Expression of human oocyte-specific linker histone
protein and its incorporation into sperm chromatinduring fertilization
Yuri Mizusawa, M.D.,a Naoaki Kuji, M.D.,a Yudai Tanaka, M.D.,b Mamoru Tanaka, M.D.,aEiji Ikeda, M.D.,c Setsuko Komatsu, Ph.D.,d Shingo Kato, Ph.D.,e and Yasunori Yoshimura, M.D.aa

Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo; b Yazaki Hospital, Kanagawa;
Department of Pathology, Keio University School of Medicine, Tokyo; d National Institute of Crop Science, Tsukuba; andeDepartment of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japanc

Objective: To investigate the expression of oocyte-specific linker histone protein (hH1FOO) in human ovaries andits incorporation into sperm chromatin after intracytoplasmic sperm injection (ICSI).
Design: Laboratory study.
Setting: University hospital.
Patient(s): Human ovarian tissues were obtained from patients at oophorectomy. Human oocytes and embryos
were obtained from infertile patients undergoing IVF and ICSI.
Intervention(s): A polyclonal rabbit antibody targeting the predicted hH1FOO protein was used for immunohistochemical analysis. Western blot analysis and the reverse transcriptase–nested polymerase chain reaction weredone to detect hH1FOO in chromatin of germinal vesicle–stage oocytes through to two-cell embryos.
Main Outcome Measure(s): The hH1FOO antibody reactivity of oocytes, ovarian tissues, and sperm chromatin
after ICSI.
Result(s): hH1FOO protein was localized in all oocytes from primordial to Graafian follicles. In unfertilized
oocytes after ICSI, the chromatin of injected sperm was condensed without hH1FOO incorporation in 45.5% of
oocytes, condensed with hH1FOO incorporation in 9%, and decondensed with hH1FOO incorporation in
45.5%. None of the oocytes contained decondensed sperm chromatin without hHFOO incorporation.
Conclusion(s): hH1FOO protein was expressed by human oocytes from primordial follicles to early embryogenesis. Sperm nuclei that were still condensed after ICSI could be separated into two categories by hH1FOO incorporation, which might provide valuable information regarding failed fertilization. (Fertil Steril 2010;93:1134–41.
2010 by American Society for Reproductive Medicine.)
Key Words: H1FOO, oocytes, fertilization, histones, decondensation, human

Eukaryotic nuclear DNA associates with histone proteins toform chromatin, which is composed of nucleosomes connected by linker DNA. A nucleosome has a protein core,
which is an octamer containing two molecules each of histones H2A, H2B, H3, and H4, with DNA wound around its
surface. It is believed that histone H1, also termed a linker
histone, binds to the linker DNA and plays an importantrole in the development and stabilization of higher-orderchromatin structure and also possibly is involved in regulating gene expression (1–4).
Received August 20, 2008; revised November 11, 2008; accepted November 24, 2008; published online January 14, 2009.
Y.M. has nothing to disclose. N.K. has nothing to disclose. Y.T. has nothing to disclose. M.T. has nothing to disclose. E.I. has nothing to disclose.
S.K. has nothing to disclose. S.K. has nothing to disclose. Y.Y. has nothing to disclose.
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (No. 17591757 and
No. 19591910), and Grant-in-Aid from the National Research Institutefor Child Health and Development (No. 18-1).
Reprint requests: Naoaki Kuji, M.D., Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku,
Tokyo 160-8582, Japan (FAX: 81-3-3226-1667; E-mail: naoaki@sc.itc.
keio.ac.jp).

1134

Although all histones are encoded by multiple genes, H1
histones show greater variability than any of the core histones. Eleven different linker histone variants have been identified in mammals, which include seven somatic variants(H1.1–H1.5, H10, and H1x), three spermatogenic variants(H1t, H1T2, and HILS1), and an oocyte-specific variant(H1FOO; H1 histone family, member O, oocyte-specific)(5). In mice, the oocyte-specific histone protein H1FOO is
expressed by oocytes from the germinal vesicle (GV) stage
through to the two-cell embryo stage (6). After intracytoplasmic sperm injection (ICSI) is performed in mice, H1FOO replaces protamine on sperm chromatin and then is replaced bysomatic H1s at the two- or four-cell embryo stage (7). A similar developmental transition also occurs in Xenopus laevis(8). Such findings suggest a significant role of H1FOO in
the regulation of gene expression during oogenesis and early
embryogenesis (9–11).
We recently characterized the transcript encoding humanoocyte–specific H1 (hH1FOO) protein, by using direct single-cell reverse transcription followed by a nested polymerase chain reaction (RT-nested PCR) (12). As well as being
expressed exclusively by oocytes, this gene product has

Fertility and Sterility Vol. 93, No. 4, March 1, 2010
Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/10/$36.00
doi:10.1016/j.fertnstert.2008.11.028

several distinctive features that are shared by oocyte-specificlinker histones from other species. First, it encodes the longest H1 histone (347 amino acids). Second, it is located on
chromosome 3q21-22 and contains five introns, whereas
the somatic H1 genes are located as a large cluster on the
short arm of chromosome 6 and lack introns. Third, its messenger RNA (mRNA) shows polyadenylation, whereas somatic H1 mRNA lacks a poly-A tail. However, theexpression and localization of the protein encoded by thismRNA have not yet been determined.
Intracytoplasmic sperm injection has proved to be an effective treatment for severe male factor infertility (13).
However, fertilization is achieved for only 60% to 70% of
injected oocytes, resulting in a significant wastage of oocytes (14, 15), and poor fertilization (failure to detect two
pronuclei [PN]) sometimes occurs after ICSI (16, 17).
Sperm chromatin is mainly associated with a sperm-specificnuclear protein, which is termed protamine, instead of withhistones. It is six times more compact than metaphase chromosomes and represents the most highly condensed chromatin found in eukaryotes (18). To achieve fertilization,
the penetrating sperm heads must undergo various morphologic and biochemical changes, including chromatin decondensation and the replacement of protamine by histones(19). Visualization of sperm chromatin decondensation
along with protamine-histone replacement might provide
useful information about the process of fertilization, as
well as about the reasons for failure of PN formation after
ICSI.
In this study, we investigated the expression and localization of hH1FOO protein in human oocytes, ovaries, and fertilized embryos by using a specific anti-hH1FOO antibody.
The relationship between morphologic changes of the sperm
head after ICSI and the dynamics of histone transfer into
sperm chromatin also were investigated by immunohistochemical analysis with use of the same antibody.

MATERIALS AND METHODSThis study was approved by the Ethics Committee of Keio
University Hospital. All samples used in this study were obtained from patients who had given written informed consent.

Human Oocytes, Embryos, and Ovarian Tissues
Human oocytes were retrieved from infertile women undergoing IVF or ICSI at the Infertility Clinic of Keio University
Hospital. Oocytes that did not display 2PN and had not initiated cleavage by 48 hours after ICSI were considered to beunfertilized. The oocytes and embryos that served as samples
for this study, including GV-stage oocytes, 3PN oocytes, arrested 2PN oocytes, and arrested two-cell embryos all werescheduled to be discarded. Specimens of normal human ovarian tissue were obtained from women of reproductive age
who underwent oophorectomy during surgery for cervicalcancer or uterine leiomyoma.
Fertility and Sterility

Polyclonal Anti-hH1FOO Antibody
Anti-hH1FOO antiserum was raised in rabbits that were immunized with a mixture of two synthetic oligopeptides corresponding to the amino acid sequences SRSPESEKPGPSHG
and KASSSKVSSQRAEA of the predicted hH1FOO protein(12). These oligopeptides were selected by using Epitope Adviser and DNASIS software (Hitachi, Tokyo, Japan) and wereconjugated to keyhole limpet hemocyanin. The antiserum
thus obtained was purified by affinity chromatography(Takara, Shiga, Japan).
Western Blotting of Ovarian Tissue and Oocytes
To obtain samples for blotting, 10 mg of ovarian tissue was
ground in sodium dodecyl sulfate (SDS) sample buffer, as described previously (20). After centrifugation at 800  g for 5
minutes, the supernatant was applied to 15% SDS–polyacrylamide gel for electrophoresis (1.0 mg of protein per lane). To
obtain oocyte protein samples, a total of 30 oocytes were subjected to three freeze-thaw cycles in SDS sample buffer andapplied to 15% SDS–polyacrylamide gel for electrophoresis.
Proteins were transferred from the gel to a polyvinylidene
difluoride membrane, which was blocked overnight in
TBST buffer (10 mmol/L tris[hydroxymethyl]aminomethane[Tris; Sigma Aldrich, St. Louis, MO] HCl, pH 7.5; 150 mmol/
L NaCl; and 0.1% polysorbate 20 [Tween 20; Sigma
Aldrich]) containing 5% skim milk powder (wt/vol). Next,
the membrane was incubated with anti-hH1FOO antibody(diluted 1:500) for 1 hour at room temperature. After washing
three times in TBST for 10 minutes each, the membrane wasincubated with a peroxidase-conjugated antibody for rabbit
immunoglobulin (diluted 1:30,000; GE Healthcare Bio-Sciences, Piscataway, NJ). Signals were detected by using an enhanced chemiluminescence Western blot detection kit (GEHealthcare Bio-Sciences).
Immunohistochemical Analysis of Ovarian Tissues
Ovarian tissue specimens were frozen in optimal cutting temperature compound, sections (6 mm thick) were cut on a cryostat, and these sections were fixed in acetone for 15 minutes atroom temperature. After blocking with 5% normal swine serum for 30 minutes at room temperature, the sections were incubated with the anti-hH1FOO antibody (diluted 1:500)
overnight at 4 C. As a negative control, the antibody was replaced by preimmune serum. Sections then were incubatedwith peroxidase-conjugated porcine anti-rabbit immunoglobulin (diluted 1:100; DakoCytomation, Glostrup, Denmark).
Color was developed with 3,30 -diaminobenzidine tetrahydrochloride (0.2 mg/mL; Dojindo Laboratories, Kumamoto, Japan) in a solution containing 50 mmol/L Tris-HCl (pH 7.4)
and 0.003% hydrogen peroxide, after which the sections
were counterstained with hematoxylin.
Reverse Transcription–nested PCR of hH1foo mRNA
About 1 mL of phosphate-buffered saline solution (PBS) containing a single unfertilized oocyte that had been previously
1135

FIGURE 1
Western blotting of extracts from unfertilized humanoocytes (left lane) and human ovarian tissue (rightlane). An identical band is seen at 36 kDa in each lane(arrow), corresponding to the molecular weight of
hH1FOO protein.

Mizusawa. Expression of human oocyte-specific H1. Fertil Steril 2010.

washed three times in drops of PBS was added to 1.0 mL of
0.2% sarcosyl solution. After brief vortexing, the lysate wasimmediately frozen in liquid nitrogen and stored at 28 C until use. The lysate was subjected directly to RT-nested PCR as
described previously (12), with use of primer pairs thatflanked an intron to avoid amplification of genomic DNA.
Fluorescence Immunohistochemical Analysis of Oocytes
and Embryos
Unfertilized oocytes and arrested embryos were fixed for 20
minutes at room temperature in a freshly prepared solution of
2% paraformaldehyde in PBS (pH 7.4). Fixed samples werethen incubated for 1 hour at room temperature in blocking solution (PBS with 10% goat serum and 0.5% Triton X-100).
After blocking, the oocytes or embryos were incubated for
1 hour at room temperature with the anti-hH1FOO antibody(diluted 1:1,500 with a solution of 0.5% Triton X-100 in
PBS). Next, the specimens were washed three times in PBS
for 15 minutes each at room temperature and incubatedwith Alexa Fluor 488 goat anti-rabbit IgG conjugate (diluted
1:1,000; Molecular Probes, Eugene, OR). After the specimens were washed again as above, DNA was stained withuse of Hoechst 33342 dye (Molecular Probes). After anotherwash in PBS, each cell was pipetted into a drop of PBS andplaced on a glass slide for examination with an Olympus
AX70 microscope (Tokyo, Japan) that was equipped withthe appropriate filter sets for epifluorescence studies.
RESULTS
Specificity of the Anti-hH1FOO Antibody
The specificity of the polyclonal anti-hH1FOO antibody wasinvestigated by Western blotting of protein extracts from 30
unfertilized human oocytes and from ovarian tissues. In
both the oocytes and the ovarian tissue samples, a single
band was detected at 36 kDa (Fig. 1), which was consistent
with the molecular weight calculated from the predicted
amino acid sequence of hH1FOO.
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Mizusawa et al.

Ovarian Localization of hH1FOO
Immunohistochemical analysis of ovarian tissue specimens
with the polyclonal anti-hH1FOO antibody is shown in Fig. 2.
The protein was localized in oocytes at all stages from primordial follicles to Graafian follicles, whereas preimmune serum
did not react with any ovarian structures. There was no reactivityof the anti-hH1FOO antibody with granulosa cells or theca cells,
confirming its specificity for hH1FOO as opposed to other histones. Although staining was more intense in the nucleus, thecytoplasm also was stained in all oocytes examined, indicatingthat cytoplasmic synthesis of hH1FOO is active from a very
early stage of oogenesis and that this protein persists in the cytoplasm.
Expression of hH1foo mRNA and Protein During Oogenesis
and Early Embryogenesis
Unfertilized human oocytes and arrested embryos were analyzed by RT-nested PCR and by fluorescence immunohistochemical analysis. As a result, hH1foo mRNA was detected
at all stages from GV-stage oocytes to two-cell embryos as
specific bands with a similar intensity (Fig. 3A). We confirmed that the nucleotide sequence of these bands was identical to that previously reported for hH1foo mRNA (12) (datanot shown).
The protein also was detected in the chromatin of oocytes
from the GV stage through to two-cell embryos (Fig. 3B). In
these experiments, at least three oocytes or embryos at eachstage were examined by RT-nested PCR and immunohistochemical analysis, with consistent results being obtained.
Replacement of Protamine by hH1FOO in Unfertilized
Oocytes After ICSI and Sperm Decondensation
Thirty-three unfertilized oocytes were obtained after ICSI
and were classified according to the reactivity of sperm chromatin with the hH1FOO antibody and the occurrence of
sperm chromatin decondensation (from slightly to totally decondensed) (Fig. 4): [1] no immunoreactivity for thehH1FOO antibody and persistence of condensed chromatin(15 oocytes, 45%), [2] immunoreactivity for hH1FOO withpersistence of condensed chromatin (3 oocytes, 9%), and[3] immunoreactivity for hH1FOO with decondensed chromatin (15 oocytes 45%). Interestingly, none of the oocytes
contained sperm that showed decondensation but were nonreactive to the hH1foo antibody.
DISCUSSION
Role of H1FOO in Oocytes
H1FOO is thought to be the predominant H1 histone associated with chromatin during oocyte growth, oocyte maturation, and early embryogenesis. On entry of a sperm into an
oocyte, protamine bound to sperm chromatin is replaced bymaternal histones, including H1FOO, in a process that is referred to as protamine-histone replacement (18, 19). Concurrently, the highly condensed sperm chromatin undergoes

Expression of human oocyte-specific H1Vol. 93, No. 4, March 1, 2010

FIGURE 2
Immunohistochemical analysis for hH1fOO in ovarian sections. Human ovarian follicles are shown at various
stages, including a primordial follicle (a), primary follicle (b), secondary follicle (c), preantral follicle (d), andGraafian follicle (e, f). Staining is detected in both the nucleus (orange or dark yellow) and the cytoplasm (yellow)of the oocytes, with its intensity being greater in the nucleus.

Mizusawa. Expression of human oocyte-specific H1. Fertil Steril 2010.

extensive decondensation to form the male PN. In fact, animal studies have shown that development of the male PN
from sperm chromatin requires the presence of maternal histones (21). In mice, H1FOO then is replaced by somatic H1 at
the two- to four-cell stage of embryogenesis (6). Because thistransition of H1 subtypes takes place simultaneously with theinitiation of zygotic development, H1FOO is thought to be involved in the transition from maternal to embryonic gene expression, resulting in remodeling of the two PN and the onset
of DNA replication (7).
Fertility and Sterility

To our knowledge, the present study is the first to demonstrate the expression of H1FOO protein in human oocytes.
Immunohistochemical analysis of human ovarian tissues revealed hH1FOO protein expression in oocytes throughout
oogenesis, even as early as the primordial follicle stage.
This finding differs from a previous report that H1FOO immunoreactivity was limited to oocytes after the primary follicle stage in mice, with no immunoreactivity being
detected in the oocytes of primordial follicles (althougha few H1foo transcripts were noted in these oocytes) (22).
1137

FIGURE 3

FIGURE 3 Continued(A) Reverse transcription–nested PCR of hH1foomRNA during oogenesis and early embryogenesis.
hH1foo mRNA was detected at all stages from GVstage oocytes to two-cell embryos as specific bands
with a similar intensity. bp ¼ base pair. (B)Fluorescence immunohistochemical analysis of an
oocyte at the GV stage, an oocyte in MII, a PN
oocyte, and a two-cell embryo (2-cell). The PN
oocyte was obtained from IVF and ICSI, and thearrested 2PN oocyte was obtained after ICSI. The GV
and MII oocytes were not exposed to sperm by either
conventional IVF or ICSI. Deoxyribonucleic acid wasvisualized by Hoechst 33342 staining, and hH1FOO
was detected by indirect immunofluorescence.
hH1FOO protein can be detected clearly in the
nucleus of the GV oocyte, MII oocyte, PN oocyte,
and two-cell embryo. All polar bodies also werepositive for both anti-hH1FOO antibody and Hoechst
33342. All of the sperm surrounding the PN oocyte,
four-cell embryo, and morula were unstained by theanti-hH1FOO antibody (some are indicated byarrows). oc ¼ oocyte chromatin; pb ¼ polar body; pn¼ pronucleus; bc ¼ blastomere chromatin.

is absent in growing oocytes, was very weak in nongrowing
oocytes within the primordial follicles of adult mouse ovaries. Although the report of Clarke et al. suggested that thesubtype transition from somatic H1 to oocyte-specific H1
may occur shortly after birth in all oocytes of the ovary (including those in the primordial follicles), further studies
will be necessary to clarify the ontogeny and role of hH1FOO
during oogenesis.

Mizusawa. Expression of human oocyte-specific H1. Fertil Steril 2010.

On the basis of such findings, those authors proposed that expression of the H1foo gene might be coupled to the process ofprimordial follicle recruitment. On the other hand, Clarke
et al. (23) reported that somatic H1 immunoreactivity, which
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Mizusawa et al.

In the present study, preovulatory oocytes in human ovarian tissue samples showed hH1FOO immunoreactivity inboth the nucleus and cytoplasm (Fig. 2), although stainingof the nucleus was much stronger than that of the cytoplasm.
It has been suggested that mouse H1FOO is mainly localized
inside the germinal vesicles, because mouse H1FOO protein
contains nuclear localization sequences but has no nuclear
export sequences (22). On the other hand, McGraw et al.
(24) found that H1FOO immunoreactivity not only was associated with the chromatin in bovine GV-stage oocytes but wasalso detected in the ooplasm, although staining of the cytoplasm was less intense compared with that of the chromatin.
It also has been reported that after removal of the GV nucleus,
H1FOO was detected in the somatic nucleus of oocytes after
nuclear transfer (7), suggesting the existence of H1FOO in
the ooplasm. On the basis of these findings, it seems that
H1FOO protein is primarily localized in the nucleus in oocytes, although there is also a relatively abundant cytoplasmic pool.

Expression of human oocyte-specific H1Vol. 93, No. 4, March 1, 2010

FIGURE 4
Fluorescence immunohistochemical analysis of oocytes that were not fertilized by ICSI. These oocytes display
three patterns of sperm chromatin immunoreactivity with the hH1foo antibody: [1] no immunoreactivity and no
decondensation (A, B), [2] immunoreactivity without decondensation (C, D), and [3] immunoreactivity withdecondensation (E, F). oc ¼ oocyte chromatin; pb ¼ polar body; sc ¼ sperm chromatin.

Mizusawa. Expression of human oocyte-specific H1. Fertil Steril 2010.

Early Incorporation of H1FOO Into Sperm Chromatin After
Penetration of the Oocyte
Studies in animals have shown that H1FOO is assembled on
sperm chromatin almost immediately after fertilization. For
example, H1FOO was detected on condensed sperm chromatin only 5 minutes after ICSI in metaphase II (MII)–arrested
mouse oocytes (6). In pigs, incorporation of H1FOO into thesperm chromatin has been reported to precede decondensation after penetration of the ooplasm (25, 26). The assemblyof H1FOO on sperm chromatin during decondensation also
seemed to occur very quickly in humans, because all swollen
sperm heads (even those that were only slightly swollen) revealed hH1FOO immunoreactivity in this study. We founda small number of unfertilized oocytes that contained condensed sperm heads with hH1FOO immunoreactivity (3/18
unfertilized oocytes containing condensed sperm heads),
which also indicated that hH1FOO assembly might occur
very early in the decondensation process.

tion (27). Among these outcomes, failure of decondensation
may arise from incomplete immobilization of sperm due toinsufficient damage to the sperm plasma membrane. However, there has been no way to determine whether sperm
have reacted with the ooplasm. Therefore, we have been unable to assess whether condensed sperm heads had not reacted because the plasma membrane was not sufficiently
damaged or had reacted but then failed to decondense. Thislatter situation is thought to arise from chromatin anomalies(28) or intrinsic oocyte defects. In this context, the performance of immunostaining for hH1FOO might provide valuable information about the cause of failed fertilization after
ICSI. Because hH1FOO exclusively exists in oocytes andnot in the sperm nucleus, hH1FOO immunoreactivity might
be able to distinguish sperm that have started to react withthe ooplasm and then failed to proceed to decondensationfrom membrane-intact and totally unreactive sperm, although
our sample size was too small to draw a firm conclusion.

A previous cytogenetic study of unfertilized human oocytes after ICSI revealed various outcomes of the injected
sperm: [1] the entire sperm was missing or was ejected outside the ooplasm, [2] sperm chromatin was undecondensed,
or [3] there was variable premature chromosome condensa-

There are two possible explanations for the existence of
hH1FOO-positive, condensed sperm nuclei. The first is that
hH1FOO has been assembled, but decondensation has not occurred because the fertilization process was arrested at a very
early stage. The second possibility is that these nuclei

Fertility and Sterility

1139

underwent decondensation, but then recondensed. In mice,natural fertilization leads to initial enlargement and dispersion of protamine, after which the decondensed sperm headdecreases in size (recondensation) and then gradually enlarges to form the male PN. It has been proposed that the recondensation of histone-containing sperm chromatin is due
to a high level of metaphase-promoting factor activity andthat oocyte activation causes this activity to decline to its
basal level, resulting in PN formation (29). Indeed, in nonactivated oocytes of pigs, there is a time-dependent increase in
the percentage of recondensed sperm nuclei (30). Sperm may
fail to decondense because the oocyte lacks decondensing activity. It has been shown that mammalian oocytes possess
sperm decondensing activity for only a short period after activation, and sperm introduced into mouse oocytes at any
time later than the first cell cycle do not undergo decondensation (31–33). Presumably, the ability of human oocytes topromote the decondensation of sperm is also time limited, because Dozortsev and De Sutter (34) reported a high incidenceof slightly swollen sperm in the cytoplasm of 1PN (i.e., activated) oocytes. Although there currently is no way to decide
which of these two explanations is correct, further observation of sperm chromatin in nonactivated (MII) oocytes, activated (1PN) oocytes, and chemically (e.g., puromycin)
activated unfertilized oocytes might help to clarify the reason
for the existence of condensed and histone-positive sperm
nuclei.
In the present study, we did not find any decondensed
sperm head without H1FOO immunoreactivity. However, it
would be interesting to search for such an alternative mechanism of failed fertilization after human ICSI in a study
with a larger sample size, along with performing experiments
on H1FOO knockout mice. So far, there have been no reports
about the detection of decondensed sperm heads without
H1FOO immunoreactivity in animals, but such sperm might
represent an alternative mechanism of failed fertilization
after ICSI.
In conclusion, we demonstrated the exclusive expressionof H1FOO protein in human oocytes from those in primordial
follicles through to early embryogenesis. During the processof fertilization after ICSI, hH1FOO seemed to be rapidly incorporated into the injected sperm nucleus. Because spermthat remained condensed after ICSI could be separated into
two categories based on H1FOO incorporation or nonincorporation, immunostaining for H1FOO might provide valuable information about the factors leading to failed
fertilization after ICSI.
Acknowledgments: The authors thank Mr. Hitoshi Abe for his kind technicalassistance. The assistance of Ms. Kazuyo Nakamura, B.A., in preparing themanuscript is also acknowledged.

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