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- Original Article -

Establishment of a high-resolution 2-D reference map of human spermatozoal proteins from 12 fertile sperm-bank donors

Ling-Wei Li, Li-Qing Fan, Wen-Bing Zhu, Hong-Chuan Nie, Bo-Lan Sun, Ke-Li Luo, Ting-Ting Liao, Le Tang, Guang-Xiu Lu

Institute of Reproduction and Stem Cells Engineering, Central-South University, Changsha 410078, China

Abstract

Aim: To extend the analysis of the proteome of human spermatozoa and establish a 2-D gel electrophoresis (2-DE) reference map of human spermatozoal proteins in a pH range of 3.5_9.0. Methods: In order to reveal more protein spots, immobilized pH gradient strips (24 cm) of broad range of pH 3_10 and the narrower range of pH 6_9, as well as different overlapping narrow range pH immobilized pH gradient (IPG) strips, including 3.5_4.5, 4.0_5.0, 4.5_5.5, 5.0_6.0 and 5.5_6.7, were used. After 2-DE, several visually identical spots between the different pH range 2-D gel pairs were cut from the gels and confirmed by mass spectrometry and used as landmarks for computer analysis. Results: The 2-D reference map with pH value from 3.5 to 9.0 was synthesized by using the ImageMaster analysis software. The overlapping spots were excluded, so that every spot was counted only once. A total of 3 872 different protein spots were identified from the reference map, an approximately 3-fold increase compared to the broad range pH 3_10 IPG strip (1 306 spots). Conclusion: The present 2-D pattern is a high resolution 2-D reference map for human fertile spermatozoal protein spots. A comprehensive knowledge of the protein composition of human spermatozoa is very meaningful in studying dysregulation of male fertility. (Asian J Androl 2007 May; 9: 321_329)

Keywords: human spermatozoal proteins; ImageMaster; mass spectrometry; overlapping narrow range; 2-D gel electrophoresis

Correspondence to: Dr Li-Qing Fan, Institute of Reproduction and Stem Cells Engineering, Central-South University, No.88, Xiangya Road, Changsha 410078, China.

Tel: +86-731-480-5322 Fax: +86-731-480-5322
E-mail: fanliqingszzx@sina.com
Received 2006-08-01 Accepted 2006-11-28

DOI: 10.1111/j.1745-7262.2007.00261.x

1 Introduction

The mammalian spermatozoa is a highly differentiated cell, produced through a complex series of morphogenetic events. In the later stages of spermatogenesis, unique cellular components, such as the acrosome, the outer dense fibers, and longitudinal columns and ribs of the fibrous sheath are formed. Also, the nuclear chromatin becomes highly condensed and transcriptionally inactive [1]. For this reason, research on spermatozoal mRNA has limits. However, with the recently developed proteomic technology, which takes 2-D gel electrophoresis (2-DE) and mass spectrometry as key techniques, we have the advantages of high through-put analysis and an integrated view of the studied target cell to study properties of spermatozoa at the molecular level. A comprehensive knowledge of the protein composition of human spermatozoa is useful to study dysregulation of male fertility.

Some previous investigations have applied 2-DE and accessory techniques to study human spermatozoal proteins, but the number of protein spots distinguished in two-dimensional maps thus far is rather low. In 1990, Naaby-Hansen et al. [2] acquired an electrophoretic map of acidic and neutral human spermatozoal proteins and obtained over 260 protein spots. In 1992, Kritsas et al. [3] obtained over 500 spermatozoal protein spots with molecular mass ranging from 12 to 105 k and isoelectric points from 5.0 to 8.5. In 1994, Xu et al. [4] acquired more than 600 spermatozoal protein spots with molecular mass ranging from 7.9 to 93.5 k and isoelectric points between 4.0 and 7.0. In 1997, Naaby-Hansen et al. [5] used isoelectric focusing (IEF)/polyacrylamide gel electrophoresis (PAGE) and non-equilibrium pH gradient electrophoresis (NEPHGE)/PAGE in the research of vectorially labeled surface proteins of human spermatozoa, and acquired a composite 2-DE image showing 1397 human spermatozoal proteins that belong to the membrane protein fraction. In 2005, Daniel et al. [6] identified more than 1 760 human sperm proteins by liquid chromatography and tandem mass spectrometry. In the present study, to obtain a high resolution 2-DE map of human spermatozoal proteins, we used multiple overlapping narrow immobilized pH gradients (IPG) and computerized 2-D reference map synthesis.

2 Materials and methods

2.1 Preparation of spermatozoa

The semen samples were collected from twelve eligible sperm donors (aged 21.38 ± 2.39 years) from the Human Sperm Bank Department of Citic Xiangya Reproductive and Genetic Hospital (Hunan, China), during the period of March to June 2005. All donors underwent a standardized screening protocol, which included physical examination, review of medical and family history, and infectious disease screening according to the standards of the Ministry of Health of China. After a 3_5 days abstinence period, ejaculates were collected via masturbation, and only the ejaculates meeting the criteria of the World Health Organization (WHO) for normal semen parameters were included in this study [7]. All donors have proven fertility, and donated sperm five times at intervals of 4.11 ± 0.56 days. This was approved by the National Human Reproduction Ethic Investigation Committee, and all donors gave written consent.

After liquefaction, the semen were separated by Percoll (Sigma, Missouri, USA) density centrifugation as described by Naaby-Hansen et al. [5] with some modifications. In brief, the sample was overlaid on a two-layer Percoll density gradient consisting of 90% and 45% isotonic Percoll solutions prepared in Ham's F-10 medium (HyClone, Utah, USA), the latter forming the upper layer. After centrifugation at 300 × g for 30 min at room temperature, the sperm pellet was collected at the bottom of the 90% layer, then washed with PBS three times by centrifugation at 450 × g for 10 min at room temperature. All samples showed > 90% spermatozoa with good motility. The spermatozoa were pooled and frozen immediately in the liquid nitrogen until use.

2.2 Solubilization of spermatozoa

All samples were prepared at the same time. Spermatozoa were routinely solubilized in lysis buffer consisting of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L Tris, 75 mmol/L DTT, and 2 mmol/L PMSF, as previously described but with some modifications (75 mmol/L DTT instead of 65 mmol/L DTT, additional 40 mmol/L Tris) [8, 9]. This was followed by centrifugation at 10 000 × g for 30 min at 4ºC. After protein concentration determination by the 2-D Quant Kit (Amersham Bioscience, Uppsala, Sweden), the supernatant was applied to the first dimensional electrophoresis or stored in _80ºC freezer for further use. In addition, parts of the supernatant were processed by the 2-D Clean Up Kit (Amersham Bioscience, Uppsala, Sweden) for protein precipitation, or by trichloroacetic acid (TCA) in acetone solution for use in pH 6_9 IPG strips [9].

2.3 2-D gel electrophoresis

The first dimensional electrophoresis was performed on an Ettan IPGphor II isoelectric focusing apparatus (Amersham Bioscience, Uppsala, Sweden). The broad range pH 3_10 IPG strip (24 cm long) and the narrower range (pH 6_9) strip and acidic narrow pH range strips cover only one pH unit, 3.5_4.5, 4.0_5.0, 4.5_5.5, 5.0_6.0 and 5.5_6.7, respectively. The strips are commercially available from Amersham Bioscience. The rehydration buffer containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 40 mmol/L DTT, 0.5% IPG buffer and a few grains of bromophenol blue was used. For the pH 6_9 IPG strip, rehydration buffer was modified with additional 10% isopropanol and 5% glycerol, and with replacement of DTT by Destreak (Amersham Bioscience, Uppsala, Sweden) [9_11]. The sample-loading amount and electrophoresis-running parameters are given in detail in Table 1.

After the isoelectric focusing electrophoresis, the IPG strips were equilibrated in the equilibration solution (6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate [SDS] and 50 mmol/L Tris-HCl, pH8.8) containing 1% DTT for 15 min and then in the same solution without DTT but with 2.5% iodoacetamide (IAA) for 15 min [9]. The second dimensional electrophoresis was carried out in a homogeneous SDS-PAGE (12.5%) on Ettan DALTsix electrophoresis unit (Amersham Bioscience, Uppsala, Sweden). The electrophoresis conditions were modified to 15ºC, 2.5 watt (W)/gel for 30 min and then 20 W/gel for 4 h.

The protein pattern was visualized by mass spectrometry-compatible silver staining, or Coomassie Brilliant Blue (CCB) staining, which is better than the former for the MS analysis [12]. All the stained gels were scanned at 300 dpi resolution using ImageScanner (Amersham Bioscience) and digitized and analyzed using 2-D ImageMaster software (Amersham Bioscience). Parameters of detection of spots by the software are as follows: Smooth 4, MinArea 6 and Saliency 2.00000.

2.4 In-gel digestion

Some mass spectrometry-compatible silver and CCB-stained spots correspondent among different strips were randomly selected, including the spots acting as the landmarks and cut from the gels for data analysis. The silver-stained spots were destained by 30 mmol/L potassium ferricyanide and 100 mmol/L sodium thiosulfate for 2 min and the CCB stained spot samples by 50% acetonitrile (made up with 25 mmol/L NH₄HCO₃), respectively. All destained spots were dried in a vacuum pump, reduced by 10 mmol/L DTT (made up with 50 mmol/L NH₄HCO₃) for 30 min at 56ºC, followed by the replacement of DTT and alkylation for 30 min in dark with 55 mmol/L IAA solution (made up with 50 mmol/L NH₄HCO₃), then dehydrated in 100 mmol/L NH₄HCO₃ and digested by adding the trypsin digestion work solution (T 6567; Sigma, Missouri, USA)) and incubation for 15_18 h at 37ºC.

2.5 TOF/TOF mass spectrometry (MS) analysis

MS analysis was performed according to Myung et al. [13] after in-gel digestion. The extracted peptides were directly applied onto a target (AnchorChip, Bruker Daltonics, Germany) that was loaded with α-cyano-4-hydroxy-cinnamic acid (CHCA) (Bruker Daltonics, Germany) matrix thin layer. The mass spectrometer used in this work was an Ultraflex TOF/TOF (Bruker Daltonics, Germany) operated in the reflector for matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) peptide mass fingerprint (PMF) or in LIFT mode for MALDI-TOF/TOF with a fully automatic mode using the FlexControl software. Database searches were performed through Mascot using combined PMF and tandem mass spectrometry (MS/MS) datasets via BioTools 2.2 software (Bruker Daltonics, Germany). Two parameters were modified according to Myung et al. [13], a maximum of four instead of three precursor ions per sample were chosen for MS/MS analysis, and MS/MS tolerance of 1.0 Da instead of 0.5 Da for MS/MS search.

3 Results

3.1 Analysis of 2-D gels

The samples loaded for electrophoresis in pH 3_10, 6_9, and acidic narrow pH gels, contained 0.5 mg, 0.8 mg, and 1.5 mg of protein, respectively. Analysis of spots with mass spectrometry was performed after silver-staining. After establishment of a reliable protocol, the analysis of all 2-D maps revealed excellent reproducibility. All the kinds of pH 2-DE were repeated at least five times, and the best 2-D patterns were used for analysis. 2-D maps of different pH gradient ranges of human spermatozoal proteins are shown in Figures 1 and 2. The number of spots detected by software analysis in pH 6_9 gel, each narrow pH range gel, and the corresponding zones of the pH 3_10 gel, are presented in the histogram of Figure 3.

Total of 1 306 distinct protein spots were detected in the broad pH range 2-DE map. Compared to the map, the sensitivity was enhanced by using narrower and narrower pH IPG strips. About 168 distinct spots were resolved in the pH 3.5_4.5 2-D gel compared to only 75 spots in the corresponding zones of the pH 3_10 2-D gel; 572 in the pH 4.0_5.0 2-D gel compared to only 191 spots; 1 463 in the pH 4.5_5.5 2-D gel compared to 313; 1 370 in the pH 5.0_6.0 2-D gel compared to 344; 1 121 spots in the pH 5.5_6.7 2-D gel compared to only 347; and 1 061 in the pH 6_9 2-D gel compared to only 631.

Our results also show that, in comparison with broad pH range IPG strips, in the narrow pH range gels not only the spots became further apart, but several visually single spots were divided into two or more protein spots [14]. These apparent improvements are shown in Figure 4. There are a total of 5 755 spots detected from the different pH gradient 2-D maps, excluding the broad pH range map. Of these 5 755 spots, approximately 3 872 independent spots could be detected on the composite narrower and narrow pH range gels after excluding the overlapping spots (1 883) by data analysis.

3.2 Protein identification from 2-D gel spots by MS

To confirm the visually identified landmarks for different pH range gels, we selected several correspondent spots from the silver-stained gels and CCB-stained gels that were used in the data analysis. Both PMF and MS/MS were performed and all the outlined spots (selected as shown in Figure 1, the CCB-stained gel is not shown) have been identified and listed in Table 2.

3.3 Composing a 2-D reference map of human fertile spermatozoal proteins

All spermatozoal proteins were extracted at the same time, to try to exclude experimental variation. First, the identical landmarks between two consecutive pH range gels (for example, pH 4.0_5.0 and pH 4.5_5.5) were matched, and the other overlapping identical spots were deleted. Then, the composite 2-D reference map that has a new large pH range (for example, pH 4.0_5.5) was composed. Other consecutive pH range gels can be composed in the same way. Finally, a 2-D reference map of human fertile spermatozoal proteins in the pH range 3.5_9.0 was composed through image analysis by using 2-D ImageMaster software (Figure 5). In the composite narrower and narrower pH ranges map 3 872 independent spots could be detected, excluding the overlapping spots (1 883). The results of 2-D gel were reproductive in several times, so we think this 2-D reference map could be used for comparison with 2-D analysis of other sperm samples by different researchers.

4 Discussion

The 2-DE is a useful technique in proteomics, giving reproducible and high-resolution data of complex protein mixtures [14, 15]. In the present study, we have attempted to improve resolution, to obtain more protein spots in a 2-D pattern of human spermatozoal proteins, by employing currently available overlapping acidic narrow pH range IPG gels strips. Unfortunately, the narrow-pH range gels strips in the basic range are still unavailable from Amersham Bioscience. We achieved a high resolution 2-DE map of human spermatozoal proteins, identifying a total of 3 872 distinct protein spots by using multiple overlapping narrow IPG. To our knowledge, the present high resolution 2-D reference map contains the highest number of human spermatozoal protein spots reported so far.

For the pH 6_9 IPG strip, the replacement of the charged reductant DTT in the rehydration solution by DeStreak or by uncharged reductant tributyl phosphine (TBP) can significantly reduce horizontal streaking, resulting in 2-D maps with spot patterns more simplified and reproducible [15, 16]. Our experiments also showed significant improvement by using TBP, especially by using DeStreak (data not shown). There was still much horizontal streaking in pH 6_9 2-DE map, and we suppose that this streaking may come from insolubility, migration or precipitation of some proteins [11, 17], and high sample-loading may also contribute to the horizontal streaking.

It is very difficult to acquire a perfect 2-D pattern for the pH 3.5_4.5 IPG strip in our experiments (shown in Figure 2). According to previous studies [18], µsol-IEF (a sample-prefractionation method) may result in a 2-DE pattern rather better, with the major interfering proteins removed by the prefractionation procedure. This method can separate proteins using much higher protein-loading, such as on the extreme acid narrow pH range IPG gels, and obtain a good 2-D pattern. Furthermore, the detection of low abundance proteins was greatly enhanced by using the prefractionation method. The method can also improve the result for the 2-DE pattern of other pH gradient gels. We are now applying this to improve the 2-D map for pH 3.5_4.5 IPG strip.

Enrichment of spots can be obtained by loading different amounts of protein. High-sample loading can enrich more proteins that have a low abundance, while low sample-loading can reduce spot trains and hence acquire better separation of the proteins that have a high abundance. We obtained more than 1 350 spots of human spermatozoal proteins from broader pH range IPG strip (18 cm in length) of pH 3_10 by using different amounts of protein loading in our study (data not shown).

It is to be noted that the present computerized 2-D reference map cannot be regarded as perfect. First, there are still some flaws in the 2-D technique, such as the inherent complexity of the procedure, and the precision of analysis is dependent on the operator to some degree. Second, the 2-D analysis software still needs further improvement. In addition, the identification by MS of the landmark picked out from the pH 3.5_4.5 2-D gel is not convincing; the spot could not be identified by MS analysis and hence is not listed in Table 2, and cannot be used in data analysis. To avoid the repeated spots between pH 3.5_4.5 gel and pH 4.0_5.0 gel to be added to the composite gel, some independent spots were left out of the analysis subjectively. On all accounts, the computerized 2-D map of human spermatozoal proteins needs improvement.

We obtained 16 identified spots by MS, including four proteins of proteasome subunits (spot No. s 5, 10, 13 and 15). The human sperm proteasome plays an important role in fertilization [19]. A comprehensive knowledge of the protein composition of human fertile spermatozoa is useful in elucidating cellular processes at the level of the proteomics and studying dysregulation of male fertility. Differential extraction of proteins from spermatozoal cytoplasm and nucleus is difficult because of the complex cyto-architecture of the spermatozoa. In fact, the protein spots from the present 2-D pattern represent a very small part of the total proteins of the whole cell. In addition, because of differential mRNA splicing and extensive co-translational and post-translational modifications of proteins, more proteins and variant forms are expressed than the number of expressed genes [20]. So further work needed to study the proteome of human spermatozoa.

Acknowledgement

This work was supported by two grants from the National Science Foundation of China (No. 30170480 and No. 30470884). We would like to thank Professor Guang-Yin Lu for enthusiastic reading and amending of the manuscript. We would like to thank Mr Ji-Xian Xiong, College of Life Science at the Hunan Normal University for enthusiastic support of technology and theory of MS. We are also very grateful to collaborators in our laboratories for help and valuable discussions and suggestions during the course of this work.

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