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RESEARCH |
2 glycoprotein in sperm motility is mediated by changes in cyclic AMPShanghai Key Laboratory for Reproductive Medicine, Department of Histology and Embryology, School of Medicine, Shanghai Jiao Tong University, Shanghai, China and 1 Shanghai Jiai Genetics and IVF Institute-China USA Center, Shanghai, China
Correspondence should be addressed to Z Ding; Email: zding{at}shsmu.edu.cn
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Abstract |
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2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the ß3-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (P<0.05 and P<0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway. |
Introduction |
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Sperm motility is generated by the extremely long flagellum that comprises >90% of the length of a mammalian sperm (Vernon & Woolley 2004, Turner 2006). Such motility is regulated by the surrounding environment; obvious changes in motility include activation, hyperactivation, and chemoattraction in various species (Ahmad et al. 1995, Ho & Suarez 2001, Wang et al. 2001, Wade et al. 2003). The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway and the calcium signaling pathway are generally recognized as the two signaling pathways most central to the regulation of mammalian sperm motility (Brokaw 1987, Wishart & Ashizawa 1987, San Agustin & Witman 1994, Wade et al. 2003). Even though the role of these signaling pathways in sperm motility has been demonstrated in many experiments, the specific underlying molecular mechanisms have never been fully explored.
Zn-2-glycoprotein (ZAG) is a 43 kDa soluble glyco-protein, originally isolated for human plasma in 1961, which was named for its tendency to precipitate with zinc salts and its electrophoretic mobility in the 2-globulins (Burgi & Schmid 1961, Sanchez et al. 1999). It has subsequently been detected in various physiological and pathological fluids (Jirka et al. 1978, Ohkubo et al. 1990, Bundred et al. 1991, Sanchez et al. 1997). Acting as a lipid-mobilizing factor, ZAG is expressed and secreted in mammalian adipose tissue and is markedly upregulated with cancer cachexia (Hale et al. 2001, Sanders & Tisdale 2004, Tisdale 2004). The expression level of ZAG is regulated through tumor necrosis factor- and the peroxisome proliferator-activated receptor-
nuclear receptor (Bao et al. 2005).
Recently, it has been reported that ZAG activity in rodents is mediated via the ß3-adrenoreceptor, with upregulation of the cAMP pathway (Russell et al. 2004, Tisdale 2004, McDermott et al. 2006). In a previous study, we reported that ZAG was primarily related to sperm forward motility in human semen (Ding et al. 2007). Our data supported the fact that ZAG in human seminal plasma might be integrated with -1-antitrypsin (AAT) and was capable of initiating forward motility in immature immotile spermatozoa. However, it is still unclear whether ZAG is present in human spermatozoa, and what the relationship is between ZAG and sperm motility.
According to the above introduction, ZAG is supposed to be a special factor related to human sperm motility. There is a possibility that the signal transduction pathway of ZAG in adipose tissue will be similar to that in the spermatozoa. Thus, in the present study, we prepared polyclonal antibodies against recombinant human ZAG. Laser scanning confocal microscope (LSCM) and flow cytometry (FCM) were then employed to identify ZAG protein in human spermatozoa. Furthermore, these antibodies were applied to the experiments of sperm motility monitored by computer-assisted sperm analysis (CASA), together with the investigation of intracellular cAMP and PKA levels.
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Results |
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). A 345 bp fragment was amplified from human cDNA by PCR and separated on a 1% agarose gel (Fig. 1B).The target fragment of ZAG was then cloned into the prokaryotic expression vector, which was named pET-28a(+)/ZAG (Fig. 1C).This construct is predicted to encode a 143 amino acid-containing recombinant protein with molecular weight of 
18 kDa. It was successfully overexpressed by inducing with IPTG; this 18 kDa protein was absent in non-induced cells, shown on 15% SDS-PAGE gel (Fig. 2A). The recombinant protein was purified and then confirmed by SDS-PAGE analysis (Fig. 2A) and Western blotting using anti-His monoclonal antibody (MAB) (Fig. 3A).
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). Anti-ZAG antibodies (without sodium azide) were then purified from crude rabbit sera, confirmed by Western blotting (Fig. 3A) and stored at 4 °C for functional analysis in vitro.
Identification and localization of ZAG in spermatozoa
Using indirect immunofluorescence, spermatozoa staining with/without fluorescence were discerned and counted. The fluorescence intensity and the ratio of fluorescent sperm to non-fluorescent sperm were significantly higher in spermatozoa stained with ZAG antibody than in the control group (stained with normal rabbit IgG; P<0.001; Fig. 4A and B). The results were confirmed using LSCM (Fig. 4C), LS-50B luminescence spectrometer (Fig. 4D), and further confirmed by the fact that ZAG was distinctly detected from human sperm proteins by Western blot (Fig. 3B). Furthermore, intense immunoreactivity was localized in the head and tail region of spermatozoa, while the most intense immunoreactivity was observed in the pre-equatorial region covering the acrosome, neck, and middle piece of flagellum (Fig. 5).
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). Sperm and forward motility indices were evaluated after 0, 60, 90, and 120 min of incubation in the different experimental conditions described above using CASA. The following parameters were measured: ratio of motile sperm and straight line velocity (VSL; time-average velocity of a sperm head along the straight line between its first and last positions). At 90 or 120 min of incubation, the motility and VSL of spermatozoa treated with the rabbit anti-ZAG IgG (50.0 µg) were significantly lower than those from sperm treated with normal rabbit IgG or those treated with Tyrode solution with 3% BSA alone (Fig. 6B and C). These results indicated that anti-ZAG antibodies were able to reduce swim-up spermatozoa motility in vitro.
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). For the measurement of intracellular PKA activity, the fluorescence intensity measured in the assay is inversely correlated with kinase activity. Thus, the PKA activity of swim-up spermatozoa incubated with anti-ZAG IgG was significantly lower than that in sperm treated with normal rabbit IgG (Fig. 7B). Therefore, both intracellular cAMP levels and PKA activity in human sperm were reduced by the anti-ZAG antibodies.
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Discussion |
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In this study, we try to explore the possible relationship between ZAG and sperm motility. Since previous studies have demonstrated the presence of ZAG in seminal and prostate fluid (Ohkubo et al. 1990, Ahlgren et al. 1995), there is a possibility that ZAG was adsorbed to the sperm. Furthermore, ZAG was reported to be capable of initiating forward motility in immature immotile spermatozoa, integrated with AAT (Ding et al. 2007). Thus, it was reasonable to investigate whether ZAG affects the motility of sperm individually. We cloned the gene encoding human ZAG and generated the relevant polyclonal antibodies. We not only identified these antibodies in the tail region of spermatozoa by immunofluorescence but also found that they reduced sperm motility. In addition, the anti-ZAG antibodies decreased intracellular cAMP levels and PKA activity.
In the regulation of mammalian spermatozoa motility, the cAMP/PKA signaling pathway is usually recognized as one of the most significant signaling pathways. This pathway is also known to play a major role in intracellular signaling during mammalian sperm capacitation (Brokaw 1987, Si & Okuno 1995, Fraser & Osiguwa 2004). The cAMP-dependent phosphorylation of flagellar proteins is at least partially responsible for the initiation and maintenance of activated sperm motility in mammals (San Agustin & Witman 1994). In mice with a targeted deletion of the sperm-specific isoform of the catalytic (C) subunit of PKA, male infertility is highly correlated with poor sperm motility (Skalhegg et al. 2002). Thus, it is very likely that one mechanism of cAMP action is to affect sperm motility via activation of PKA. On the other hand, cAMP has also been associated with gated ion channels, involved in the calcium signaling pathway. Therefore, increased intracellular cAMP through both the cAMP/PKA and calcium signaling pathways could lead to hyperactivated motility via upregulation of the flagellar wave (Ahmad et al. 1995, Ho & Suarez 2001).
However, how does ZAG affect the cAMP/PKA signaling pathway in human sperm? The cAMP signaling pathway is reported to be involved in the process of lipolysis in adipocytes in many mammals, for which the ß-adrenergic receptor is directly induced by ZAG (Russell et al. 2002, 2004, Sanders & Tisdale 2004, Tisdale 2004, McDermott et al. 2006). The crystalline structure of ZAG resembles a class I major histocompatibility complex (MHC) heavy chain, but, unlike other MHC-related proteins, ZAG does not bind the class I light-chain ß2-microglobulin (Sanchez et al. 1997, 1999). ZAG structure includes a large groove analogous to the class I MHC peptide-binding grooves. However, instead of a peptide, ZAG’s groove contains a non-peptidic compound that might be implicated in lipid catabolism under normal or pathological conditions (Delker et al. 2004, McDermott et al. 2006). The mechanism involved in lipid catabolism is that ZAG can stimulate lipolysis in adipocytes through the ß3-adreno- receptor, which directly results in the stimulation of adenylate cyclase and an increase in intracellular cAMP (Russell et al. 2004, Tisdale 2004, Russell & Tisdale 2005). Interestingly, ß-adrenergic receptors have already been detected in human sperm (Adeoya-Osiguwa & Fraser 2005, 2007, Adeoya-Osiguwa et al. 2006), and ß3-adrenoreceptor also has a similar localization to ZAG. Thus, we presume that ZAG is able to stimulate cAMP signaling via the ß-adrenoreceptor in human sperm, a potential pathway of motility regulation. This could explain why anti-ZAG antibodies can inhibit sperm motility, together with the decreasing of intra-cellular cAMP and PKA levels.
In conclusion, our current investigations demonstrate that ZAG is present in human spermatozoa, and observed mostly in the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum. ZAG may be a novel factor involved in the regulation of sperm motility, mediated by activation of the cAMP/PKA signaling pathway.
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Materials and Methods |
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Prokaryotic expression and purification of ZAG
Human cDNA was kindly gifted by Dr Min Wang from the Shanghai Key Laboratory for Reproductive Medicine (Shanghai, China). The segment of ZAG was amplified with the forward primer 5'-GCGGATCCGGAAGGTTTGGTTGTGA-GAT-3' (containing BamHI site, underlined) and the reverse primer 5'-GCAAGCTTTCCCTGGGTAGAAGTCGTAG-3' (containing HindIII site, underlined). The PCR product was purified from the agarose gel and cloned into the pMD18-T vector (TaKaRa Biotech, Dalian, China) to transform Escherichia coli (E. coli, strain DH16B). The recombinant plasmid was digested with BamHI and HindIII, and ligated into the pET-28a(+) expression vector (Novagen, Darmstadt, Germany) at the same restriction site. The target fragment of ZAG was confirmed by restriction enzyme digestion, PCR, and sequencing. The new recombinant prokaryotic expression vector was named pET-28a(+)/ZAG, and propagated in E. coli BL21(DE3) host cells cultured with 0.1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) for 5 h at 37 °C with gentle shaking. Bacterial cells were harvested by centrifugation (5000 g) for 15 min at 4 °C, and disrupted by gentle ultrasonic treatment (50 W, 20 sx10) with a 50% duty cycle on ice. Recombinant protein was purified based on its His6-tag by affinity chromatography using a Ni2+-NTA His-binding resin (Pierce, Rockford, IL, USA; Joshi & Puri 2005). For further purification, the recombinant protein was separated on preparative electrophoresis, and the corresponding band was cut from the gel and eluted in PBS. The eluted protein was freeze-dried in a Heto Freeze Dryer (Thermo Electron Corporation, Waltham, MA, USA), and its identity was confirmed by 15% SDS-PAGE analysis and Western blotting using anti-His MAB.
Production of polyclonal rabbit anti-ZAG antibodies
Two healthy white New Zealand rabbit males (6 months old, body weight 2.5 kg; purchased from Shanghai SLAC Laboratory Animal Co., Shanghai, China) were accommodated in the animal facility for at least for 1 week prior to immunization. The pre-immunized rabbits’ blood was obtained, and the serum was separated and kept at –80 °C for later use (negative control). The rabbits were immunized a total of three times with the recombinant ZAG protein and the adjuvants to raise antibodies (Hu et al. 2002). The titer of the antiserum was measured using an indirect ELISA at 405 nm using an Anthos Zenyth 1100 multimode detector (Anthos Labtec Instruments GmbH, Wals, Austria) with a 5-s pre-read shake. Finally, the anti-ZAG IgG was purified through immunoaffinity chromatography from crude rabbit sera, using the ImmunoPure (G) IgG Purification kit (Pierce). In the meantime, normal rabbit IgG was purified from pre-immunized rabbit sera.
Preparation of spermatozoa and sperm proteins
Semen samples were collected via masturbation after 3–5 days of sexual abstinence from young healthy donors (20–30 years of age). Individual semen samples were allowed to liquefy at 37 °C, and the mature spermatozoa were separated from seminal plasma, immature germ cells, and non-sperm cells by Percoll density ingradient centrifugation (Fraser & Osiguwa 2004). In sperm motility inhibition experiments, motile, intact spermatozoa were obtained using the swim-up method, as previously described (Bronson & Fusi 1990).
Purified spermatozoa were solubilized in a lysis buffer containing: 8 M urea, 1.5% Triton X-100, and 1 mM phenyl methyl sulfonyl fluoride. The sperm suspension was then vortexed for 10 min at 4 °C and kept in a rotating ice bath for at least 2 h in order to allow proteins to dissolve completely. After protein dissolution, samples were ultrasonicated (30 W, 10 sx3, VC600, Sonics and Materials, Newtown, CT, USA) and centrifuged at 12 000 g for 20 min to remove the sediment. Protein concentration was determined by the BCA Protein Assay Kit (Pierce), using BSA to generate the standard curve.
Western blot analysis
For Western blot analysis, samples containing 20 µg protein were separated on 15% SDS-PAGE gel (denatured). Proteins were then transferred to PVDF membranes (GE Healthcare, Waukesha, WI, USA) using a semi-dry transfer apparatus (Bio-Rad), and membranes were blocked with 5% BSA. Immunoblotting was performed with primary antibody at a suitable dilution in Tris-buffered saline containing 0.1% Tween 20 and 5% BSA overnight at 4 °C. Blots were then incubated with a goat–anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) at a 1:1000 dilution for 1 h at room temperature. Signals were detected by ECL (ECL Plus, GE Healthcare), following the manufacturer’s protocol.
Indirect immunofluorescence of human spermatozoa
For the immunofluorescence studies, fresh human spermatozoa were harvested over a discontinuous 47/90% Percoll density gradient and subsequently washed thrice with PBS. The sperm solution (2x105 sperm/ml) was then added onto slides, fixed with acetone for 30 min. All subsequent incubations were performed in a humid chamber. The slides were blocked in 5% BSA in PBS for 30 min at room temperature, and later incubated either with the purified rabbit anti-human ZAG antibody (1:500) overnight at 4 °C or with normal rabbit IgG (1:500 dilution) as negative control. After washing, samples were incubated with a secondary antibody linked with fluorescein isothiocyanate (FITC; Biosource, Burlingame, CA, USA) diluted 1:300 for 3 h at 37 °C. Finally, the fluorescent-stained sperm samples were viewed and counted under a LSCM (Carl Zeiss LSM-510, Jena, Germany).
Flow cytometry (FCM)
Purified spermatozoa were washed twice with HEPES buffer (pH 7.3) containing 1% BSA and 0.5 mM CaCl2, and then adjusted to the concentration of 1x106 cells/ml. Cells were incubated with rabbit anti-human ZAG antibody diluted 1:1000 overnight at 4 °C or with normal rabbit IgG (1:500 dilution) as negative control. After samples were washed twice with HEPES buffer, FITC-labeled goat anti-rabbit antibody diluted 1:1000 was used as a secondary antibody and incubated for 1 h at room temperature. Finally, fluorescence was measured by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), and the percentage of fluorescent cells was analyzed using CellQuest software (Becton Dickinson). The same treated samples were also analyzed on a LS-50B luminescence spectrometer (Perkin–Elmer, Norwalk, CT, USA) to measure fluorescent intensity at 510 nm (490 nm excitation).
Measurement of sperm kinematics
Donors’ sperm isolated by the swim-up method were collected, washed, and resuspended in Tyrode solution with 3% BSA. Before the experiment of kinematics, the sperm concentration was adjusted to 10x106/ml. In total, 35 individual sperm samples from different healthy donors were used in this motility experiment. Swim-up spermatozoa were incubated in the presence or absence of prepared and purified rabbit anti-ZAG antibodies (without sodium azide) or normal rabbit IgG at different concentrations (1, 10.0, 50.0, and 100.0 µg) for up to 2 h at 37 °C. Sperm and forward motility indices were evaluated after 0, 60, 90, and 120 min of incubation with the antibodies using CASA (Hamilton-Thorn Research, Beverly, MA, USA).
Measurement of intracellular cAMP and PKA levels
For the measurement of intracellular cAMP and PKA levels in human spermatozoa, 18 individual swim-up spermatozoa populations (every six individual samples for each experiment and three experiments were repeated totally) were incubated with or without rabbit anti-ZAG antibodies (or added cAMP at the concentration of 15 pmol/108 cells) for 2 h at 37 °C. Subsequently, the samples were washed with PBS and centrifuged for 20 min at 4 °C. Sediments were then collected and assayed for intracellular cAMP using cAMP Immunoassay kit (R&D Systems, Minneapolis, MN, USA), as well as for PKA activity using ProFluor PKA Assay (Promega), according to the manufacturer’s instructions.
Statistical analysis
For each sample, kinematical parameters were expressed as the median of analyzed sperm tracks per man. Data were recorded and analyzed using the SAS 8.2 statistical software. Sperm kinematics was analyzed using the Student–Newman–Keuls one-way ANOVA test. P<0.05 was considered statistically significant. Values reported were mean ± S.E.M.
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Footnotes |
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References |
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