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RESEARCH |
Shanghai Key Laboratory for Reproductive Medicine, Departments of Histology and Embryology and 1 Department of Patho-Physiology, School of Medicine, Shanghai Jiao Tong University, No. 280, Chong Qing Rd. (South), Shanghai 200025, China
Correspondence should be addressed to Z Ding; Email: zding{at}shsmu.edu.cn
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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; Jones 1998). While the caudal epididymis acts as a sperm reservoir, the caput and corpus are responsible for sperm maturation (Moore 1998, Gatti et al. 2004, Franca et al. 2005, McLean 2005). Mammalian spermatozoa undergo morphological, biochemical, and physiological modifications within the epididymis, which has a very active secretory and reabsorption function (Cooper 1998, Holland & Nixon 1998, Dacheux et al. 2003, 2005, Toshimori 2003). Throughout its length, the epididymis is able to create sequential changes in the composition of luminal fluid, e.g. ions, solutes, proteins, and lipids (Kirchhoff et al. 1998, Nixon et al. 2002, Saez et al. 2003, Frenette et al. 2004, Gatti et al. 2005). The epithelial cells of the epididymis form a barrier to create a unique microenvironment in the lumen, where interactions between epithelial cells and spermatozoa take place via the fluid (Amann et al. 1993, Dacheux et al. 2003, 2005, Chan & Zhang 2005, Sullivan et al. 2005). Thus, the interactions between sperm and the various molecules in the luminal fluid strongly indicate that these complex interactions are crucial for sperm maturation.
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In this study, we employed high-resolution two-dimensional gel electrophoresis (2DE) and mass spectrometry to investigate protein variations in rat spermatozoa as they traverse the epididymis. We found that several proteins, including the ERp29 precursor, initially reported in mammalian spermatozoa, were modified/modulated significantly while undergoing epididymal maturation. These findings may provide crucial information for not only investigating the modification of sperm membrane, but also elucidating the mechanism of sperm maturation through the epididymis.
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Materials and Methods |
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Animals and isolation of epididymal sperm
Animal experiments were conducted according to the International Guiding Principles for Biomedical Research Involving Animal, as promulgated by the Society for the Study of Reproduction. Adult male Sprague–Dawley rats (aged 90–120 days) were purchased from the Shanghai Laboratory Animal Center (Jiu-Ting, Shanghai, China). Caput and caudal spermatozoa were extracted from every four rats for each experiment. Immediately, after the animals were killed, the epididymides were separated, and fat and overlying connective tissue carefully dissected out. Epididymal spermatozoa from caput and caudal segments were obtained following the method previously described by Acott and Hoskins (Acott & Hoskins 1978). Spermatozoa were collected and centrifuged at 1000 g for 15 min. To remove cytoplasmic droplets, extraneous materials, and cells, spermatozoa were centrifuged through a 10–50% discontinuous Percoll gradient. Finally, sperm concentration was assessed by hemocytometry. Contamination by somatic cells in each sperm preparation was <0.1%.
Protein extraction and concentration
Spermatozoa were homogenized in analysis buffer containing 1.5% Triton X-100 and 1 mM PMSF. 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. Ultrasonification (50 W, 10 sx3 times, VC600, Sonics and Materials, Inc., Newtown, CT, USA) and centrifugation (12 000 g, 10 min) were then applied to get rid of the sediment. Membrane protein was isolated with the ReadyPrep Protein Extraction Kit (membrane I). For isoelectric focusing electrophoresis (IEF), the soluble fraction was desalted and concentrated through centrifugal filter units (Centricon Centrifugal Filter Unit with Ultracel YM-10 membrane; Millipore, Billerica, BA, USA), and then processed with the ReadyPrep 2D cleanup kit. The concentration of solubilized proteins was determined using the Reagent Compatible and Detergent Compatible protein assay kit.
2D gel electrophoresis and imaging analysis
Aliquots of 200 µg protein sample were diluted in ~300 µl rehydration buffer containing 8 M urea, 2% m/v CHAPS, 25 mM dithiothreitol, 0.2% Bio-lyte (3–10 pI range), and 0.002% bromophenol blue. The sample was then applied onto a 17 cm immobilized pH gradient strip with a linear range of pH 3–10, covered with mineral oil. First-dimension IEF was performed using a Protean IEF cell system at 17 °C under the following conditions: 50 V for 12 h, 250 V for 30 min, 500 V for 30 min, 1000 V for 1 h, 10 000 V for 2 h, and 10 000 V for 50 000 Vh(Vhour). After equilibrating and alkylating, the electrophoresed strips were sealed onto the top of the second-dimension gels (12% SDS-polyacrylamide gel) with 0.5% agarose. Silver staining using previously described modifications was followed (Rabilloud et al. 1992).
In this study, we repeated the 2DE experiment three times in order to confirm spot patterns before proceeding with further analysis. Replicates were scanned with a GS-800 calibrated densitometer using standardized parameters, and gel images were processed by PDQuest 7.2.0 software. Spot densities were determined after normalization, based on the total spot volumes on the gel. Protein spots, with significant changes in densities (paired t-test, P<0.05) in a consistent direction (increase or decrease), were considered to be different and selected for further identification.
In-gel digestion and peptide extraction
Selected protein spots were cut out from the 2DE gels using the Gelpix spot-excision robot (Genetix, Hampshire, UK), and washed three times with Milli-Q water. According to the manufacturer of ZipPlate micro-solid phase extraction (SPE) plate, gel pieces were transferred into ZipPlate micro-SPE plate wells (Millipore) and incubated in a silver de-staining solution (equal volumes of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate) in a dark chamber for 20 min at room temperature. After being washed twice with Milli-Q water, gel pieces were washed twice in 25 mM ammonium bicarbonate/5% acetonitrile (ACN), 25 mM ammonium bicarbonate/50% ACN, and 100% ACN. When the gel pieces were dried, proteins were digested overnight with 10 µl of trypsin (10 ng/µl, mass spectrometry grade, Promega) in 25 mM ammonium bicarbonate at 37 °C. Peptide fragments, extracted with 0.2% trifluoroacetic acid (TFA) for 30 min, were applied onto the C18 resin and then desalted with 0.2% TFA. Finally, the tryptic peptide mixtures were recovered by centrifugation at 1750 g for 15 s with 5 µl elution solution containing 50% ACN/0.1% TFA.
Mass spectrometry
Tryptic peptides were lyophilized and suspended in 2 ml of a matrix solution containing 10 mg/ml 
-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN and 0.1% TFA. A 0.7 ml aliquot was spotted onto the MALDI sample target plate. Peptide mass spectra were obtained from a MALDI-TOF/TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA, USA). Prior to real sample acquisition, six spots were calibrated for signal and parameter optimization. The peptide mass fingerprinting (PMF) was gained in the mass range of 800–4000 Da with ~3000 laser shots. To acquire spectra with mass accuracy of <25 ppm, trypsin autolysis peaks were employed for internal calibration. Five of the most intense peaks, excluding those from the matrix, background, trypsin autolysis, acrylamide, or keratin peaks, were selected for subsequent MS/MS data acquisition. Then, the collision-induced energy was adjusted to 561 027 Torr for MS/MS spectra acquisition.
Protein identification was processed and analyzed by Global Protein Server Workstation (Applied Biosystem), using MASCOT software of Matrix Science. Mass tolerance was limited to 50 ppm. Results from both the MS and MS/MS spectra were accepted as ‘good identification’ when the global protein server (GPS) score confidence was >95%.
Western blotting
SDS-PAGE was conducted on 25 µg solubilized sperm protein using 12% polyacrylamide gels. Separated proteins were then transferred to PVDF membranes (GE Healthcare, Waukesha, WI, USA), using a semi-dry transfer apparatus (Bio-Rad). Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% BSA. Immunoblotting was performed with rabbit polyclonal ERp29 antibody (Abcam, Cambridge, MA, USA) at 1:1000 dilution overnight at 4 °C. After washing with TBS, membranes were then incubated with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP, DakoCytomation, Glostrup, Denmark) at a 1:4000 dilution for 1 h at room temperature. Finally, signals were detected by ECL (ECL Plus, GE Healthcare Waukesha). In the meantime, ß-actin served as the internal control.
Immunofluorescent staining
Tissue samples were obtained from rat epididymis, and snap frozen with Tissue-Tek optimal cutting temperature (OCT) compound (Fisher Scientific Co., Pittsburgh, Pennsylvania, USA). The segments of epididymal caput and cauda were cut into 6 µm thick frozen sections. Caput and caudal spermatozoa were collected and smeared onto separate slides. Tissue sections and sperm slides were fixed in ice-cold acetone for 30 min, and then blocked with 5% BSA in PBS for 30 min at room temperature, and later incubated either with rabbit anti-rat ERp29 antibody (1:500 dilution) in PBS overnight at 4 °C or with normal rabbit IgG (1:500 dilution) as control. Samples were washed with PBS and incubated with a secondary antibody linked with fluorescein isothiocyanate (1:300 dilution; Rockland, Gilbertsville, PA, USA) for 3 h at 37 °C. Digital photographs of fluorescent sections were viewed under a laser scanning confocal microscope (LSCM) and analyzed using the image analysis KS400 version 3.0 software packages (Carl Zeiss LSM-510; Carl Zeiss Vision, Munchen, Germany). The ratios of high fluorescence area (190–255 gray level) in total fluorescence area (110–255 gray level) were calculated and compared. After taking photographs, the immunofluorescence-stained tissue sections were re-stained with hematoxylin/eosin (HE) and photos were taken again for cell discrimination.
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). Spot densities were determined, with normalization based on total spot volumes on the gels. Protein spots, with significant changes in densities (paired t-test, P<0.05) in a consistent direction (increase or decrease), were judged to be deregulated, and were cut out for identification. Forty-three such deregulated spots were located, of which 24 were up-regulated and 19 down-regulated. An example is shown in Fig. 4A, where the spot was significantly up-regulated in caudal spermatozoa.
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, Table 1). We were able to identify seven unique proteins with high confidence (Fig. 3; Table 1). However, 16 spots representing seven unique proteins meant that some proteins were represented by at least two distinct spots on the 2DE gels. As summarized in Table 1, each of these seven proteins is characterized by its predicated MW, pI, mean fold, Swiss-Prot accession number, and regional abundance.
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). In order to further confirm results of 2DE, western blot analyses were performed using poly-antibodies. Immunoreactions with the ERp29 precursor and ß-actin antibodies were observed on PVDF membranes containing proteins from the caput and caudal sperm (Fig. 4B). The blotting intensity for the ERp29 precursor increased significantly on the PVDF membranes that contained proteins from the caudal sperm compared with those from the caput, with ß-actin as an internal control. In western blotting of membrane proteins, the ERp29 precursor content was also higher in the caudal sperm than in the caput (Fig. 4C). These results verified that the ERp29 precursor is expressed mainly on caudal sperm.
Localization of ERp29 precursor in epididymis and spermatozoa
In order to further confirm the localization of the ERp29 precursor on epididymal spermatozoa, indirect immunofluorescent staining was performed using the anti-rat ERp29 antibody. Intense immunoreactivity was observed on the surfaces of whole caudal spermatozoa, including the sperm head (Fig. 5C). Caput spermatozoa, however, showed only a faint fluorescence on the anterior region of the tail (neck and middle piece, Fig. 5A). On the other hand, with the normal rabbit IgG as control, no evident reaction was detected on either the caput or caudal sperm (Fig. 5B and 5D). The immunofluorescence-stained epididymal frozen sections, re-stained with HE, demonstrated that ERp29 was localized in cytoplasm of epididymal epithelia (Fig. 6). In the same slide, the fluorescence intensity was significantly higher in the caudal epididymis than in the caput, measured and analyzed by LSCM and KS400. As the negative control, fluorescence was not detected in the sections incubated with normal rabbit IgG (figures not shown).
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Discussion |
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ERp29, a 29 kDa protein, was first revealed during proteomic investigations of mammalian tissues (Demmer et al. 1997, Mkrtchian et al. 1998, Hubbard & McHugh 2000). The ERp29 precursor has a signal peptide at the N-terminus that is cleaved proteolytically upon entry into the endoplasmic reticulum (ER). Mature ERp29 (25.6 kDa, Mr=29 000 on SDS-PAGE) has an N-terminal domain homologous to the thioredoxin-like domains in protein disulfide isomerase (PDI), and a C-terminal domain with similarities to the P5 subfamily of PDI (Hubbard et al. 2000, Liepinsh et al. 2001, Sargsyan et al. 2002a, 2002b, Mkrtchian & Sandalova 2006); a KDEL-variant motif at the C-terminus specifies retention in the ER. ERp29 is expressed ubiquitously in mammalian tissues, and homologous proteins have been identified in organisms as primitive as the fruit fly (Shnyder & Hubbard 2002, MacLeod et al. 2004, Park et al. 2005). These proteins are regarded to be specifically associated with the ER, mitochondria, Golgi, nuclei, or cell membrane (Huang et al. 2002). In humans, ERp29 is encoded by a single gene on chromosome 12 that has been highly conserved during mammalian evolution (Hubbard & McHugh 2000, Mkrtchian & Sandalova 2006). As ERp29 lacks the double cysteine motif, essential for PDI redox activity, it has been suggested that it plays a role in protein maturation and/or secretion, related to the chaperone function of PDI (Liepinsh et al. 2001, Mkrtchian & Sandalova 2006), primarily in the production of endomembrane and secretory proteins (Shnyder & Hubbard 2002, MacLeod et al. 2004). ERp29 is considered to be another major reticuloplasmin, thus adding distinct functionality to the ER machinery (Hubbard & McHugh 2000, MacLeod et al. 2004, Hermann et al. 2005). All available data point to its important role in the formation of early secretory pathways within the ER (Mkrtchian et al. 1998, Hubbard et al. 2000, Liepinsh et al. 2001, Sargsyan et al. 2002a, 2002b, Shnyder & Hubbard 2002, MacLeod et al. 2004, Hermann et al. 2005, Park et al. 2005, Mkrtchian & Sandalova 2006), possibly by participating in the folding of proteins. ERp29 assists in the protein folding as well as in the secretion of the secretory/plasma membrane proteins under the close cooperation with other ER chaperones and the ER stress signaler, PKR-like ER kinase (Park et al. 2005).
ERp29 also has distinctive regulatory and biochemical properties, including a distinct lack of calcium-binding activity (Demmer et al. 1997, Hubbard et al. 2000, Liepinsh et al. 2001, Shnyder & Hubbard 2002, MacLeod et al. 2004, Hermann et al. 2005). Further evidence for ERp29’s unique nature includes its novel tertiary structure (Liepinsh et al. 2001), lack of classical ER stress-response elements (Sargsyan et al. 2002a, 2002b), ability to bind other ER proteins, and mediating membrane penetration or fusion (Magnuson et al. 2005). However, the functional characterization of ERp29, e.g. its specific molecular function, is far from completion and remains unknown.
In our 2DE map and mass spectrometry analyses, the ERp29 precursor was initially detected on rat spermatozoa. The quantity of the protein extracted from both spermatozoa and sperm membrane were dramatically increased during epididymal transit, as confirmed by western blot assay. Using indirect immunofluorescence, intense immunoreactivity was clearly observed on the whole tail of caudal spermatozoa, while only faint fluorescence was seen on the anterior region of the tail (neck and middle piece) of caput spermatozoa. In order to reveal the potential source of the protein on spermatozoa, immunofluorescence and HE staining for frozen sections demonstrated that ERp29 expression in rat epididymal epithelia was gradually increasing during the spermatozoa transit. ERp29 appears to be a secretory protein and might be absorbed by spermatozoa during epididymal transit. The PSORT II analysis program (http://www.psort.org/) predicted the four most probable localizations for ERp29 as being: extracellular (44.4%), mitochondrial (22.2%), ER (22.2%), and vacuolar (11.1%). These clues reveal that the ERp29 precursor might not only be an essential functional component of ER machinery, but also have its molecular function on the sperm membrane. ERp29 function may be comparable with that of calreticulin, which is another resident protein of the ER, present in the acrosome of both round spermatids and mature sperm, that is regarded to be an important factor in signal transduction in the reproductive system (Nakamura et al. 1993, Naaby-Hansen et al. 2001, Park et al. 2001, Ho & Suarez 2003). Importantly, immunofluorescence and subcellular fractionation studies showed co-localization of ERp29 with calreticulin in the ER (Ferrari et al. 1998). On the other hand, caudal spermatozoa have an intact mitochondrial sheath, whose membrane maturation also needs the ERp29 involvement. Induction of caudal spermatozoa motility is likely dependent on a transient increase in cytosolic Ca2+ followed by a secondary increase in cyclic AMP. The rate of calcium transport into the sperm relies on the ability of mitochondrial Ca2+ uptake to maintain an adequate gradient for plasmalemma Ca2+ influx, whereas the rate of such transport is dependent in turn on the rate of aerobic respiration fueled by various substrates (Hoskins et al. 1978, Breitbart et al. 1990). Although ERp29 cannot bind calcium, it may have interactions with calcium-binding molecular chaperones on the membrane of mitochondrial sheath, e.g. calnexin or calreticulin, and indirectly influence calcium transport. Thus, ERp29 or its precursor could be presumed to be involved in the regulation of such cell functions such as sperm motility and acrosome reaction, as well as membrane maturation in sperm/organelle.
Mere identification of spermatozoa ERp29, however, is not sufficient to identify its entire biological function(s) in this event. Additional studies are still warranted to further clarify its roles. Undoubtedly, such endeavors may greatly help us to better understand and elucidate the mechanism(s) of the protein function and sperm maturation during epididymal transit.
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Acknowledgements |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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