated by testosterone [47] and downregulated by estrogens [48]. This response is in agreement with both the decrease in estradiol concentration found in the proximal segments of epididymis [49] and the important and well-known effect of androgens on epididymal duct protein expression [50]. Again, the origin of epididymosomes via their apocrine secretion by epididymal epithelial cells argues in favor of the possible transfer of ABCG2 protein from the former to the latter compartment. Moreover, the high ABCG2 protein expression pattern observed in cauda epididymis corresponds to the microcompartmentalization of ABCG2 in the apical pole of these cells, which is the region involved in the apocrine secretion of epididymosomes. On the other hand, ABCG2 has been reported to be specifically localized in the apical membrane of polarized cells, where it has been involved in primary functions, such as detoxification and multidrug transport [19]. Likewise, ABCB1 [42] and ABCG1 [51] have been localized in the apical pole of epididymal epithelial cells in different epididymal segments in rat and mouse, respectively. These observations suggest that by analogy with the epididymis, one must consider the alternative hypothesis that the periacrosomal localization of ABCG2 in maturing spermatozoa might arise from the apical expression of that protein in the elongating spermatids. According to that view, the role of epididymosome-associated ABCG2 would thus remain to be determined. Furthermore, the above considerations raise the possibility that ABCG2 might play a role in detoxification in the epididymis.

At the time of ejaculation, cauda epididymal spermatozoa move into seminal plasma, where changes associated with the early events of capacitation have been shown to take place [24]. The fact that protection afforded by cholesterol sulfate is no longer required and this conjugate is excreted from sperm cells suggests that ABCG2 might also become futile at or near that stage. In fact, we observed that although ABCG2 is still present in ejaculated spermatozoa in agreement with the results published by Scharenberg et al. [21], it appears to be nonfunctional, compared with parallel determinations in epididymal spermatozoa. This result is not consistent with those results reported by Scharenberg et al. [21], who showed that ABCG2 is functional in ejaculated spermatozoa using a similar experimental approach.

Because the inclusion of ABCG2 in membrane lipid raft microdomains [52, 53] and the presence of cholesterol in the plasma membrane [10, 11] are necessary for ABCG2 activity in somatic cells, the presence of this pump in lipid rafts was determined in epididymal and ejaculated spermatozoa. However, because ABCG2 was indeed found to be localized in lipid rafts during their transit through the epididymis as well as after ejaculation, the lack of ABCG2 pump activity could not be attributed to a mislocalization of this transporter. In support of these results, we observed that ABCG2 was always present in the acrosomal region of the plasma membrane, where cholesterol [54] and membrane lipid raft domains [55] are localized in ungulates.

The functionality of ABCG2 has also been correlated to its tyrosine phosphorylation. This modification favors membrane presentation and might be one of the molecular conformations that protect this protein from degradation [13]. We observed that whereas plasma membrane ABCG2 is phosphorylated in epididymal spermatozoa, this is not the case for spermatozoa after ejaculation, a finding that might also explain the higher intracellular ABCG2 contents found in ejaculated spermatozoa. Immunoreactive ABCG2 present in the plasma membrane fraction of epididymal sperm was found under isoforms with different molecular weights. This might be explained by the different detergent that was used to extract proteins, which likely preserves ABCG2 homodimers [56] and homotetramers [57], or by differences arising from the glycosylated state. However, N-glycosylation has been shown to be dispensable for ABCG2 expression, trafficking to the plasma membrane, or overall function [58].

At the time of ejaculation, epididymal proteins are exposed to seminal plasma. In the current study we have demonstrated that the seminal plasma fraction containing prostasomes could decrease the amount of ABCG2 extractable with Triton X-100 from ejaculated spermatozoa. We considered the hypothesis that prostasomes might induce ABCG2 dephosphorylation, based on previous reports that among the phosphatases found in the human prostasome proteome, prostatic acid phosphatase behaves as a protein tyrosine phosphatase [59]. Because tyrosine dephosphorylation causes a redistribution of ABCG2 to other intracellular compartments, it might account for the inability of Triton X-100 to extract this transporter at the plasma membrane level. We further observed that a 1-h incubation of ejaculated sperm with prostasomes was necessary to observe a reduction in plasma membrane ABCG2 content. Although early capacitation-associated processes, such as cholesterol removal from ejaculated spermatozoa, occur relatively soon after ejaculation [24], it has been demonstrated that prostasome proteins are transferred to spermatozoa after a 1-h contact with prostasomes [60].

In summary, ABCG2 is present in the acrosomal region of bull spermatozoa from spermiation in the testis until ejaculation. Although sperm ABCG2 is functional during epididymal transit, molecular modifications that occur at the time of ejaculation inactivate it. Results presented in this study suggest that dephosphorylation of its tyrosine residues promotes its partial dissociation from the plasma membrane and loss of function. Because these changes are accompanied by prostasome-mediated ABCG2 partial removal, it is suggested that these membranous vesicles could have a role in the regulation of the molecular function of sperm ABCG2 after ejaculation. These results support the hypothesis that ABCG2 may have an important role in the translocation of plasma membrane cholesterol sulfate during epididymal maturation.

ACKNOWLEDGMENT

The authors wish to thank Alliance Semex Inc. (CIAQ Inc., St-Hyacinthe, QC, Canada) for providing the fresh semen samples and for technical assistance in flow cytometric analysis of the samples. We are also grateful to S. Goupil, E. Poirier, and C. Lachance for their very helpful technical assistance. Drs. Pierre Leclerc and Richard Poulin are acknowledged for critical reading of the manuscript.

Footnotes

1

Supported by grants from the Canadian Institutes for Health Research and the Natural Sciences and Engineering Research Council of Canada (R.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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