Molecular Human Reproduction Vol.11, No.9 pp. 677–681, 2005
Advance Access publication September 28, 2005

doi:10.1093/molehr/gah217

Fine resolution of human sperm nucleoproteins by
two-dimensional electrophoresis
Tsuyoshi Yoshii1, Naoaki Kuji1,4, Setsuko Komatsu2, Kazuhiro Iwahashi1, Yudai Tanaka1,
Hiroyuki Yoshida1, Akira Wada3 and Yasunori Yoshimura1
1

Department 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, Japan4

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@sc.itc.keio.ac.jp

Key words: histone/human/nucleoprotein/protamine/spermatozoa

Introduction
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 havereported changes in relative amounts of P1 and P2 between fertile andinfertile 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 thatsome 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 ininsufficient 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. Nonequivalent 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 andaccurate view of changes in composition of these proteins associatedwith 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 verysmall. 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 thoroughscavenging. 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.

Materials and methods
Samples
Semen samples were collected from male volunteers demonstrated to be fertile
by a history of producing offspring. Semen ejaculates obtained at the hospitalby 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 hof obtaining the sample. Spermatozoa numbering 50 × 106 per volunteer were
washed three times with M199 (Invitrogen, Carlsbad, CA) (using 10 min ofcentrifugation at 1600 g each time) and twice with phosphate-buffered saline(PBS; using 5 min of centrifugation at 1600 g each time). Pellets were thenkept frozen (−80°C) until use.

Extraction of nuclear proteins
Sperm nuclear basic protein was extracted according to the method describedby Balhorn et al. (1977); with minor modification. Briefly, the sperm pellet© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For
Permissions, please email: journals.permissions@oupjournals.org
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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, weanalysed these proteins on a gel sheet by a radical free, highly reducing method based on Kaltschmidt and Whittmann’s twodimensional 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-specifichistone 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.

T.Yoshii et al.
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 toobtain 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 thenpelleted 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 thenpelleted by centrifugation at 14 500 g, washed with acidified acetone, anddried 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.

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 offive 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 thespot, 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 forall spots present in the gel.

spots, which were more basic and of lower molecular weight,
appeared in the sperm nucleoprotein pattern; these were thought torepresent protamines.
Sperm nucleoproteins were separated into 12 spots. Amino acidsequence 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 fivespots with the same N-terminal amino acid sequence (ARYRXXR;
Table I); the amino acid corresponding to ‘X’ could not be determinedby 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.

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 weredetected 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 acidresidues were identified by on-line high-performance liquid chromatography(120A; Applied Biosystems). Amino acid sequences obtained were comparedwith those of known proteins with the web-accessible search program FastA(http://www.dna.affrc.go.jp).

Results
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 theelectrophoretic 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

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Figure 2. Sperm nucleoproteins spots analysed by radical-free, highly reducing (RFHR) two-dimensional electrophoresis. Human sperm nucleoproteins
were separated into 12 spots with high reproducibility. Protamines in humansperm nucleoproteins were resolved distinctly, located essentially along two
diagonals representing protamine 1 (P1) and protamine 2 (P2) groups. P1 consisted of five P1 forms, whereas group P2 consisted of six P2-related proteins(HPI1, HPI2, HPS1, HPS2, HP2 and HP3). All P2-related proteins, representing HP2, HP3 and their precursors, were separated clearly from each other.

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Electrophoresis

Figure 1. Gel patterns of nucleoproteins compared between sperm and HeLa
cells. A typical electrophoretic analysis of the nuclear proteins extracted fromhuman sperm (A) and HeLa cells (B) by the RFHR two-dimensional electrophoresis method is shown. In contrast to the electrophoretic pattern for somatic(HeLa) cells, which includes only histone without protamines, sperm showedspots similar in position to histones of HeLa cells but also smaller, more basic
spots thought to be protamines.

Fine resolution of sperm nucleoproteins
Table I. Amino acid sequences for each spot
Spot

N-terminal aminoacid sequence

Protein

1
2
3
4
5
6
7
8
9
10
11
12

XEVSSKGATISXKGFVRYRVRSLSERSHEV
HGQEQ
QGLSP
SPEHVEVYERTHGQ
RTHG
GQSHYRRR
ARYR…
ARYR…
ARYR…
ARYRXXR…
ARYR…

TH2B (testis-specific histone H2B)
HPI1 (human protamine 2)
HPI2 (human protamine 2)
HPS1 (human protamine 2)
HPS2 (human protamine 2)
HP2 (human protamine 2)
HP3 (human protamine 2)
Human protamine 1
Human protamine 1
Human protamine 1
Human protamine 1
Human protamine 1

The five protamine 1 (P1) spots were found to have the same N-terminal
amino acid sequence (ARYRXXR). The amino acid ‘X’ could not be
determined by Edman degradation and may correspond to the cysteine residueof P1. Differing locations of these five spots presumably represent differencesin posttranslational modification

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 insequence 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.
Densitometry results obtained by averaging the quantitation fromtwo gel samples are shown in Figure 4. Relative amount of TH2B
extracted in this study was 282.6 as compared to the total amount ofanalysed nucleoprotein spots, 7364.8. Even considering the amount ofseveral 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 inour 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 P2related 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.

Discussion
Approximately 85% of human sperm nuclear histones are replaced byprotamines during late spermatogenesis (Barone et al., 1994) accompanied by a dramatic change in the structure of chromatin (FuentesMascorro 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 aminoterminal extension of one to four residues (Gusse et al., 1986; McKay
et al., 1986; Sautiere et al., 1988; Arkhis et al., 1991; Oliva andDixon, 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 aminoacids (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 theamino 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 ofhistones 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

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Figure 3. Amino acid sequence of each spot separated by RFHR twodimensional electrophoresis in comparison with reported human protaminesequences. Amino acid sequences of protamines reported in previous studies
and protamines analysed in this study are shown. Amino acid sequences ofprotamine 2 (P2)-related proteins corresponded to portions of the prm2 gene(accession number p04554) sequence. HPI1, HPI2, HPS1, HPS2, HP2 andHP3 corresponded to segments 2–, 22–, 34–, 37–, 46–, and 49– of prm2. HP4(45– of prm2), not detected in this study, was thought to be extremely low inquantity.

Figure 4. Protein expression level of protamine 1 (P1) and protamine 2 (P2).
After Coomassie Brilliant Blue (CBB) staining, the gel was scanned and preprogrammed spot quantification was performed using Image Master 2D Elite
software. Relative quantities averaged from two gel samples were calculated.
The ratio P1/P2, measured as in previous studies, was 1.02, representing good
agreement. In addition, the relative quantity of all P2-related forms (HPI1,
HPI2, HPS1, HPS2, HP2 and HP3) was determined as 1.000, 0.6000, 1.116,
2.083, 3.267 and 2.455, respectively.

T.Yoshii et al.

680

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 toinclude 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 incomposition 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 fromincomplete 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 isthought 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 humansperm 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 spermwith diminished penetration capacity, whereas patients lacking P2 but
having increased P2 precursors showed significantly diminished sperm
penetration (Carrell and Liu, 2001). Intriguingly, however, alteredamounts 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 alongwith 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 anindication of abnormal late-stage spermiogenesis in general (Carrell andLiu, 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.

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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 forunderstanding spermatogenesis as well as pathogenesis and diagnosis
in many cases of male infertility. However, sperm nucleoproteins arehighly basic and have such a small molecular size that fine separationand precise quantification are difficult even using an acid-urea-Triton
gel or a pH-gradient gel. For this reason, no reports have described theprecise 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 Wittmannmethod (1970) with the aim of separating and quantifying proteinsthat 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, theproteins are separated mainly by their charge in acid-urea gels in thefirst 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 gelsand 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, toconcentrate 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 becamemuch 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; inhumans 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 havereported a relationship between abnormal amounts of protamines andlate 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 thisdrawback; 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 mightsuggest that multiple forms of P1 with different degrees of C-terminal
phosphorylation were separated sensitively or differing formation ofintermolecular disulphide bridges occurred. We have tried to analyse

Fine resolution of sperm nucleoproteins
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 maleinfertility.

Acknowledgements
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 helpof 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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