Mol. Hum. Reprod. Advance Access originally published online on March 21, 2007
Molecular Human Reproduction 2007 13(5):307-316; doi:10.1093/molehr/gam012
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Identification and localisation of SERCA 2 isoforms in mammalian sperm
1 Département d'Obstétrique/Gynécologie, Centre de Recherche en Biologie de la Reproduction, Université Laval and Ontogénie et Reproduction, Centre de recherche du CHUQ-CHUL, 2705 boul. Laurier, Sainte-Foy, Québec, QC, Canada G1V 4G2
2 To whom correspondence should be addressed at: Tel: +1-418-525-4444 ext. 46267; Fax: +1-418-654-2765; E-mail: pierre.leclerc{at}crchul.ulaval.ca
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Abstract |
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Upon binding to the egg's zona pellucida, capacitated spermatozoa will undergo a calcium-dependent exocytotic event called acrosome reaction. During this process, Ca2+ depletion from internal stores is followed by an important rise in [Ca2+]i due to a massive Ca2+ influx. Previous reports have shown that the acrosome can act as a Ca2+ store and that depletion of thapsigargin-sensitive stores induces acrosome exocytosis in capacitated spermatozoa from different mammalian species. The effect of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCAs), suggests the presence and implication of SERCA in the active Ca2+ uptake during mammalian sperm capacitation. Although the presence of a thapsigargin-sensitive Ca2+-ATPase has been debated, the aim of this study was to clearly determine whether SERCAs are present in mammalian spermatozoa. Using three different anti-SERCA 2 antibodies, mono- and polyclonal, which recognised the same protein, we successfully identified and localised SERCA 2 in human, mouse and bovine sperm. Western blot analysis suggests that more than one SERCA 2 splice variant are present, one detected in the fraction containing the outer acrosomal membranes and another one present in the subcellular fraction containing the sperm midpiece. These results were confirmed by indirect immunofluorescence where SERCA 2 was observed in the acrosome and midpiece regions of human sperm. SERCA 2 immunohistochemical studies on human testis and PCR-amplification of mRNA encoding for each SERCA 2 splice variant in spermatogenic cells support the presence of this Ca2+-ATPase family in mature spermatozoa. In this paper, we clearly demonstrate, for the first time, the presence of SERCA 2 in mammalian sperm.
Key words: Ca2+-ATPase/calcium/capacitation/spermatozoa/intracellular stores
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Introduction |
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Calcium gradients are of major importance to spermatozoa. They are crucial during sperm transit in the female genital tract so that the spermatozoa can use Ca2+ as a signalling molecule in the different pathways occurring prior to or leading to fertilisation. Upon binding to the egg's zona pellucida, capacitated spermatozoa will undergo a calcium-dependent exocytotic event called acrosome reaction. During this process, Ca2+ depletion from internal stores is followed by an important rise in [Ca2+]i due to a massive Ca2+ influx from the extracellular medium (O'Toole et al., 2000; Rossato et al., 2001). It was previously observed during sperm capacitation that there is a smaller increase in the intracellular free Ca2+ concentration compared with the net Ca2+ uptake (Handrow et al., 1989; Baldi et al., 1991). These findings suggest that part of the calcium influx during capacitation is accumulated in intracellular stores in order to be emptied when acrosomal exocytosis is triggered (Baldi et al., 1991). However, in mature sperm cells, there is no endoplasmic reticulum (ER) or Golgi apparatus, which are usually known to act as internal Ca2+ stores in somatic cells. Many potential stores have been suggested to play a role in [Ca2+]i in mammalian sperm such as the cytoplasmic droplet, the redundant nuclear envelop and the mitochondria in the midpiece (Kuroda et al., 1999; Naaby-Hansen et al., 2001; Ho and Suarez, 2003). More evidence, however, points toward the acrosome, a Golgi-derived lysosome-like vesicle that lies in the anterior region of the sperm head. In fact, Ca2+ has been shown to accumulate in the acrosome of human, boar, mouse and ram sperm (Berruti and Franchi, 1986; Watson and Plummer, 1986; Rossato et al., 2001; De Blas et al., 2002; Herrick et al., 2005). Receptors for inositol trisphosphate (IP3), normally found on Ca2+ internal store membranes of different somatic cells, have also been localised at the acrosome in many mammalian species (Walensky and Snyder, 1995; Dragileva et al., 1999; Kuroda et al., 1999; Naaby-Hansen et al., 2001; Ho and Suarez, 2003). Furthermore, calreticulin, a Ca2+-binding protein localised within Ca2+ stores, has been detected in the acrosome of sperm from different species as well (Nakamura et al., 1992a,b; Naaby-Hansen et al., 2001; Ho and Suarez, 2003).
As in somatic cells, intracellular sperm Ca2+ concentration must be tightly regulated, which strongly suggests that Ca2+-ATPases play an important role in sperm function. There are three large families of Ca2+-ATPases in which different genes encode different isoforms. The transcript from each isoform can also be alternatively spliced in its 3' sequence, resulting in tissue-dependent transcripts that will display a unique C-terminal region upon translation. The families are named plasma membrane Ca2+-ATPase (PMCA 1–4, ATP2B1–4), which exports Ca2+ outside the cells (Shull and Greeb, 1988; Verma et al., 1988; Greeb and Shull, 1989; Strehler et al., 1990; Carafoli, 1994; Strehler and Zacharias, 2001), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA 1–3, ATP2A1–3) and secretory pathway Ca2+-ATPase (SPCA, ATP2C1), which both sequester Ca2+ within intracellular organelles (MacLennan et al., 1985; Brandl et al., 1987; Gunteski-Hamblin et al., 1988; Lytton and MacLennan, 1988; Burk et al., 1989; Gunteski-Hamblin et al., 1992; Ton et al., 2002; Shull et al., 2003; Van Baelen et al., 2003; Wuytack et al., 2003). Previous reports have shown that the Ca2+-ATPase PMCA 4 is expressed in the principal piece of the sperm tail and is essential for hyperactivated motility and male fertility (Okunade et al., 2004; Schuh et al., 2004). Although it plays an essential role in sperm motility and regulation of intracellular Ca2+ levels, the presence of PMCA 4 does not explain the accumulation of Ca2+ within intracellular stores.
Other reports have shown that the acrosome can act as a Ca2+ store and that depletion of thapsigargin-sensitive stores induces acrosome exocytosis in capacitated spermatozoa from different mammalian species (Meizel and Turner, 1993; Walensky and Snyder, 1995; Spungin and Breitbart, 1996; Llanos, 1998; Dorval et al., 2002). Therefore, the effect of thapsigargin, a specific inhibitor of SERCA (Thastrup et al., 1990), suggests the presence and the potential implication of SERCA in the active Ca2+ uptake leading to the filling of intracellular stores during mammalian sperm capacitation. In fact, thapsigargin-binding sites have been localised to the acrosome, the post-acrosomal region and the midpiece of human sperm (Rossato et al., 2001). This suggests the presence of Ca2+ stores to those regions and that Ca2+ uptake would putatively be mediated by SERCA pumps. On the other hand, the presence of SERCA in sperm is jeopardised by the fact that concentrations of thapsigargin used to promote an increase in intracellular Ca2+ or induce acrosome exocytosis are 10–300 times higher than the concentrations used in somatic cells (Treiman et al., 1998). Furthermore, previous reports failed to detect SERCA in sperm but were able to detect SPCA suggesting that SPCA would be the only Ca2+-ATPase present in mature sperm (Harper et al., 2005; Gunaratne and Vacquier, 2006).
In the present study, it is clearly demonstrated, for the first time, that members of the SERCA 2 family are present in mature mammalian sperm. This supports a possible role for this Ca2+-ATPase in Ca2+ sequestration in internal stores.
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Materials
The enhanced chemiluminescence (ECL) kit, protein G sepharose beads and Percoll used for washing sperm were purchased from GE Healthcare Bio-Sciences Inc. (Baie d'Urfé, QC, Canada). Monoclonal anti-SERCA 2 antibody (IID8; BIOMOL Research laboratories Inc., Plymouth Meeting, PA, USA), which was raised against purified canine cardiac sarcoplasmic reticulum, is specific for all SERCA 2 isoforms (Jorgensen et al., 1988). Monoclonal anti-SERCA 1 (A52), which recognises the amino acids 657–672 (Clarke et al., 1989), anti-SERCA 3 (PL/IM430), which was raised to highly purified platelet intracellular membranes and recognises human SERCA 3 (Hack et al., 1988; Poch et al., 1998), and polyclonal anti-SERCA 2 and 3 (N1) antibody, which was raised against the purified fragment encompassing amino acids 362–704 of rat SERCA 2 but recognises both SERCA 2 and SERCA 3 products (Chandrasekera and Lytton, 2003), were generously provided by Dr J. Lytton (University of Calgary, AB, Canada). Polyclonal anti-SERCA 2 (N-19) is an affinity purified goat polyclonal antibody raised against a peptide at the N-terminus of SERCA 2 of human origin, which was supplied from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary antibodies raised against mouse, rabbit and goat immunoglobulin G (IgGs) and conjugated to either horse-radish or fluorescein isothiocyanate (FITC) were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). Lectin from Arachis hypogaea (peanut) conjugated to horse-radish peroxidase (PNA-HRP), anti-bleaching agent 1,4-diazabicyclo[2.2.2]octane (DABCO), deoxyribonuclease I (DNAse I), Hoechst 33342, protease inhibitors and mouse, rabbit and goat IgGs that were used as controls were obtained from Sigma-Aldrich (St Louis, MO, USA). VECTASHIELD Mounting Medium with DAPI was purchased at Vector Laboratories (Burlingame, CA, USA). Trizol, poly d(T)12–18 primer and trypsin were supplied by Invitrogen (Carlsbad, CA, USA). RQ1 RNase-free DNase, SuperScript III and RNasin were purchased from Promega (Madison, WI, USA). Taq DNA polymerase was acquired at QIAGEN (Mississauga, ON, Canada) and phusion DNA polymerase at New England Biolabs Inc. (Pickering, ON, Canada). Micro BCA Protein Assay kit was purchased at Pierce Biotechnology Inc. (Rockford, IL, USA). BD Falcon 40 µm nylon mesh sieve was supplied by BD Biosciences (Bedford, MA, USA). Immobilon-P polyvinylidene difluoride (PVDF) 0.45 µm pore size and Microcon YM-10 Centrifugal Filter Unit (10 kDa exclusion limit) was supplied by Millipore (Billerica, MA, USA) and X-ray films were from Fuji (Tokyo, Japan). All other chemicals were of analytical grade.
Preparation of human spermatozoa
Ejaculates were obtained by masturbation from healthy volunteers after a minimum of 2 days of sexual abstinence. All sperm donors gave informed written consent and ethical approval was obtained from the hospital (CHUQ) and Laval University ethical committees for research on human subjects. Only the samples with normal sperm parameters according to the World Health Organization (1999) criteria were used. After liquefaction, the semen was layered on top of a gradient composed of 2-ml fractions each of 20%, 40%, and 65% and 0.1 ml of 95% Percoll made isoosmotic in a HEPES-buffered saline (HBS; 25 mM HEPES, 130 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 14 mM fructose, pH 7.6) and was centrifuged (30 min, 1000 x g) to wash the spermatozoa from the seminal plasma. Sperm cells at the 65–95% interface and within the 95% Percoll fraction were collected, counted to evaluate sperm concentration and washed once by centrifugation (5 min, 500 x g) in HBS.
Preparation of bull spermatozoa
Freshly ejaculated bull semen was collected at an artificial insemination facility (CIAQ Inc., St-Hyacinthe, QC, Canada) and kindly donated by l'Alliance Semex Canada. The semen was maintained at 23°C until arrival at the laboratory (within 2.5 h). The semen (1 ml) was first diluted, with 5 ml of HBS (10 mM HEPES pH 7.2, 150 mM NaCl), then washed from seminal plasma by two centrifugations (500 x g, 10 min). The supernatant was discarded, and spermatozoa were resuspended in 5 ml HBS, centrifuged under the same conditions and, after discarding the supernatant, sperm concentration was evaluated using a hematocytometer. Part of the washed sperm were used for indirect immunofluorescence and the other part was solubilised in sample buffer [62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% (w/v) Sodium dodecyl sulphate (SDS), 100 mM DTT and 0.01% (w/v) bromophenol blue], heated at 100°C for 5 min and processed for SDS–polyacrylamide gel electrophoresis (PAGE) and western blot. Particulate material was removed by centrifugation (10 000 x g, 5 min, 4°C).
Preparation of mouse spermatozoa
Adult CD-1 male mice were obtained from Charles River Canada. Animals were killed by asphyxia in a CO2 chamber in agreement with the recommendations of the Canadian Council on Animal Care. An incision was made on the cauda epididymis, and low pressure was applied with forceps on the epididymis to expel spermatozoa. The cells were resuspended in phosphate-buffered saline (PBS; 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4) for 30 min at 37°C, then washed twice by centrifugation at room temperature (20 min, 150 x g). The final pellet was resuspended in HBS (10 mM HEPES, pH 7.2, 150 mM NaCl), and sperm concentration was evaluated using a hematocytometer. Murine spermatozoa were processed as the bovine ones.
Subcellular fractionation
Subcellular fractions of Percoll-washed human sperm were obtained through the membrane isolation procedure described by Noland et al. (1983). The complete procedure was performed at 4°C. Spermatozoa were supplemented with protease inhibitors [250 µM phenylmetheylsulphonyl fluoride (PMSF), 10 µg/ml each of aprotinin, pepstatin A and leupeptin] and subjected to nitrogen cavitation at 500 psi for 10 min. The suspension was collected slowly and was next centrifuged 10 000 x g for 20 min to separate the pellet of cavitated sperm from the supernatant. Supernatant was further centrifuged at 100 000 x g for 1 h to isolate the plasma membrane fraction (pellet) and cytosolic fraction (supernatant). Aliquots were taken at each step of the procedure. Proteins from the cytosolic fraction were concentrated using a Microcon YM-10 Centrifugal Filter Unit. Proteins were solubilised in sample buffer and heated at 100°C for 5 min. Particulate material was removed by centrifugation (10 000 x g, 5 min, 4°C). Protein concentration was determined using the Micro BCA Protein Assay kit after precipitation by trichloroacetic acid to get rid of the detergents and reducing agents. The presence of SERCA 2 was investigated by western blot on equal amounts of proteins from each fraction.
Solubilisation and immunoprecipitation
Washed human spermatozoa were resuspended in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS and 0.5% deoxycholate) or in an equivalent buffer without the SDS and deoxycholate but added with 0.5% Nonidet P-40, for 30 min on ice and vortexed every 5 min. Each detergent solution contained protease inhibitors (250 µM PMSF, 10 µg/ml each of aprotinin, pepstatin A and leupeptin). Solubilised proteins were collected by a 20 min centrifugation at 10 000 x g at 4°C. Anti-SERCA antibodies (1 µg; IID8 or N1) were added and the suspension was incubated overnight at 4°C with end-over-end rotation. Protein G sepharose beads were added and incubated with rotation for 2 h at 4°C. The immune complex associated with the beads was precipitated by centrifugation. The beads were washed three times by centrifugation (3 min at 4500 x g at 4°C) with the solubilisation buffer and then resuspended with sample buffer. SERCA 2 was detected by western blot analysis, using an anti-SERCA antibody different from the one used for the immunoprecipitation.
Western immunoblotting
Solubilised proteins in sample buffer were separated by 7.5% SDS–PAGE under reducing conditions (Laemmli, 1970) and electro-transferred on PVDF or nitrocellulose membranes (Towbin et al., 1979). Non-specific binding sites were blocked by incubating the membrane in TBST [20 mM Tris, pH 7.4, 0.9% (w/v) NaCl, and 0.1% (v/v) Tween 20] containing 5% (w/v) dry skim milk for 1 h. For the western blot done with the N-19 anti-SERCA 2 antibody, the membranes were blocked with TBST supplemented with 3% bovine serum albumin (BSA). The membranes were next incubated with the polyclonal antibody N1 (SERCA 2 and 3), for 2 h at room temperature, or with the monoclonal PL/IM430 (SERCA 3), the monoclonal IID8 (SERCA 2) or the polyclonal N-19 (SERCA 2), overnight at 4°C. After extensive washes in TBST, the membranes were incubated in the presence of the corresponding HRP-conjugated secondary antibody for 1 h at room temperature, washed again then processed for detection. Positive bands were detected by ECL on X-Ray films. Human heart tissue was provided by the hospital pathology department and used as positive control for SERCA 2. To assess the subcellular fraction containing outer acrosomal membranes, the nitrocellulose membrane was blocked in TBST supplemented with 1% BSA and 1 mM CaCl2, incubated with PNA-HRP and washed in the same solution, and the positive signal was detected as for the immunoblots.
Indirect immunofluorescence
Thirty five microliters of a 20 x 106 cells/ml suspension of washed human, bull and mouse sperm were deposited on poly-L-lysine coated coverslips and allowed to settle for 30 min at room temperature. Spermatozoa were then fixed for 15 min in 3.7% formaldehyde in PBS, rinsed with PBS, permeabilised for 10 min in 0.2% Triton X-100 in PBS and rinsed again with PBS. Nonspecific sites were blocked with PBS supplemented with 5% non-immune rabbit serum. Samples were then incubated overnight at 4°C with the goat polyclonal SERCA 2 antibody (N-19) diluted in the blocking solution, rinsed with PBS and incubated with a FITC-labelled rabbit anti-goat secondary antibody. Non-immune goat IgGs were used as control instead of the primary antibody. Following washes with PBS, the coverslips were mounted on slides with 90% glycerol containing an antifading agent (1.5% DABCO). Immunofluorescence was detected by epifluorescence microscopy with a UV light.
Isolation of human haploid (1 N) and tetraploid (4 N) testicular germ cells
Normal human testes recovered from a 26-year-old man registered in our local organ transplantation programme. This donor was a victim of accidental death and did not have pathologies that could affect his reproductive function. Tissues were collected while artificial circulation was maintained to preserve organs and tissues assigned for transplantation. A piece of human testis was taken underneath the tunica albuginae and rinsed in D-PBS (6.8 mM CaCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 0.5 mM MgCl2, 137 mM NaCl, 8.1 mM Na2HPO4, pH 7.4). The biopsy was minced with scissors, and 0.1% (w/v) trypsin, MgCl2 (final 10 mM) and DNAse I (2 µg/ml) were added to the mixture, which was incubated at 37°C for 10 min with vigorous shaking to facilitate cell dissociation. Fetal bovine serum (8% final) was next added to quench the trypsin activity. The seminiferous tubules were further dissociated by several up-and down-pipetting motions. The preparation was filtered on a 40 µm nylon mesh sieve. The cells were centrifuged for 15 min at 250 x g, the pellet was resuspended in 15 ml D-PBS supplemented with DNAse I and centrifuged for another 15 min at 250 x g. The resulting pellet was resuspended in 10 ml of 0.4 M sodium citrate, pH 2.35 (made in DEPC water) and incubated overnight at room temperature. The cells were centrifuged again and resuspended in 10 ml of 0.4 M sodium citrate, pH 4.5 and incubated overnight at 4°C. The cell preparation was centrifuged again for 15 min at 500 x g, and the pellet was resuspended in Isoton II solution (10 mM HEPES, 147 mM NaCl, 5 mM KCl, 1 mM EDTA, 0.2 g/l phenoxyethanol, 0.1% BSA, pH 7.4). Hoechst 33342 was added (1.5 µg/ml final), cells were incubated at 37°C for 1 h, then haploid (1 N) or tetraploid (4 N) testicular germ cells were sorted according to their DNA content using an Epics Elite ESP flow cytometer (Beckman Coulter; Miami, FL, USA) equipped with a HeCd laser (Omnichrome, Chino, CA, USA) with an excitation wavelength of 325 nm. The sorted haploid and tetraploid populations were pure at 98.3% and 91.6%, respectively, according to cell DNA content.
Reverse transcription–polymerase chain reaction
Total RNA was isolated from human haploid and tetraploid cells and testis using Trizol. The RNA, 100 ng, was reverse transcribed with SuperScript III in a 20 µl reaction mixture [containing the supplier's buffer, 10 mM DTT, 500 µM deoxynucleotides triphosphate (dNTPs) and 40 U RNasin], using a poly d(T)12–18 primer. PCR amplification was done in a 50 µl final volume using 2 µl of the reverse transcription product with 200 µM dNTPs and 6 µM of each primer (see Table I). For SERCA 2a and 2c, the specific amplification was performed using 0.5 U Taq DNA polymerase under the following conditions: 40 cycles of 30 s of denaturation at 94°C; 30 s of annealing at 61°C and 1 min extension at 72°C, followed by an additional 10 min of extension at 72°C. For the amplification of SPCA 1, GAPD2 and SERCA 2b, phusion DNA polymerase was used. PCR conditions for SPCA 1 were: 40 cycles of 10 s of denaturation at 98°C; 30 s of annealing at 62°C and 30 s extension at 72°C, followed by an additional 10 min of extension at 72°C; for SERCA 2b and GAPD2, the PCR conditions were: 40 cycles of 10 s of denaturation at 98°C; 30 s of annealing at 65°C and 15 s extension at 72°C followed by an additional 10 min of extension at 72°C. PCR products were visualised on agarose gel containing ethidium bromide. Special care was taken to design primers at gene intron/exon boundaries and, under the conditions used, no genomic DNA was amplified.
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Immunohistochemistry
Human testes fixed in a commercial formaldehyde solution (ACP, Montreal, QC, Canada) and embedded in paraffin were provided by the hospital pathology department. Immunohistochemistry was performed on 5 µm-thick sections. The slides were deparaffined and rehydrated through successive incubation in ethanol baths with decreasing concentrations. At the end, they were bathed in water. The testis sections were washed in PBS. Antigen retrieval was achieved by incubation of the slides for 10 min in a boiling bath containing 10 mM sodium citrate pH 6.0. Non-specific sites were blocked with 1% donkey serum in PBS for 1 h. The slides were then incubated overnight at 4°C in the presence of the N-19, a polyclonal antibody directed against SERCA 2. After being washed with PBST (PBS supplemented with 0.05% of Tween 20), the sections were incubated with a FITC conjugated donkey anti-goat IgG for 1 h at room temperature. Slides were next subjected to five washes in PBST and finally mounted in VECTASHIELD Mounting Medium containing DAPI. Immunofluorescence was visualised by epifluorescence microscopy with a UV light.
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Results |
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SERCA 2 is present in human spermatozoa
In preliminary experiments, we investigated the presence of SERCA 1, 2 and 3 by western blot experiments on protein extracts from human spermatozoa. We were unable to detect SERCA 3 using PL/IM430, a monoclonal antibody that recognises all SERCA 3 isoforms, and the results with a monoclonal antibody directed against SERCA 1 were unconvincing (data not shown). As shown in Fig. 1A, SERCA 2 was detected in human sperm with both polyclonal (N1) and monoclonal (IID8) antibodies that recognise all splice variants of SERCA 2 in human tissues. Although the polyclonal N1 antibody recognises both SERCA 2 and 3, the absence of detection of SERCA 3 with the specific monoclonal antibody strongly suggests that the protein detected is SERCA 2. The latter is by far the most widespread of all SERCA isoforms, SERCA 2b being recognised as ubiquitous. Since SERCA 2 is a highly conserved protein within different species, we sought to detect this enzyme in mouse, bovine and human spermatozoa. As shown in Fig. 1B, SERCA 2 detection with another polyclonal antibody (N-19) showed two different bands in each species with masses ranging from 105 to 115 kDa, which is in agreement with the reported molecular weight of SERCA 2a (110 kDa), 2b (115 kDa) and 2c (110 kDa) isoforms (Lytton and MacLennan, 1988; Gelebart et al., 2003). Furthermore, as a positive control, we tested our antibodies on human heart protein extracts along with the sperm protein samples and the same 105–115 kDa protein bands were obtained (data not shown). Cardiac tissues are known to strongly express SERCA 2 (Zarain-Herzberg et al., 1990).
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To further confirm the presence of SERCA 2 in our western blot analyses, we performed an immunoprecipitation assay on human sperm extracts. As shown in Fig. 2A, the SERCA 2 isoform with the highest masse is not soluble in RIPA buffer in the presence or absence of ionic detergents. However, immunoprecipitation was possible with the most abundant form of SERCA 2 detected in sperm. For this immunoprecipitation assay, we used both the polyclonal (N1: anti-SERCA 2 and 3) and the monoclonal (IID8: anti-SERCA 2) antibodies, both of which recognise SERCA 2. We then probed the immunoprecipitate with an antibody different from the one used for the immunoprecipitation. As shown in Fig. 2B, both antibodies immunoprecipitated SERCA 2 and recognised the immunoprecipitated protein. We also used another polyclonal antibody (N-19) to detect SERCA 2 in the immunoprecipitates, and this antibody also recognised the same protein as the other antibodies (data not shown). Therefore, these results support and confirm the presence of SERCA 2 found in our western blot analysis.
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Our next step was to determine the subcellular localisation of SERCA 2 in human sperm. As shown in Fig. 3A, the two SERCA 2 bands previously detected in the total sperm extracts are found in different subcellular fractions. The heaviest band (110 kDa) is detected in the membrane fraction (lanes 2), which contains outer acrosomal membranes (Fig. 3B). On the other hand, the other SERCA 2 band (105 kDa) appears mostly in the cavitated sperm fraction (Fig. 3A; lane 4). When this fraction was sonicated to separate the flagellum from the sperm head, the fractions containing the midpiece portion of the flagellum, either the heads or the flagellae, were positive for SERCA 2 (data not shown). Furthermore, SERCA 2 was absent from the cytosolic fraction that was used as a control because SERCA 2 is known to have at least 10 transmembrane domains. The presence of outer acrosomal membranes (Fig. 3B; lanes 2) was assessed by using PNA-HRP, which was shown to bind to the outer acrosomal membrane (Flesch et al., 1998).
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The cellular localisation of SERCA 2 was next assessed by indirect immunofluorescence. In human as well as in mouse and bovine sperm, SERCA 2 is detected in the acrosome and midpiece areas (Fig. 4). However, the signal observed in the midpiece appears to be restricted to human sperm, as a similar signal was detected in bovine and murine sperm when non-immune goat IgGs were used (data not shown). These observations confirmed that in those species, SERCA 2 is restricted to the acrosomal area of the sperm cell. Similar results, although weaker, were obtained in human sperm using the monoclonal SERCA 2 antibody IID8 (data not shown). The localisation of SERCA 2 in human sperm is in agreement with the results obtained by western blots in the different subcellular fractions (Fig. 3).
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Testicular expression of SERCA 2 during spermatogenesis
The developmental expression of SERCA 2 during spermatogenesis was next assessed by immunohistochemistry on human testis sections. SERCA 2 is expressed in both the seminiferous tubules and the interstitial tissues (Fig. 5). This result is not surprising since this protein is known to be ubiquitously expressed. However, within the seminiferous tubules, it can be easily observed that SERCA 2 protein expression begins at the primary spermatocyte stage and that the signal becomes more intense in round and elongating spermatids. The protein is barely detectable in spermatogonia.
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SERCA 2 splice variants in human testis, haploid (1 N) and tetraploid (4 N) cells
Because our antibodies do not discriminate between the different SERCA 2 splice variants, RT–PCR experiments with specific primers listed in Table I were done to determine which one is present in human spermatids and putatively expressed in mature spermatozoa. The presence of SERCA 2a and 2c, which only differ by the addition of an intronic sequence in the 3'-termini portion of SERCA 2c, was investigated in the same PCR reaction as described previously (Gelebart et al., 2003). Each splice variant is expressed in the testis (Fig. 6). SERCA 2a and 2c seem to be expressed at specific stages of spermatogenesis, with SERCA 2a transcript being detected only in tetraploid cells (primary spermatocytes) and SERCA 2c mRNA being observed only in haploid cells (round to elongated spermatids). SERCA 2b, on the other hand, was found to be expressed in both spermatogenic cells, although expressed in higher levels in haploid cells. The detection of SPCA 1 in both haploid and tetraploid cells by RT–PCR procedures was not surprising as the protein has been shown in mature human sperm (Harper et al., 2005) (Fig. 6). As shown in Fig. 6, higher levels of the GAPD2 transcript, the human spermatogenic cell-specific variant of glyceraldehyde 3-phosphate dehydrogenase (Welch et al., 2000), were found in haploid cells than in tetraploid cells, which confirm the enrichment of the two populations. In mouse, GAPD2 transcript is expressed specifically in round and elongating spermatids (Welch et al., 1992).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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The aim of this study was to clearly determine whether the Ca2+-ATPases of the SERCA family are present in mammalian spermatozoa. To the best of our knowledge, this is the first identification and localisation of SERCA 2 in human, mouse and bovine sperm. Furthermore, western blot analysis on sperm extracts from these different species showed that more than one splice variant of SERCA 2 is present. SERCA 2 has three known splice variants, SERCA 2a, 2b and 2c (Lytton and MacLennan, 1988; Gelebart et al., 2003). In human sperm, at least two splice variants migrating at different speeds in electrophoresis gels are observed. One isoform was detected in the fraction containing outer acrosomal membranes, whereas the other was present in the subcellular fraction containing the sperm midpiece. Indirect immunofluorescence confirmed these results in human sperm, as SERCA 2 was observed in the acrosome and midpiece region, whereas only the acrosomal staining was specific in mouse and bovine sperm. The confirmation of this identification in human sperm was further supported by the use of three different anti-SERCA 2 antibodies in western blot analysis on total sperm protein extracts and in the immunoprecipitation assay. These results strongly suggest the presence of SERCA 2 in mammalian sperm. In addition, the detection of SERCA 2 in spermatocytes and spermatids by immunohistochemical methods and the PCR-amplification of mRNA encoding for each of the reported SERCA 2 splice variants in haploid and tetraploid spermatogenic cells are in perfect agreement with the presence of this family of Ca2+-ATPases in mature spermatozoa.
Despite the controversy on the presence/absence of SERCA in mature sperm, much evidence points to its presence. Thapsigargin, a tumour-promoting drug known to specifically inhibit SERCAs causing an increase in [Ca2+]i through Ca2+ depletion from internal stores and capacitative Ca2+ entry (Thastrup et al., 1990), has been shown by different laboratories to induce a rise in human sperm [Ca2+]i (Blackmore, 1993; Meizel and Turner, 1993; Perry et al., 1997). This major Ca2+ increase leads to the acrosome reaction. Thapsigargin is known to have only specific effects on SERCAs and not on other Ca2+-ATPases (Thastrup et al., 1990). However, the concentrations of thapsigargin required to affect [Ca2+]i or promote the acrosomal exocytosis were 10–300 times higher than the concentrations used in somatic cells (Treiman et al., 1998), which was an argument against the presence of SERCAs in sperm. In addition, previous reports failed to detect SERCAs in human and sea urchin mature sperm (Harper et al., 2005; Gunaratne and Vacquier, 2006). However, even though the present report clearly demonstrated that SERCA 2 is present in mammalian sperm, this strongly suggests that sperm contains both thapsigargin-sensitive and -insensitive stores, this latter containing other Ca2+-ATPases from other families such as SPCA 1, the presence of which has been demonstrated in human spermatozoa (Harper et al., 2005).
The debate that non-selective doses of SERCA inhibitors are needed to induce the acrosome reaction in capacitated sperm may partly be explained by the fact that sperm populations are heterogeneous and that not all spermatozoa undergo capacitation at the same time. Therefore, the acrosome reaction induced by thapsigargin or other inducers would only affect a fraction of the sperm population. In fact, in many reports in which physiological inducers, such as ZP3, have been used to induce sperm acrosome reaction, only part of the sperm population responded. It was suggested that the non-responsive group would reflect inefficient in vitro sperm capacitation (Arnoult et al., 1999; O'Toole et al., 2000). In a previous report from our laboratory, the addition of thapsigargin to a capacitated sperm suspension resulted in two subpopulations, the low (LR) and high (HR) responsive sperm according to their intracellular free Ca2+ concentration (Dorval et al., 2003). LR population showed weak increase, whereas HR population showed high levels of Ca2+ in response to thapsigargin treatment. Furthermore, the number of sperm within the HR populations increases during capacitation, which indicated that sperm progressively acquire the ability to undergo a net Ca2+ influx in response to thapsigargin during capacitation. Moreover, even though the [Ca2+]i was elevated in all the sperm cells within the HR population, not all sperm experienced exocytosis of the acrosome suggesting that Ca2+ is not the only factor implicated in the acrosome reaction and that sperm do not all capacitate at the same time. This is also in agreement with the fact that in sperm, SERCA 2 may not be the only Ca2+-ATPase playing a role in Ca2+ accumulation. SPCA 1, a thapsigargin-insensitive Ca2+-ATPase, has been demonstrated in human sperm using bis-phenol, an inhibitor of both thapsigargin-sensitive and insensitive Ca2+-ATPases (Harper et al., 2005). In that report, both thapsigargin and bis-phenol induced sperm acrosome reaction, which also supports the presence of thapsigargin-sensitive and-insensitive stores in mature spermatozoa. SERCA 2 and SPCA 1 could act together in sperm intracellular store Ca2+ store accumulation. This represents a crucial event during sperm capacitation for the Ca2+-dependent regulation of hyperactive motility and acrosome reaction (O'Toole et al., 2000; Jungnickel et al., 2001; Ho and Suarez, 2003; Harper et al., 2004), the two major functions of spermatozoa prior to fertilisation.
Immunodetection in whole sperm extracts as well as in the different subcellular fractions confirms the presence of SERCA 2 and suggests that more than one SERCA 2 splice variant is present. In humans, the SERCA 2 gene yields primary transcripts that are processed in a tissue-specific manner, giving rise to three isoenzymes with unique C-terminal domains that are identical up to amino acid 993, but differ in their carboxyl terminus. SERCA 2 is by far the most widespread and evolutionary the oldest of all SERCA isoforms. Although we could not discriminate at the protein level the identity of SERCA 2 splice variants in spermatozoa, all of them were detected in the RNA isolated from human testis. Normally, SERCA 2a (997 amino acids) is expressed in cardiac/slow twitch skeletal muscle and SERCA 2b (1046 amino acids) is known to be expressed ubiquitously (Gunteski-Hamblin et al., 1988; Lytton and MacLennan, 1988). SERCA 2c was recently detected in human monocytes and foetal heart via RT–PCR (Gelebart et al., 2003), but information on its tissue-expression pattern and functional characterisation remains elusive (Gelebart et al., 2003). The results obtained in our PCR experiments suggest that SERCA 2c expression occurs post-meiotically, in support for the presence of this variant in mature sperm. SERCA 2b appears to follow a similar expression pattern in the testis. In addition, more investigations are needed to determine whether the expression of SERCA 2a stops during spermatogenesis. However, these two observations remain to be confirmed at the protein level.
Using fluorescence-conjugated thapsigargin, it has previously been reported that this SERCA inhibitor binds to the acrosomal region, suggesting that this subcellular compartment is a thapsigagin-sensitive Ca2+ store (Rossato et al., 2001). Using SERCA 2 antibodies, our results show similar localisation to the acrosome of human bovine and mouse spermatozoa. Human sperm also displayed SERCA 2 signal in the midpiece, which again, is in agreement with the reported binding of thapsigargin to human sperm (Rossato et al., 2001). Subcellular human sperm fractions confirmed that at least two different SERCA 2 isoforms are present, one being in the fraction containing outer acrosomal membranes and the other to the fraction containing the sperm midpieces.
According to their molecular weights, the isoform present in the outer acrosomal membrane could putatively be SERCA 2b, whereas SERCA 2a and 2c could localise to the midpiece region. SERCA 2b is usually found in the ER of most cell types and is considered the housekeeping isoform. It is also found to be associated with IP3 gated Ca2+ stores (Gunteski-Hamblin et al., 1988; John et al., 1998). The extended carboxyl terminus in SERCA 2b contains a highly hydrophobic stretch, representing an 11th transmembrane segment. Consequently, the extreme C-termini of SERCA 2a/2c and SERCA 2b are situated at opposite sides of the ER membrane with the SERCA 2b tail protruding into the lumen (Campbell et al., 1992). This suggests that these isoforms could be regulated differently on their C-terminus tail. In fact, it was observed that calreticulin, a Ca2+-binding chaperon of the endoplasmic reticulum, could modulate SERCA 2b but not 2a activity in Xenopus oocytes (John et al., 1998). As SERCA 2, calreticulin is present in the acrosomal region of human sperm (Naaby-Hansen et al., 2001). Calreticulin inhibits SERCA 2b when the stores are filled, and therefore, the present study suggests that this Ca2+-ATPase could be negatively modulated at the end of capacitation, prior to the acrosome reaction, when the stores are filled with Ca2+.
The presence of SERCA 2 to the midpiece region of human sperm is intriguing but suggests that this Ca2+-ATPase could play a role in sperm motility. Ca2+ is known to play a crucial role in sperm hyperactivation. Sperm acquisition of hyperactivated motility is critical for transport through the female tract and for fertilisation (Ho and Suarez, 2001a). It has also been demonstrated that the SERCA inhibitor thapsigargin induces hyperactivation of bovine sperm by promoting Ca2+ release from IP3 receptor-gated internal stores. Similarly, thimerosal, an IP3 receptor agonist, also causes an increase in intracellular Ca2+ levels and stimulates hyperactivation (Ho and Suarez, 2001b). The store shown to be implicated in this Ca2+ accumulation is composed of the redundant nuclear envelops (Ho and Suarez, 2003), which are present in the neck region of sperm. In our study, it is not known whether this structure is still present in the head/midpiece fraction where we detected SERCA 2. However, there are reasons to believe that sperm mitochondria would also be implicated in hyperactivated motility. Recently, it was suggested that the role of mitochondria would be in the maintenance (Gilabert and Parekh, 2000; Hoth et al., 2000; Gilabert et al., 2001) and modulation (Parekh, 2003) of the capacitative Ca2+ entry in many cell types. Whether SERCA are involved in these activities remain to be established. In addition, it was shown that the ER Ca2+ refilling upon IP3 treatment depends on a Ca2+ cross-talk between the ER and the mitochondria, which delivers entering Ca2+ towards the ER and the cytoplasmic compartment (Malli et al., 2005). This emphasizes that the role of SERCA 2 in the sperm midpiece region requires further investigation.
In this report, we clearly demonstrate that SERCA 2 is present in mammalian sperm. It is localised to the acrosomal region of the sperm head, and in human, it is also present in the midpiece. At least two SERCA 2 splice variants are found in human spermatozoa. Our results support the hypothesis that the acrosome acts as an intracellular Ca2+ store and that SERCA 2 is responsible, at least partially, for the Ca2+ sequestration during capacitation. Although the mechanisms modulating SERCA 2's activity during capacitation remain to be investigated, the present results are in agreement with the presence of another Ca2+ store, located at the neck or midpiece region of mature spermatozoa.
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The authors wish to thank Dr J. Lytton, University of Calgary, for his generous gift of A52, N1 and PL/IM430 antibodies. We are thankful to Dr M. Dufour for his help on sorting the cells based on their ploidy. Study supported by a grant from Canadian Institutes of Health Research (CIHR); C.L. and V.D. are supported by a studentship from CIHR and Fonds de la Recherche en Santé du Québec (FRSQ), respectively; and P.L. is supported by a Chercheur-Boursier scholarship from FRSQ.
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