Mol. Hum. Reprod. Advance Access originally published online on September 28, 2005
Molecular Human Reproduction 2005 11(9):677-681; doi:10.1093/molehr/gah217
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Fine resolution of human sperm nucleoproteins by two-dimensional electrophoresis
1Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, 2Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba, 3Department of Physics, Osaka Medical College, Takatsuki, Osaka, Japan
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. E-mail: naoaki{at}sc.itc.keio.ac.jp
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Abstract |
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Human sperm nucleoproteins consist of protamines and histones. Changes in composition of these proteins are thought to correlate with spermatogenesis and may be involved in some instances of male infertility. We sought to separate sperm nucleoproteins including variants of protamine using an improved two-dimensional electrophoretic method, with the aim of comprehensively analysing all sperm nucleoprotein constituents. After extracting nuclear basic proteins from the sperm of normal volunteers, we analysed these proteins on a gel sheet by a radical free, highly reducing method based on Kaltschmidt and Whittmann’s two-dimensional electrophoresis. Basic proteins from sperm nuclei were separated clearly into 12 spots. By amino acid sequence analysis, these spots corresponded to protamine 1 (P1)- (five spots), protamine 2 (P2)-related proteins (six spots) and testis-specific histone H2B (one spot). The N-terminal amino acid sequences of the six P2-related proteins were compatible with those of HPI1, HPI2, HPS1, HPS2, HP2 and HP3, and quantitative comparison could be performed. In conclusion, human sperm nucleoproteins including all P2-related variants could be analysed quantitatively with high resolution on a single electrophoretic gel.
Key words: histone/human/nucleoprotein/protamine/spermatozoa
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Introduction |
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Several groups of investigators have suggested that a sperm maturation defect resulting from incomplete replacement of nuclear histones might be associated with human infertility. In sperm from fertile men, protamine 1 (P1) and protamine 2 (P2) can be found at a ratio of approximately 1:1 (Balhorn et al., 1988
); in contrast, various studies have reported changes in relative amounts of P1 and P2 between fertile and infertile men (Chevaillier et al., 1987; Balhorn et al., 1988; Bach et al., 1990; Blanchard et al., 1990; Belokopytova et al., 1993; de Yebra et al., 1993; Bench et al., 1998; de Yebra et al., 1998) or in patients pursuing IVF programs (Colleu et al., 1997; Khara et al., 1997; Carrell and Liu, 2001; Steger et al., 2001, 2003). Subsequent studies have reported that some infertile men have diminished amounts of P2 in sperm that may reflect incomplete processing of P2 precursors (de Yebra et al., 1998).
In the above studies, separation of sperm nucleoproteins containing protamine had been carried out using one-dimensional (1-D) polyacrylamide gels (de Yebra and Oliva, 1993). However, 1-D electrophoresis has the drawback of overlaps between some protein fractions, resulting in insufficient separation of protamine variants or forms of protamine posttranslationally modified by acetylation or phosphorylation. No reports have delineated human sperm nucleoprotein composition including all P2-related variants based upon isolation on a single gel sheet. Non-equivalent two-dimensional (2-D) electrophoresis analysis is appropriate to this task; a single spot corresponds to a single protein. Separating sperm nucleoproteins in this manner would provide a more complete and accurate view of changes in composition of these proteins associated with male infertility. To our knowledge, however, many studies concerning sperm nucleoproteins have not yet made use of 2-D electrophoresis.
Precise separation of sperm nucleoproteins has been difficult because they are highly basic, rich in disulphide bridges, and very small. With an improved method of the radical-free, highly reducing (RFHR) 2-D electrophoresis developed in this laboratory (Wada, 1986a,b), quantitative yield and separation ability for such proteins have improved greatly. Disulphide bridging is blocked by maintaining the gels under highly reducing conditions, whereas artefactual modification of proteins by free radicals in the gels is avoided by thorough scavenging. In this study, we sought to separate sperm nucleoproteins with high resolution on one electrophoretic gel sheet to quantitatively analyse histones, P1, P2 and their precursors.
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Samples
Semen samples were collected from male volunteers demonstrated to be fertile by a history of producing offspring. Semen ejaculates obtained at the hospital by masturbation were kept at 37°C until complete liquefaction had occurred. Standard semen analysis (semen volume, sperm concentration, total sperm count and progressively motile sperm concentration) was performed within 1 h of obtaining the sample. Spermatozoa numbering 50 x 106 per volunteer were washed three times with M199 (Invitrogen, Carlsbad, CA) (using 10 min of centrifugation at 1600 g each time) and twice with phosphate-buffered saline (PBS; using 5 min of centrifugation at 1600 g each time). Pellets were then kept frozen (–80°C) until use.
Extraction of nuclear proteins
Sperm nuclear basic protein was extracted according to the method described by Balhorn et al. (1977); with minor modification. Briefly, the sperm pellet was suspended with minimal sonication in 0.9 ml of 10 mM dithiothreitol (DTT) and 0.05 M trisaminomethane–HCl (Tris–HCl) at pH 8.0 for incubation at 4°C for 15 min. Cetyltrimethylammonium bromide (CTAB) was added to obtain a final concentration of 1%, and the sample was incubated for an additional 30 min at 4°C to allow dissolution of sperm tails. The heads were then pelleted by centrifugation at 3000 g, washed twice in 1% CTAB in 0.01 M Tris–HCl at pH 8.0, and then washed twice in Tris–saline. The purified sperm nuclei were dissolved in 0.1 ml of 5 M guanidine hydrochloride (Gdn-HCl)-0.01 M Tris at pH 8.0, in which the chromatin was allowed to expand at 4°C for 30 min. After sonication, the chromatin was diluted and urea, 2-mercaptoethanol (MSH), and sodium chloride (NaCl) were added to give final concentrations of 0.5 M Gdn-HCl, 3 M urea, 0.5 M MSH and 2 M NaCl. Following dissociation of the proteins at 4°C for 60 min, DNA was precipitated by the addition of HCl to a final concentration of 0.5 N. After precipitation at 4°C for 60 min, the DNA was pelleted by centrifugation at 14 500 g for 10 min. The nucleoproteins were precipitated from the appropriate pooled fractions with 20% trichloroacetic acid at 4°C for 30 min. The precipitate was then pelleted by centrifugation at 14 500 g, washed with acidified acetone, and dried in vacuo and stored at –30°C until 2-D gel analysis. As controls, nucleoproteins of HeLa cells were also extracted in the same manner as above except for the process of dissolution of sperm tails.
Electrophoresis
Extracted nucleoproteins (approximately 0.5 mg/gel) were analysed by the RFHR method of 2-D gel electrophoresis as previously described (Wada, 1986a,b), except that 6 M urea was replaced with 7 M urea, the acrylamide concentrations of the 0-D and 1-D gels were lowered from 8 to 7.2%, and gels 2-mm thick were used in place of 3-mm gels. Apparatus for RFHR 2-D electrophoresis was obtained from Nippon Eido (Tokyo, Japan).
Protein densitometry
Experiments using 2-D electrophoresis for the determination of the relative protamine amounts were carried out in two separate runs using the mixture of five samples. Coomassie Brilliant Blue (CBB) R-250 was used to stain the gel, which then was scanned using a flatbed scanner (Amersham Biosciences, Upsala, Sweden). Preprogrammed spot quantification was performed using Image Master 2D Elite software (version 2.0, Amersham Biosciences). Specifically, spot detection, spot measurement and background subtraction were performed. Following automatic spot detection, gel images were carefully edited. The quantity represented by a protein spot was expressed as the volume of the spot, which was defined as the sum of the intensities of all pixels making up the spot. To correct for variability of CBB staining and to reflect the quantitative variations in intensity of protein spots, spot volumes were normalized for all spots present in the gel.
Amino acid sequence analysis
Following separation by electrophoresis, proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Pall Bio Support Division, Port Washington, NY) using a semidry transfer blotter (Nippon Eido) and were detected by CBB staining. Stained protein spots were excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (Procise 494, Applied Biosystems, Foster City, CA, USA). Edman degradation was performed according to the manufacturer’s standard program and released phenylthiohydantoin derivatives of amino acid residues were identified by on-line high-performance liquid chromatography (120A; Applied Biosystems). Amino acid sequences obtained were compared with those of known proteins with the web-accessible search program FastA (http://www.dna.affrc.go.jp).
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Results |
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In each semen specimen, sperm nuclear basic proteins were separated by RFHR 2-D electrophoresis with high reproducibility. Typical separations of nucleoproteins isolated from human sperm (A) and somatic (HeLa) cells (B) are shown in Figure 1. Based on a comparison of the electrophoretic pattern with that of the somatic cells, which consists of only histone without protamines, one spot was recognized in a position similar to that of the histone of HeLa cells. Several additional spots, which were more basic and of lower molecular weight, appeared in the sperm nucleoprotein pattern; these were thought to represent protamines.
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Sperm nucleoproteins were separated into 12 spots. Amino acid sequence analysis showed that these spots corresponded variously to P1-family proteins, P2-related proteins, and testis-specific histone H2B (TH2B; Figure 2, Table I). Protamines in human sperm nucleoproteins resolved well on the gels, located essentially along respective diagonals representing two groups, group P1 and group P2 (Figure 2). Group P1 represented P1-family proteins and was composed of five spots with the same N-terminal amino acid sequence (ARYRXXR; Table I); the amino acid corresponding to ‘X’ could not be determined by Edman degradation and may represent the cysteine residues of P1. Group P2 consisted of six spots. These proteins corresponded to HPI1, HPI2, HPS1, HPS2, HP2 and HP3 respectively, because the N-terminal amino acid sequences were compatible with differences in N-termini between the reported testicular P2 variants (Table I). All P2-related molecular forms including P2 and its precursors were separated clearly from one another. Such resolution was highly reproducible under the conditions of electrophoresis described here.
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Amino acid sequences of human protamines reported in the UniProt database (http://www.ebi.uniprot.org/index.shtml) and previous studies (McKay et al., 1985, 1986; Ammer et al., 1986; Gusse et al., 1986) and the protamines that we analysed in this study are shown in Figure 3. The amino acids of these P2-related proteins corresponded well in sequence to the gene prm2 (accession number p04554). Specifically, N-terminal amino acid sequence of HPI1, VRYRVRSLSERSHEV, corresponded to residues 2– of prm2, that of HPI2, HGQEQ, corresponded to residues 22– of prm2, that of HPS1, QGLSP, corresponded to residues 34– of prm2, that of HPS2, SPEHVEVYERTHGQ, corresponded to residues 37– of prm2, that of HP2, RTHG, corresponded to residues 46– of prm2, and that of HP3, GQSHYRRR, corresponded to residues 49– of prm2. HP4 (45–of prm2), not detected in this study, is thought to be present only in extremely small quantities.
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Densitometry results obtained by averaging the quantitation from two gel samples are shown in Figure 4. Relative amount of TH2B extracted in this study was 282.6 as compared to the total amount of analysed nucleoprotein spots, 7364.8. Even considering the amount of several small spots near the TH2B spot, the total amount of histone analysed in this study accounted for less than 15%, of the previously reported quantity (Gatewood et al., 1987). In addition, the ratio of P1 to P2, measured in the same manner as in the previous 1-D electrophoresis study that seemed not to recognize spot 8, spot 9 or spot 10 in our study, was HP1/(HP2+HP3) = (spot 11 + spot 12)/(spot 6 + spot 7) = (1099.1 + 1051.6)/(1199.9 + 901.6) = 1.02. The value of P1/P2 (1.02) overlapped greatly with ratios reported in a study concerning fertile men (Balhorn et al., 1988). Relative quantification of all P2-related forms, HPI1:HPI2:HPS1:HPS2:HP2:HP3 resulted in 367.3/367.3:220.3/367.3:409.9/367.3:765.2/367.3:1199.9/367.3:901.6/367.3 = 1.000:0.6000:1.116:2.083:3.267:2.455.
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Discussion |
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Approximately 85% of human sperm nuclear histones are replaced by protamines during late spermatogenesis (Barone et al., 1994
) accompanied by a dramatic change in the structure of chromatin (Fuentes-Mascorro et al., 2000; Brewer et al., 2002). In humans, sperm nuclear proteins include two families of protamines, P1 and P2, which differ in molecular mass, amino acid composition and sequence. The P1 family is represented by HP1 with different degrees of phosphorylation and is related to other mammalian P1 protamines. The P2 family includes HP2, HP3 and HP4 proteins, which differ only by an amino-terminal extension of one to four residues (Gusse et al., 1986; McKay et al., 1986; Sautiere et al., 1988; Arkhis et al., 1991; Oliva and Dixon, 1991; Chauviere et al., 1992; Bianchi et al., 1994). HP1, HP2, HP3 and HP4 have 50, 57, 54 and 58 amino acids, respectively (Figure 3). HP1 is synthesized as a mature protein product. In contrast, P2 family proteins are synthesized initially as a precursor protein of 102 amino acids (Balhorn, 1989; Alimi et al., 1993a) directed by a single-copy gene located on chromosome 16 in humans (Domenjoud et al., 1990). P2 family proteins attain mature form by proteolytic cleavage of the amino terminus (Balhorn, 1989). 1-D polyacrylamide gel electrophoresis has detected minor components of human sperm nucleoproteins showing a rate of migration that is intermediate between those of histones and protamines (Sautiere et al., 1988). These proteins therefore have been called intermediate basic proteins, designated HPI1, HPI2, HPS1 and HPS2 (Gusse et al. 1986). Because intermediate basic proteins, which have lengths of 66–100 residues, share a common C-terminal domain identical to the amino acid sequence of HP2, HP3 and HP4 (Sautiere et al., 1988; Martinage et al., 1990; Alimi et al., 1993a), these four intermediate basic proteins are thought to be the immediate precursors of HP2, HP3 and HP4.
Precise separation and quantification of nucleoproteins in mature sperm, including minor P2 precursors, is considered important for understanding spermatogenesis as well as pathogenesis and diagnosis in many cases of male infertility. However, sperm nucleoproteins are highly basic and have such a small molecular size that fine separation and precise quantification are difficult even using an acid-urea-Triton gel or a pH-gradient gel. For this reason, no reports have described the precise quantitative composition of all human sperm nucleoproteins including all HP2 precursors based on analysis of one gel. The RFHR 2-D electrophoresis method in this study was developed (Wada, 1986a,b) as an improvement of the Kaltschmidt and Wittmann method (1970) with the aim of separating and quantifying proteins that are small, basic and rich in disulphide bridges. Thus, the method is well suited to the analysis of human sperm nucleoproteins.
Extremely small basic proteins are difficult to separate because their mobility is exceedingly high. In RFHR 2-D electrophoresis, the proteins are separated mainly by their charge in acid-urea gels in the first dimension, whereas in the second dimension separation is based mainly on mobility rate (Wada, 1986a,b). Preruns using radical scavengers were carried out to eliminate residual free radicals from gels and thereby decrease loss of proteins caused by such radicals. The sample charging electrophoresis, before the first dimensional electrophoresis, also was designed to limit protein loss and, in addition, to concentrate a dilute sample solution as a sharp band. The second dimensional electrophoresis was carried out at a more acidic pH, 3.6, to obtain better separation of extremely small basic proteins. With these modifications, quantitative yield and reproducibility became much better. In addition, protamines rich in cysteine residues migrated together with charged reductants, which served in this RFHR method to avoid formation of artefactual disulphide bridges during migration, whereas protamines were separated after modifying cysteine residues with iodoacetate in the previous study (de Yebra and Oliva, 1993). Because human nucleoproteins could be separated and purified without molecular modification by our method, these purified protamine variants should contribute importantly to analysis of protamine function.
The ratio of P1 to P2 in mature sperm varies between species; in humans it is approximately 1.0 (Balhorn et al., 1988). In addition, previous studies have reported differences in amounts of P1 and P2 between fertile and infertile men (Chevaillier et al., 1987; Balhorn et al., 1988; Bach et al., 1990; Blanchard et al., 1990; Belokopytova et al., 1993; de Yebra et al., 1993, 1998; Bench et al., 1998) or patients undergoing IVF (Colleu et al., 1997; Khara et al., 1997; Carrell and Liu, 2001; Steger et al., 2001, 2003). Further, some groups have reported a relationship between abnormal amounts of protamines and late spermiogenetic defects (Barone et al., 1994; Khara et al., 1997; Bench et al., 1998; de Yebra et al., 1998; Carrell and Liu, 2001). Although the P1/P2 ratio had been measured by analysing spots separated by 1-D electrophoresis in these previous studies, 1-D electrophoresis does not always completely eliminate overlap between protein fractions. Our RFHR 2-D electrophoresis eliminated this drawback; separation and quantitative yield were greatly improved. Nucleoproteins in sperm from fertile men were separated into 12 spots on a single gel; these spots were shown to be HP1, HPI1, HPI2, HPS1, HPS2, HP2, HP3 and TH2B, with P1 being separated into five spots with the same N-terminal amino acid sequence (ARYRR). This might suggest that multiple forms of P1 with different degrees of C-terminal phosphorylation were separated sensitively or differing formation of intermolecular disulphide bridges occurred. We have tried to analyse these five spots by mass spectrometry or Western blotting, but have not yet succeeded, probably because of the high basicity or small molecular size of protamine. P2-related proteins were separated into six spots; their N-terminal amino acid sequences were compatible with those of HP2, HP3 and four reported basic proteins of size intermediate between histones and protamines (HPI1, HPI2, HPS1 and HPS2) with different N-termini (Roux et al., 1988; Sautiere et al., 1988; Alimi et al., 1993a,b) (Table I). Quantification of protein spots was performed automatically using Image Master 2D Elite software. As a result, total human sperm nucleoprotein analysed was found to include 4% TH2B, a major histone in spermatozoa. This relative amount was less than in a previous report (Gatewood et al., 1987), in which approximately 15% of histone remained unreplaced in mature sperm nuclei; we suspect that our methodology for nuclear protein extraction was not optimal for analysis of histone, or that relative collection of protamine was increased by our method. On the other hand, the P1/P2 ratio, eliminating the small amounts of spots 2, 3, 4, 5, 8, 9, 10 in this study which might not be recognized by the previous 1-D electrophoretic method, was 1.02, which corresponded to the ratio reported by other studies in fertile men (Balhorn et al., 1988).
Concerning the relationship between male infertility and changes in composition of sperm nucleoproteins resulting from late spermatogenetic defects, de Yebra et al. (1998) reported that some infertile men have diminished amounts of P2 in their sperm possibly resulting from incomplete processing of P2 precursors, and they have an increased amount of putative P2 precursors. Accordingly, the composition of P2 precursors, such as HPI1, HPI2, HPS1 and HPS2 in mature sperm is thought to be important for analysis of spermatogenetic mechanisms. Previous sperm nucleoprotein quantification by 1-D electrophoresis analysis in previous studies has failed to determine the entire protein complement of mature sperm including all protamine precursors, presumably because of overlap of proteins; 2-D electrophoresis eliminated this problem. Our investigation of P2 precursor content of mature sperm of fertile men showed a relative quantification of HPI1:HPI2:HPS1:HPS2:HP2:HP3 of 1.000:0.6000:1.116:2.083:3.267: 2.455. To the best of our knowledge, this report is the first to quantify sperm nuclear basic proteins, especially P2 variants, in mature human sperm on one gel. In the future, pathogenesis of spermiogenetic defects may be better understood by specimens from infertile men.
Abnormal amounts of protamine were found to be common in sperm with diminished penetration capacity, whereas patients lacking P2 but having increased P2 precursors showed significantly diminished sperm penetration (Carrell and Liu, 2001). Intriguingly, however, altered amounts of protamine did not affect sperm decondensation following ICSI or subsequent embryo growth, implantation rates, or pregnancy rates. Thus, decondensation and pronuclear formation can proceed adequately when P2 is not detectable (Carrell and Liu, 2001). Because replacement of protamine by histone occurs late in spermiogenesis along with acrosome formation, membrane remodelling, and other functionally important morphologic and biochemical events, altered protamine expression is thought to reflect a wide range of spermiogenetic defects. Specifically, altered protamine expression appears not to be directly related to or causative of diminished fertilization ability but represents an indication of abnormal late-stage spermiogenesis in general (Carrell and Liu, 2001). Although the mechanism of histone replacement by protamine is not clear, a strong relationship is apparent between abnormal amounts of protamine and late spermiogenetic defects including diminished fertilization capacity. In addition, posttranslational modification or posttranslational cleavage events may be responsible for the observed variation in protamine expression. Using our sensitive RFHR 2-D electrophoretic method, even protein alterations such as posttranslational forms can be clearly separated and quantified. Thus, ejaculated semen specimens could be used to predict fertilization ability.
In conclusion, this study demonstrated that sperm nucleoproteins, especially all P2-related proteins from ejaculated semen specimen could be separated clearly with high resolution and relatively quantified on one electrophoretic gel by RFHR 2-D electrophoresis. Application of this method to specimens from infertile men might prove useful in determining pathogenesis and specific diagnosis in male infertility.
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Acknowledgements |
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This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (No. 15790910). The help of Ms. Kazuyo Nakamura, B.A. in preparing the manuscript is acknowledged.
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Submitted on June 15, 2005; accepted on July 25, 2005.
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