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From the Centre de Recherche en Biologie de la Reproduction and Département d'Obstétrique-Gynécologie, Faculté de Médecine, Université Laval, Quebec City, Quebec, Canada.
| Correspondence to: Dr Robert Sullivan, Unité d'Ontogénie-Reproduction, Centre de Recherche du Centre Hospitalier de l'Université Laval (CHUQ), 2705 boulevard Laurier, T1-49, Quebec City, Quebec, Canada G1V 4G2 (e-mail: robert.sullivan{at}crchul.ulaval.ca). |
| Received for publication August 25, 2008; accepted for publication February 16, 2009. |
Estrogen is found in high concentrations in the excurrent duct, where it
regulates the expression of genes involved in water reabsorption. Estrogen
sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific
sulfonation with a high affinity for estrogens. Because sulfated estrogens do
not bind to estrogen receptors, they are considered to be hormonally inactive.
EST may thus determine where along the male tract estrogenic environment
predominates. Sulfotransferase activity increases along the epididymis and may
also play a role in sperm physiology during the epididymal transit. Using a
bovine model, we investigated the distribution of EST along the excurrent duct
and the possibility that sterols associated with spermatozoa can be substrates
of this enzyme. Reverse transcription polymerase chain reactions showed that
mRNA encoding EST was expressed in the testis and all along the
epididymis. A highly specific antiserum was raised against the bovine
recombinant EST and used in Western blots and immunohistologic studies.
Western blots of tissue homogenates showed that EST was localized all along
the excurrent duct with a higher signal in the caput and corpus epididymidis.
EST was detectable in the intraluminal compartment only in the caput
epididymidis, where it was associated with epididymosomes and spermatozoa. EST
was undetectable in different fractions of fluids collected in the cauda
segment. In immunohistologic studies, EST was restricted to the acrosomal
region of the caput, but not the cauda epididymal spermatozoa, and detectable
in the cytoplasm of the epithelium bordering the lumen all along the
epididymis as well as in the rete testis and vas efferens. This enzyme was
also associated with the nucleus in the caput and corpus as well as with the
apical membrane of the corpus epididymal epithelium. When recombinant EST was
incubated in vitro in the presence of caput and cauda spermatozoa, it was able
to add sulfate to sperm membrane cholesterol. Our study shows that EST is
present in both the intracellular and intraluminal compartments of the
epididymis, suggesting that this enzyme plays different roles along the
excurrent duct.
Key words: Reproductive tract, sperm maturation, cholesterol, cholesteryl sulfate
High concentrations of testosterone reach the intraluminal compartment in
the proximal region of the epididymis. Testosterone is rapidly reduced by 2
steroid 5
-reductase isoforms, assuring a strong androgenic environment
in the proximal region of the excurrent duct
(Viger and Robaire, 1996). The
androgens support high protein synthesis and secretion activities of
epididymal principal cells necessary for sperm maturation and transport. In
man, estradiol is secreted by the testis and is found in high concentration in
semen (Claus et al, 1985;
Claus et al, 1992). It may also
play a role in the female genital tract, at least in porcine species
(Claus et al, 1987). Estrogen
in the male reproductive tract is thought to be synthesized by Leydig cells
(Hess et al, 2001) and by
epididymal spermatozoa (Hess et al,
1995), both cells having aromatase activity. Estrogen present in
the male tract regulates the expression of genes involved in water
reabsorption mainly in the vas efferens as well as in the epididymis
(Hess et al, 1997;
Hess, 2000;
Hess et al, 2001;
Bili
ska et al,
2006).
Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes specific sulfonation with a high substrate affinity for estrogens, including estrone and estradiol (Gamage et al, 2006). Because sulfated estrogens do not bind to estrogen receptors, they are considered to be hormonally inactive (Song et al, 1995). Leydig cells are characterized by a high EST activity (Hobkirk and Glasier, 1992; Song et al, 1997), which is under luteinizing hormone and androgen control (Song, 2001). EST activity has also been reported in principal cells of the distal epididymis as well as in the luminal epithelium and smooth muscle of the vas deferens in mouse (Tong and Song, 2002). Because EST is involved in estrogen metabolism, its expression in the male reproductive tract determines where along the tract an estrogenic environment predominates.
Sulfotransferase activity increases along the epididymis as a consequence of its local synthesis (Bouthillier et al, 1984). Cholesterol (CHO) and desmosterol are the major sulfated sterols in the epididymis of human and hamster, respectively. During epididymal maturation, these sulfated sterols accumulate in the plasma membrane covering the acrosome of spermatozoa (Legault et al, 1979; Langlais et al, 1981). Because sulfated sterols are potent inhibitors of capacitation (Bleau et al, 1975), it is hypothesized that they provide protection against premature release of sperm acrosomal proteases within the male tract (Roberts, 1987). EST may also be involved in sperm physiology during the epididymal transit. This is based on previous observations from our laboratory showing that EST is secreted by epididymal epithelial cells in primary culture and binds to sperm when the latter are exposed to the primary cell culture medium (Reyes-Moreno et al, 2002). Using a bovine model, we investigated the distribution of EST along the excurrent duct and the possibility that sterols associated with spermatozoa can be substrates of this enzyme.
Materials and Methods
Biologic Material
Epididymides from sexually mature bulls were obtained from a slaughterhouse
(Frenette et al, 2002).
Immediately after slaughtering, testicles were kept on ice and brought to the
laboratory within 2 hours. Epididymides were dissected and cleared from
connective tissues. Only epididymides with swollen appearance and motile cauda
epididymal spermatozoa were used in this study. Upon arrival, small pieces of
tissues were dissected on ice and processed for RNA extractions. For
immunohistologic studies, pieces of tissues were fixed in 4% paraformaldehyde
in phosphate-buffered saline (PBS) for 24 hours before embedding in paraffin.
Tissues from different epididymal segments were homogenized in 25 mM Tris
buffer (pH 7.5) containing 50 mM NaCl and 1 mM phenyl methyl sulfonyl fluoride
(PMSF), followed by centrifugation at 20 000 x g for 30
minutes. The supernatant protein solutions were used for Western blot
analysis. Protein concentrations were determined by the Bradford method
(Bio-Rad protein assay kit; Bio-Rad Laboratories Inc, Hercules,
California).
Fluid from the cauda epididymidis was collected by retrograde flushing, and fluid from the caput was obtained by neatly cutting a few tubules and applying pressure to the proximal portion of the dissected caput. Only intraluminal fluids devoid of blood or tissue contamination were further processed. Epididymosomes presenting with a reddish color were discarded. Fluid from the corpus segment was not collected because of the difficulty of obtaining uncontaminated samples. Spermatozoa and epididymosomes were prepared from caput and cauda epididymal fluids as previously described (Frenette and Sullivan, 2001). Briefly, epididymal fluids were diluted with 150 mM NaCl, spermatozoa were pelleted by centrifugation at 700 x g for 5 minutes and washed twice. The supernatants were centrifuged twice at 3000 x g for 20 minutes, and then the supernatants (epididymal fluids [F]) were ultracentrifuged at 120 000 x g for 2 hours to pellet epididymosomes. The supernatants were considered the soluble protein fractions (S) secreted in the intraluminal compartment of the caput or cauda epididymidis. The pellets were resuspended in 150 mM NaCl and submitted to chromatography on a Sephacryl S-500HR gel (Pharmacia, Baie d'Urfé, Canada). The void volumes were ultracentrifuged at 120 000 x g for 2 hours to pellet epididymosomes (Frenette et al, 2006). This method of epididymosome preparation yields a pure suspension of vesicles with diameters ranging from 20 to 500 nm (Rejraji et al, 2002; Frenette et al, unpublished data).
Detection of EST Transcripts in Testicular and Epididymal Tissues
Reverse-transcribed EST cDNAs from testis and different epididymal
segments were amplified using 5'-CTC CTT CAT GTC TTC ATA GA-3' and
5'-ATT ACC TGG AAT GTA GCA C-3'. The amplified fragment of 356 bp
was directly sequenced by the core facilities of our institution. A
glyceraldehyde-3-phosphate dehydrogenase cDNA fragment was used as a
control.
Cloning and Protein Production of Bovine Recombinant EST
Total RNAs extracted from bovine testicular tissues were
reverse-transcribed using SuperScript II (Invitrogen Corp, Carlsbad,
California). 5'-CGA TGA GTT CTT CCA AAC CAT CC-3' and 5'-CCT
AGA TCT TAG TTC GGA AC-3' forward and reverse primers, respectively,
were designed to amplify a full-length cDNA of bovine EST. The
polymerase chain reaction (PCR) conditions were as follows: initial
denaturation at 95°C for 5 minutes followed by 40 cycles of denaturation
at 94°C for 1 minute, annealing at 55°C for 1 minute, and extension at
72°C for 1 minute. The PCR product was purified using the QIAquick PCR
purification kit, cloned in pGEM-T (Promega Corp, Madison, Wisconsin), and
sequenced by the core facilities of our institution. Another set of primers
containing the NdeI and BamHI restriction sites in the
forward and reverse primers, respectively, were used to amplify the pGEM
clone, and the amplified EST cDNA was transferred into the pET16b
plasmid (Novagen, La Jolla, California).
BL21 CD+ bacteria were transformed with the pET16b-EST plasmid, and His-tagged recombinant protein overexpression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were sonicated in 20 mM Tris, 500 mM NaCl (pH 8.0) (buffer A), and the soluble (His)10-EST (rec-EST) was purified on ProBound resin according to the supplier's instructions (Invitrogen). Briefly, the clear supernatant was applied on a 1-mL ProBound resin column. The column was washed successively with 3 volumes of buffer A containing 60 mM, 100 mM, and 250 mM imidazole. Finally, the rec-EST was eluted in buffer A containing 500 mM imidazole. The purified rec-EST was dialyzed against 150 mM NaCl and stored at –80°C until used.
Anti–rec-EST Antibody Production and Purification
New Zealand albino rabbits were immunized with 300 µg of purified
rec-EST in 0.15 M NaCl mixed with an equal volume of Freund complete adjuvant.
Boost injections were given each 4 to 5 weeks with 150 µg of rec-EST
emulsified in incomplete adjuvant. Blood samples were collected by cardiac
puncture, and the serum was recovered after clotting. Approximately 4 mg of
purified rec-EST was covalently linked to 4 mL of CNBr-activated Sepharose
(Pharmacia). This affinity column was used for the purification of specific
anti-EST IgGs using standard procedures.
Western Blotting
Protein from 20 x 106 spermatozoa extracted with 0.3%
Triton X-100, 25 µg of protein from epididymosomes or from epididymal
fluids, and 30 µg of protein homogenates from epididymal tissues were
subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and electro-transferred onto nitrocellulose membrane. The detergent
treatment used solubilizes the total immunodetectable EST associated to
spermatozoa (data not shown). After blocking with 5% dry skim milk in PBS/0.1%
Tween, the blots were incubated for 2 hours with purified rabbit IgG
anti–rec-EST in 1.0 µg/mL of blocking solution. After several washes,
the blots were incubated for 1 hour with a goat anti-rabbit IgG coupled to
peroxidase diluted 1:2000 and revealed using ECL substrate (Amersham,
Buckinghamshire, United Kingdom). Probing of the Western blots with a
monoclonal antibody against β-actin (Sigma-Aldrich, St Louis, Missouri)
at 1:5000 dilution was used as a loading control.
To test the specificity of the antibodies, 40 µg of purified anti–rec-EST IgGs were preincubated with 0, 10, or 50 µg of rec-EST in 150 µL of PBS containing 0.1% NaN3 for 2 hours at room temperature and then overnight at 4°C. The adsorbed antibody was diluted to 1.0 µg/mL in blocking solution and used to probe Western blots as described previously.
Immunolocalization of EST
Rete testis, vas efferens, and epididymal tissues were rapidly fixed in 4%
neutralized paraformaldehyde and embedded in paraffin. Paraffin sections were
cleared by immersion in xylene and rehydrated with successive baths of
ethanol. Endogenous peroxidases were neutralized with 3% hydrogen peroxide in
methanol for 10 minutes. Six-micrometer histologic sections were blocked by
incubation with 5% goat serum in PBS and incubated at 4°C overnight with 5
µg/mL anti–rec-EST IgGs in PBS supplemented with 0.5% goat serum.
After washing, tissues sections were incubated with biotinylated goat
anti-rabbit IgG and processed for staining using the ABC Vectastain kit
(Vector Laboratories, Burlingame, California). Negative controls were
performed using purified rabbit IgGs.
Caput and cauda spermatozoa washed twice in PBS were smeared on microscopic slides, fixed with 3.7% formaldehyde in PBS, blocked with 1% bovine serum albumin in PBS, and incubated with purified EST antibody at 10 µg/mL for 2 hours at 37°C. The immune complexes were visualized with a goat anti-rabbit IgG coupled to Alexa-Fluor 568 (Invitrogen).
EST Enzymatic Activity
Purified rec-EST was incubated at 37°C for 2 hours in 500 µL of
reaction solution containing MES/PIPES (10 mM each; pH 6.9), 2 mM
MgCl2, 150 mM NaCl, 1 µCi phosphoadenosine phosphosulfate
(PAP35S; specific activity = 1.72 Ci/mmol [Perkin-Elmer; Boston,
Massachusetts]), and 10 nmol estradiol in 2% ethanol as EST's substrate. In
control experiments, rec-EST was replaced by Niemann-Pick type C2 protein
(NPC2) purified as previously described
(Girouard et al, 2008). NPC2
is secreted by the epididymal epithelium and is characterized by a CHO pocket
(Friedland et al, 2003). At
the end of incubation, the reaction mixture was extracted with 6 volumes of
CHCl3:methanol (2:1) and an aliquot was counted by liquid
scintillation.
To test the possibility that rec-EST can sulfonate sperm membrane sterols, 25 to 30 x 106 spermatozoa collected from the caput or cauda epididymal fluids were incubated in the same enzymatic reaction conditions without estradiol and ethanol. The reaction solutions containing spermatozoa were extracted twice with 6 volumes of CHCl3:methanol (2:1), dried under nitrogen stream, and analyzed by high-performance thin-layer chromatography (HPTLC) using methyl acetate: isopropanol:chloroform:methanol:0.25% KCl in water (25:25: 25:10:9) as migration solvent (Vitiello and Zanetta, 1978). The 35S incorporation was visualized by exposing the plate with Kodak x-ray film. The plates were colored by dipping in a solution of 10% CuSO4 (wt/vol) and 8% H3PO4 (vol/vol) and heated at 180°C for 15 to 30 minutes. Phospholipids like sphingomyelin, phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine as well as CHO and cholesteryl sulfate (CH-S) were run on HPTLC as controls.
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At the transcriptional level, EST was detectable in testicular tissues as well as all along the epididymis. The amplified fragment appeared to be slightly more intense in the caput and corpus epididymidis (Figure 1).
When induced with IPTG, BL21CD+ bacteria transformed with pET16b-EST plasmid sonicated and subjected to SDS-PAGE produced a protein of 37 kd (data not shown). The cDNA encoding rec-EST showed 100% homology with the bovine EST SULT1E1 cDNA; the first known sulfotransferase cloned (Nash et al, 1988). This rec-EST was used to produce a rabbit polyclonal antiserum. When the antiserum was used to probe tissue homogenates from the male reproductive tract, a 35-kd protein was detected in the epididymal tissue samples. An intense signal was detected in the caput and corpus epididymal tissues, decreasing in the cauda epididymidis and vas deferens (Figure 2B). EST was also detectable at a much lower level in testicular tissue homogenates (Figure 2C). The antiserum used to detect this protein was highly specific as shown by the ability of rec-EST to eliminate the immune complex signal when used to preadsorb the anti–rec-EST IgGs (Figure 2D).
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The specific anti–rec-EST IgGs were used in immunohistologic studies to investigate the cellular origin of EST along the epididymis. All along the epididymis, EST was immunodetectable at a low level in the smooth muscle layer surrounding the epididymal tubule (Figure 4). In the caput epididymidis, EST was localized in the principal cells as a uniform cytoplasmic labeling as well as in association with the nucleus. Patchy staining of the EST protein was detected in the apical regions of the caput epithelium. EST was also associated with punctate structures in the caput and corpus lumen; association with these structures was absent in the rete testis and cauda and vas deferens. In the corpus, EST was also uniformly distributed in the cytoplasm, together with strong labeling of the principal cell apical pole. Only a few nuclei were labeled. In the cauda, cytoplasmic EST was detectable at a lower intensity when compared with that in the caput and corpus epididymidis (Figure 4). In addition, the luminal epithelium of the rete testis was strongly labeled when histologic sections were probed with the anti–rec-EST antibodies. The vas efferens epithelium bordering the intraluminal compartment also expressed EST (Figure 5).
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Discussion
EST is known to be expressed by Leydig cells in murine species (Song et al, 1995; Song et al, 1997; Song, 2001; Tong and Song, 2002; Luu-The et al, 2005) and is thought to protect steroidogenic cells from estrogen-induced biochemical sequelae (Tong et al, 2004). EST-encoding cDNA can be amplified from bovine testicular tissue homogenates. Very low levels of testicular EST are detectable by Western blots, probably because Leydig cells represent a low percentage of the testicular tissue mass. In bovine, the weak expression of testicular EST compared with its expression along the epididymis suggests that EST originating from the gonads contributes modestly to the EST detected in high concentration in the epididymal lumen. EST has also been shown to be expressed in the epithelium of the mouse corpus and cauda epididymidis but not in the caput regions (Tong and Song, 2002). In bovine epididymis, unlike what is found in the mouse, EST is detectable by immunohistochemistry all along the excurrent duct, with a higher intensity in the proximal regions. This enzyme belongs to the class of cytosolic sulfotransferases responsible for the metabolism of small endogenous substrates such as natural and synthetic steroids (Gamage et al, 2006). As in murine species, bovine EST is detectable in the cytoplasm of principal cells all along the epididymis. In the caput, it is also observed in some nuclei, and strong staining is associated with the apical membrane of principal cells in the corpus epididymidis. This variation of subcellular localization along the epididymis suggests that EST may play different functions along the excurrent duct.
Estrogen plays a major role in the male reproductive tract, regulating water reabsorption especially in the proximal portion of the excurrent duct (Hess et al, 1997; Hess, 2000; Hess et al, 2001). This correlates with high concentrations of estradiol in the intraluminal compartment of the excurrent duct (Hess et al, 2001) as well as in semen (Claus et al, 1987; Claus et al, 1992). For many years, sulfation has been considered a major metabolic pathway for circulating estrogens (Strott, 1996). Sulfated estrogens have greatly reduced receptor-binding affinity (Gamage et al, 2006). High EST expression in the male reproductive tissues thus suggests that this enzyme plays a role in male tissue protection against estrogenic activity once water reabsorption is completed (Tong et al, 2004; Luu-The et al, 2005). On the other hand, sulfated sterols are more hydrophilic and may exist as a circulating reservoir that can be hydrolyzed in the active hormone (Strott, 1996). This could be the reason why EST is strongly expressed in the rete testis. Leydig cells are responsible for the synthesis of testicular estrogens owing to their aromatase activity. Whether the rete testis metabolizes estrogens to protect the excurrent duct from the estrogenic environment or provides a reservoir of hydrolyzable estrogens remains to be determined. The vas efferens may also be involved in this process because they also strongly express EST.
Cytoplasmic EST is expressed all along the excurrent duct, and an intraluminal EST form is detected in the caput epididymidis but not in the cauda, suggesting that this enzyme plays a different role in the proximal part of the epididymis. In this segment, EST present in the intraluminal fluid fraction is associated with the epididymosomes, the soluble protein fraction, and at a lower level, the spermatozoa. The EST in the caput epididymal intraluminal compartment is probably the result of apocrine secretion activity by the principal cells of this segment. This process consists of the formation of cytoplasmic blebs at the apical membrane of the epithelial cells. These blebs detach from the cells and release their content in the intraluminal compartment when they disintegrate (Aumuller et al, 1999). This process is well described in the epididymis and is involved in the secretion of cytoplasmic components, including small membranous membrane vesicles named epididymosomes (Hermo and Jacks, 2002; Rejraji et al, 2002; Sullivan et al, 2005). Apocrine secretion is probably involved in the secretion of the principal cell cytoplasmic form of EST as well as the one associated with epididymosomes. Epididymosomes are involved in the transfer of some selected epididymal secreted proteins to the transiting spermatozoa (Saez et al, 2003; Sullivan et al, 2007). These small vesicles may be responsible for the association of EST with spermatozoa in the caput epididymidis. The intraluminal EST in the caput epididymidis may play a different role than the one played by the cytoplasmic EST present all along the excurrent duct. Its transient association with spermatozoa in the proximal region of the epididymis suggests that it may be involved in sperm maturation.
EST shows a high affinity for estradiol and estrone (Falany, 1997). In fact, bovine rec-EST shows a much greater (>100x) affinity for estradiol than for CHO in an enzymatic in vitro assay (data not shown). Even though it is weak, its affinity for CHO may be relevant to the epididymis physiology. In fact, the enzyme can sulfonate membrane CHO of caput as well as cauda spermatozoa. Sterol sulfonation has been known for many years to be part of the maturation process of spermatozoa during their transit along the epididymis (Roberts, 1987). CH-S stabilizes plasma membranes, protecting spermatozoa against sequelae that can occur during their transit along both male and female genital tracts (Legault et al, 1979; Langlais et al, 1981). The involvement of epididymal EST in sperm membrane CHO sulfonation would be restricted to the caput segment of the epididymis even though rec-EST is able to sulfonate cauda epididymal sperm CHO in an in vitro assay. Interestingly, the association of EST to caput spermatozoa is restricted to the acrosomal cap. If the sperm-associated EST is involved in CHO sulfonation, this may represent a mechanism of sperm protection against premature acrosome reaction or loss as already proposed by Roberts (1987). At least in humans, sterol sulfate is localized on the sperm plasma membrane, mostly in the region of the acrosome (Langlais et al, 1981). Once sterol sulfonation is completed, the EST-sperm association would not be required anymore, explaining why cauda spermatozoa are devoid of this enzyme. We cannot exclude however that other sulfotransferases are involved in sperm membrane CHO metabolism within the epididymis. It remains that the sulfotransferase involved in sulfonation of sperm membrane CHO occurring during the epididymal transit has to be present in the intraluminal compartment to be part of this process. The dual distribution of EST in the caput epididymidis supports the involvement of this enzyme in sperm maturation. EST functions along the epididymis may be even more complex if we consider its nuclear localization and its strong association with the apical membrane of corpus epididymal principal cells.
Based on EST tissue distribution, this study suggests that this enzyme plays different roles in the epididymis. The cytoplasmic EST distributed all along the excurrent duct would be responsible for estrogen metabolism, whereas the intraluminal EST restricted to the caput epididymidis could be involved in sperm CHO sulfonation occurring during the maturation process.
Acknowledgments
We wish to acknowledge Dr R. Oko from Queen's University (Kingston, Ontario, Canada) for advice in identification of rete testis and vas efferens histologic sections, as well as Mrs Julie Girouard for her help in preparing the biologic material.
Footnotes
Supported by grants from Natural Sciences and Engineering Research Council of Canada and L'Alliance Boviteq Inc (St-Hyacinthe, Quebec, Canada) to R.S. and P.L.
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