BBRC
Biochemical and Biophysical Research Communications 304 (2003) 351–357
www.elsevier.com/locate/ybbrc

Structure and expression of the human oocyte-speciﬁc histone H1
gene elucidated by direct RT-nested PCR of a single oocyte
Yudai Tanaka,a Shingo Kato,b,* Mamoru Tanaka,aNaoaki Kuji,a and Yasunori YoshimuraabaDepartment of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

Received 24 March 2003

Abstract
Oocyte-speciﬁc histone H1 is expressed during oogenesis and early embryogenesis. It has been described in mice and somenonmammalian species, but not in humans. Here, we identiﬁed the cDNA in unfertilized human oocytes using direct RT-nested
PCR of a single cell. Sequencing of this cDNA indicated an open reading frame encoding a 347-amino acid protein. Expression was
oocyte-speciﬁc. Homology was closest with the corresponding gene of mouse (H1oo; 42.3%), and, to lesser extent, with that of
Xenopus laevis (B4; 25.0%). The gene, named osH1, included ﬁve exons as predicted by the NCBI annotation project of the humangenome, although the actual splicing site at the 30 end of exon 3 was diﬀerent by 48 nucleotides from the prediction. The presence ofpolyadenylation signals and successful ampliﬁcation of cDNA by RT-PCR using an oligo(dT) primer suggested that the osH1mRNA is polyadenylated unlike somatic H1 mRNA. Our technique and ﬁndings should facilitate investigation of human fertilization and embryogenesis.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Human; Oocyte; Oocyte-speciﬁc histone H1

Histones are the major protein constituents of chromatin in eukaryotic cell nuclei. Two types of histone
proteins exist: the core histones H2A, H2B, H3, and H4and the linker histone H1. An octamer of the core histones intertwined with 146 bp of DNA forms a component of the nucleosome core particle. The linker
histone H1 is involved in sealing DNA joining adjacentnucleosome core particles to maintain higher-order
packaging of chromatin. In mammals, each of theseclasses except for H4 is subdivided into several subtypes[1]. The most divergent class of histones is the H1 protein family, which consists of eight subtypes in mammals: the somatic variants H1a, H1b, H1c, H1d, H1e,and H10 ; the testis-speciﬁc variant H1t; and the oocytespeciﬁc variant H1oo [2–6].
Oocyte-speciﬁc H1 is characteristic in a number ofways. First, it is expressed only during oogenesis andearly embryogenesis and then replaced by somatic H1*
Corresponding author. Fax: +81-3-5360-1508.
E-mail address: skato@sc.itc.keio.ac.jp (S. Kato).

subtypes shortly after fertilization [6–8]. This replacement may loosen the condensed chromatin structure,
resulting in a change in transcriptional pattern from theoocyte to the embryo [9–11]. Second, unlike most othersubtypes, it contains both introns and a poly(A) tail, andlacks characteristic motifs in the promoter regions thatare shared by the other H1 genes [12]. Finally, in comparison with other subtypes, sequence homology ofoocyte-speciﬁc H1 genes between diﬀerent organisms are
relatively low.
All H1 subtypes except for oocyte-speciﬁc H1 havebeen characterized at the levels of protein and gene inboth humans and mice [5,6]. Oocyte-speciﬁc H1 has
been identiﬁed in four species; csH1 in the sea urchin[13], B4 in Xenopus laevis [7,14], H1M in the zebraﬁsh[15], and H1oo in the mouse [6]; but it has not yet been
identiﬁed in humans. Oocyte-speciﬁc H1 may play animportant role in forming the male pronucleus during
fertilization, since remodeling of sperm chromatin requires replacement of protamine by oocyte-speciﬁc H1.
Thus, identiﬁcation and characterization of the human0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0006-291X(03)00610-7

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Y. Tanaka et al. / Biochemical and Biophysical Research Communications 304 (2003) 351–357

oocyte-speciﬁc H1 gene would be prerequisite forelucidation of human infertility.
Both clinical and technical constraints limit genomic
and protein investigation in human oocytes. Since oocytes usable for research are limited to those failing toinitiate cleavage for more than 2 days after retrieval, theamount of available sample is absolutely insuﬃcient forthe conventional analysis of gene expression at themolecular level. Therefore, we developed a new method
for investigation of gene expression in a single oocyte
and used it to identify and characterize the humanoocyte-speciﬁc histone H1 gene.

Materials and methods
All procedures were approved by the Ethics and Legal Committee of
Keio University Hospital. Specimens used in our research were obtained from informed and consenting patients with written agreements.
Cells. Human oocytes were retrieved from infertile women byroutine methods to carry out intracytoplasmic sperm injection (ICSI)
according to the standard procedure at the Infertility Clinic of Keio
University Hospital. After incubation for a few hours, a spermatozoon
was injected into each oocyte. If cell cleavage was not initiated after at
least 48 h, an oocyte was considered unfertilized and used in our
program if the donor couple approved.
Specimens of normal human ovarian tissue were obtained fromconsenting women of reproductive age who were undergoing oophorectomy in the course of surgery for uterine cervical cancer or uterine
leiomyoma at Keio University Hospital. Human ovarian granulosa cells
were collected from aspirated follicular ﬂuid during the oocyte retrieval
procedure. Brieﬂy, the ﬂuid was centrifuged at 1500 rpm for 5 min. The
cell pellet was washed twice by suspension and centrifugation.
MOLT-4 cells (a human T-cell line) were maintained at 37 °C in
RPMI 1640 medium supplemented with 10% fetal bovine serum, 0.1 M
L -glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin in an
atmosphere containing 5% CO2 .
Human semen was obtained with consent from healthy men who
had come to the outpatient clinic to have their sperm examined. The
semen was processed using polyvinylpyrrolidone-coated colloid media
for puriﬁcation of sperm.
RNA preparation. RNA in a single human oocyte was analyzed
directly by our newly established method. An unfertilized oocyte selected from sperm-injected oocytes by aspiration was transferred into
1.0 ll of 0.2% sarcosyl solution to give a ﬁnal volume of 2.0 ll. This
concentration of sarcosyl was enough to break down cell membrane ofoocytes without inhibiting reverse transcription after a 10-fold dilution. After suspending and agitation, the cell lysate was immediately
frozen in liquid nitrogen and then stored at )28 °C in a deeper freezer
until use. The lysate was directly subjected to RT-nested PCR as described below. Total RNA was isolated from human ovarian tissue,
human ovarian granulosa cells, MOLT-4 cells, and sperm using the
RNAeasy kit (Qiagen, Hilden, Germany) according to the manufacturerÕs instructions. Isolated RNAs were stored at )28 °C. RNAs fromother human tissues (breast, colon, kidney, liver, lung, skeletal muscle,and spleen) were purchased from Stratagene (CA, USA).
RT-nested PCR. Sixteen primers were designed according to the
genomic sequence [AC080007; Homo sapiens 3q BAC RP11-529F4(Roswell Park Cancer Institute Human BAC Library)] and the mRNA
sequence [XM_067747; the gene similar to the H1 histone family(oocyte speciﬁc)] predicted by the NCBI (National Center for Biotechnology Information) annotation system (Table 1). The RNA wasconverted to cDNA using ThermoScript reverse transcriptase (Invitrogen, CA, USA) and RNAsin (Promega, WI, USA) according to theTable 1
Sequences of primers designed for RT-nested PCR
Primers

Sequence

MR3U
MR4U
GM2U
GM2BU
GM2D
GM1Da
MR5U
MR6U
MR4D
MR3Da
MR7U
MR8U
MR2D
MR1Da
MR8D
MR7Da

50 -GGTGAGGGGTCTGCTGGCTG-30
50 -GTCTGCTGGCTGCACCTGTC-30
50 -GGAGGCCCGAGCCACAGCAG-30
50 -TCCCGGTGGGACGCCGCCAC-30
50 -CGAGGAGGCCACGGCGCATG-30
50 -CAGTGGCCCCCCTGGCTTTG-30
50 -AATCCAGCCCAGGAAGATGG-30
50 -CTCCCAGGAGAGCGGGTGAG-30
50 -CACCTTGCCCACGTTGGGAG-30
50 -CTTTGCTGGCCTCTTGGCTG-30
50 -GGCAGGGGCCAAACACCAAG-30
50 -TGCTCCTGCTAAGGGCAGTG-30
50 -GATGAGGCCTTGATGGGCAG-30
50 -CTTCAGCCCTCTGGCTGGAC-30
50 -TAGGGCAGAGGCTCGGTCTC-30
50 -AGAGCAGTGGTTAGTTGAAG-30

aPrimers GM2D, MR1D, MR3D, and MR7D were used for reverse transcription.

manufacturerÕs instructions. The cDNA was then ampliﬁed by nested
PCR using AmpliTaq (Applied BioSystems, NJ, USA) and PCRx
Enhancer System (Invitrogen) to attain eﬃcient ampliﬁcation of GCrich sequences according to the manufacturerÕs instructions. The ampliﬁcation steps involved 30 cycles of denaturation at 94 °C for 15 s,
annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. PCR
products were analyzed by electrophoresis in 1.5% agarose gels. All theprimers listed in Table 1 were used for each oocyte. For RNAs ofovarian granulosa cells, sperm, MOLT-4, and human tissues, primers
used were GM1D for RT; GM1D and MR3U in ﬁrst-round PCR; andGM2D and MR4U in second-round PCR. As a control, RT-PCR of bactin was performed with primers, 50 -CACTCTTCCAGCCTTCC
TTCC-30 and 50 -CGGACTCGTCATACTCCTGCTT-30 , which were
designed according to the previously reported data [16]. The secondround PCR was not performed for the b-actin controls.
Sequencing. PCR products were sequenced by Takara (Shiga, Japan) using a DYEnamic ET Terminator Cycle Sequencing kit(Amersham Biosciences, NJ, USA).
Accession Number. AY158091 for the oocyte-speciﬁc histone H1
gene.

Fig. 1. Products of RT-nested PCR of a single human oocyte. Primer
pairs used (Table 1) are: (1) MR3U/GM1D, MR4U/GM2D; (2)GM2U/MR3D, GM2BU/MR4D; (3) MR5U/MR1D, MR6U/MR2D;
and (4) MR7U/MR7D, MR8U/MR8D. An HaeIII digest of UX DNA
is used for molecular weight markers (5). A contig of cDNA sequence
was established from the DNA sequences of the four products.

Y. Tanaka et al. / Biochemical and Biophysical Research Communications 304 (2003) 351–357

Results
Identiﬁcation of H1 mRNA from a single human oocyte
Since few human oocytes are available for our experiments, we chose RT-nested PCR rather thanNorthern blotting in order to identify oocyte-speciﬁc
histone mRNA. The lysate of a single oocyte was directly subjected to RT-nested PCR using four sets ofprimers, whose sequences were determined based on aputative human oocyte-speciﬁc gene (Table 1). Discrete
bands with expected lengths were detected from any setsof primers (Fig. 1). When either of sequence-speciﬁcprimer MR7U and oligo(dT) primer was used for reverse transcription, the primer set of MR7D/MR7U and
MR8D/MR8U produced an identical cDNA sequence353(data not shown). The DNA bands obtained were considered to have been derived from mRNA but not fromgenomic DNA because each of a primer set is located ineach of an exon pair ﬂanking a long intron. A contig
was constructed from DNA sequences of the PCR
products, giving a 1067-bp molecular sequence with a
1041-bp open reading frame (Fig. 2). We designated thisgene as osH1. The initiation codon was assigned asshown in Fig. 2 because it is the ﬁrst ATG downstreamof the TATA box on the putative genomic DNA. The
osH1 gene encoded a 35.8-kDa protein of 347 aminoacids. According to the GenBank genomic sequencedata, the gene includes ﬁve exons and is located on 3q2122 in contrast with the somatic H1 genes which arelocated in a large cluster on the short arm of chromosome 6.

Fig. 2. The osH1 gene. (A) Sequence of the osH1 cDNA. Numbering starts from the initiation codon. The gray-shaded sequence is the open reading
frame. The unshaded sequences are the UTR at both ends and the promoter region. Splicing sites are shown in triangles. A sequence of 48 nucleotides
not present in the NCB-predicted version is shown in bold italics. The CCAAT box, the TATA box, the cytoplasmic polyadenylation element (CPE),
the deadenylation element, and the polyadenylation signal are indicated as bold letters with underlines. (B) Schematic presentation of exons andintrons of osH1 in the genomic sequence. Exons are indicated as open boxes. Numbers between exons indicate length of introns. Locations of allprimers employed for RT-nested PCR are indicated.

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Y. Tanaka et al. / Biochemical and Biophysical Research Communications 304 (2003) 351–357

Homology with oocyte-speciﬁc H1 of other species
Alignment of amino acid sequences of oocyte-speciﬁc
H1 of human and other species was performed with theassistance of CLUSTAL W software (version 1.8) [17](Fig. 3). The osH1 gene exhibited a 42.3% identity scorewith H1oo (mouse), 25.0% with B4 (X. laevis), 25.1%
with H1M (zebraﬁsh), and 22.5% with csH1 (sea urchin). In contrast, homology of osH1 with H1b (somatic
subtype) and H1t (testis-speciﬁc subtype) was only
18.5% and 15.3%, respectively (Table 2). In general,
linker histones of the H1 variety possess a three-domain
structure including a central globular domain (consisting of Helix I, II, and III) ﬂanked by amino- and carboxy-terminal domains. Sequence homology was

greatest in the central globular domain, consistent withthe observation that linker DNA interacts with thecentral globular domain of H1 protein [5,6,12]. Whenhomology analysis was limited to the globular domain,
osH1 shared 74.3%, 57.1%, 45.1%, and 38.9% identitywith the corresponding regions of H1oo, B4, H1M, andcsH1, respectively.
Oocyte-speciﬁc expression of osH1
RT-nested PCR was performed for human ovariantissue and ovarian granulosa cells. In the ovarian tissue,
osH1 cDNA was detectable up to a 25-fold dilution,
while b-actin was detectable up to a 56 -fold dilution. In
ovarian granulosa cells, however, osH1 cDNA was not

Fig. 3. Amino acid sequence alignment of osH1 with other oocyte-speciﬁc H1s. The amino acid sequences of other oocyte-speciﬁc H1s were extracted
from the GenBank database (for Accession number, see Table 2). Identical amino acid residues are shaded black; similar amino acids are shadedbright gray; and unrelated residues are unshaded. The globular domains are underlined (Helix I, II, and III).

Table 2
Sequence similarity of osH1 protein to oocyte-speciﬁc H1 of other species and other subtypes of human H1
ProteinSequence identity (%)

GapsAmino acid overlap

H1oo (mouse)
H1M (zebraﬁsh)
B4 (Xenopus laevis)
csH1 (sea urchin)
H1t (human)
H1b (human)
H10 (human)

42.3
25.1
25.0
22.5
15.3
18.5
19.6

47
87
74
47
129
127
116

148
85
87
78
52
64
68

Alignment was performed with the assistance of CLUSTAL W software (version 1.8). Accession numbers for each proteins are: AY007195(H1oo), X13855 (B4), AF499607 (H1M), AAB48830 (csH1), NP_005314 (H1t), and NP_005312 (H1b).

Y. Tanaka et al. / Biochemical and Biophysical Research Communications 304 (2003) 351–357

355mRNA was detected up to a 125- or 625-fold dilution of
10 lg of total RNA by RT-PCR (Fig. 5). It must be
noted that the second-round PCR was performed for
osH1 but not for b-actin; therefore, the former assay
was much more sensitive than the latter.

Discussion

Fig. 4. Expression of osH1 and b-actin mRNA in human ovary andovarian granulosa cells. RT-nested PCR for osH1 was performed using
the primer pairs GM1D for reverse transcription, GM1D/MR1U forthe ﬁrst-round PCR, and GM2D/MR2U for the second-round PCR.
RT-PCR for b-actin mRNA was performed as described in Materials
and methods.

detected even in the undiluted RNA sample, while bactin was detectable up to a 125-fold dilution (Fig. 4).
The sequence of the cDNA product was identical to thatof osH1 (data not shown).
No osH1 mRNA was detected by RT-nested PCR in
10 lg of total RNAs from any human tissues tested(breast, colon, kidney, liver, lung, skeletal muscle,
spleen, lymphocytes, and sperm). In contrast, b-actin

Heterogeneity of histone H1 was ﬁrst described in1966 [18]. At the present time, eight subtypes of histone
H1 have been identiﬁed in mammals. Five of these
are collectively called the somatic types, which aredesignated H1a, H1b, H1c, H1d, and H1e [2–6]. Subtypes from H1b to H1e are present in all somatic cells,
while H1a is restricted to the thymus, testis, spleen, andpossibly lymphocytes and neurons [19]. They share ahighly conserved central globular domain and exhibit
variation in their amino- and carboxy-terminal domains[20]. The sixth subtype, known as H10 or H5, is diﬀerentiation stage-speciﬁc and has a divergent sequence inthe globular domain and the tails compared with somatic
subtypes [20]. The remaining two subtypes are known astissue-speciﬁc; testis-speciﬁc H1 (H1t) and oocyte-speciﬁc H1 (H1oo in mouse) [6,21,22]. H1oo shows little
homology with other somatic subtypes over the fulllength, while H1t exhibits some homology in the globular
domain [23].
To date, the oocyte-speciﬁc histone H1 gene has beenreported in four species of organisms; csH1 in the sea
urchin, B4 in X. laevis, H1M in the zebraﬁsh, and H1ooin the mouse [6,7,13,14]. Presence of a human homo-Fig. 5. Tissue localization of osH1 transcripts studied by semi-quantitative RT-nested PCR. Total RNAs from human colon, liver, lung, breast,
kidney, spleen, skeletal muscle, MOLT-4 cells, and sperm were diluted ﬁvefold sequentially. All original solutions contained 10 lg of total RNA.
b-Actin was detectable up to a 54 dilution in colon, 54 in liver, 55 in lung, 55 in breast, 55 in kidney, 56 in spleen, 54 in skeletal muscle, 54 in MOLT-4
cells, and 54 in sperm. osH1 was undetectable in any of these samples.

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Y. Tanaka et al. / Biochemical and Biophysical Research Communications 304 (2003) 351–357

logue of the mouse oocyte-speciﬁc H1 gene was recently
predicted by the NCBI annotation system, which had
not been proved in vivo, however. In the present study,
using our method for single-cell analysis, we foundtranscripts of the predicted gene encoding an oocytespeciﬁc linker histone H1 (osH1), as a 347-amino acidprotein with a calculated molecular mass of 35.8 kDa.
We believe that the osH1 gene belongs to the genefamily of oocyte-speciﬁc histone H1 for the following
reasons. (1) The mRNA was expressed only in the human oocyte and ovary. (2) The encoded protein had theclosest homology with H1oo in the mouse, and to lesser
extent, with B4 in X. laevis. The oocyte-speciﬁc H1s are
the longest H1 histones described to date [6]; evenamong these, osH1 appeared exceptionally long (cf.
H1oo, 304 amino acids; B4, 273; and csH1, 299).
We identiﬁed cDNA of the human oocyte-speciﬁc H1gene with PCR primers designed according to the genestructure that had been predicted by the NCBI annotation project from the human genomic sequence
through a gene prediction algorithm, GenomeScan. The
cDNA that we obtained was longer than the predicted
cDNA by 48 nucleotides at the 30 end of exon 3 (Fig.
2A). In other words, the actual 50 splice site of intron 3 islocated 48 nucleotides downstream of the predicted one.
GenomeScan was developed in 2001; its sensitivity andspeciﬁcity both have been estimated to exceed 80%,
which are better than those of any other gene prediction
programs [24,25]. Howsoever the accuracy of programs,
our result indicated that exact sequence of a computerpredicted gene should be determined by experiments at
the molecular level.
Generally, tissue-speciﬁc H1 subtypes are less conserved among organisms than somatic H1 subtypes. For
instance, in comparisons between the human and mousegenes, H1b (somatic subtype) and H1t (testis-speciﬁc
subtype) exhibited homologies of 70.1% and 59.7%,
respectively [21,22]. Even when considering these scores,
the homology score of 42.3% between mouse and human oocyte-speciﬁc H1 (osH1 and H1oo) determined inthis study was remarkably low. Oocyte-speciﬁc H1 isthought to bind less tightly to linker DNA than theother H1 subtypes because its net charge is less basic[12,14]. Stronger binding of oocyte-speciﬁc H1 withlinker DNA could interfere with its replacement by somatic H1 during embryogenesis. This need for lessstringent interaction may allow oocyte-speciﬁc H1 toacquire greater variation.
The cDNA sequence of oocyte-speciﬁc H1 involved
only 6 and 21 bp in the 50 and 30 untranslated regions(UTR), respectively. The remainder of both the UTR
and the promoter region, however, can be deduced fromthe genomic sequence in GenBank. Somatic H1 genes
generally harbor TATA boxes, CCAAT boxes, H1boxes (AAACACA), and TG boxes (TGTGT/CTA) inthe promoter [26,27]. Although the oocyte-speciﬁc H1

genes of nonhuman species lack such promoter sequences [12], the 50 ﬂanking region of the osH1 genecontains two of these promoter elements, the TATA box
at position )67 and the CCAAT box at )108, withposition 1 corresponding to the initiation of translation.
On the other hand, the 30 UTR of both osH1 andH1oo contain the consensus polyadenylation signal(AAUAAA), the cytoplasmic polyadenylation element(UUUUAU), and the deadenylation element (AUUUA)(Fig. 2A). In our analysis, osH1 cDNA was detected by
RT-nested PCR using an oligo(dT) primer for reverse
transcription. These ﬁndings indicated that the osH1mRNA is polyadenylated like other oocyte-speciﬁc H1
mRNAs, in contrast with a lack of polyadenylation insomatic H1 mRNAs.
In the process of normal fertilization, the condensed state of the sperm chromatin is loosened byreplacement of protamine by oocyte-speciﬁc H1, followed by formation of the male pronucleus, which
eventually leads to cleavage [28]. In the clinical practice of in vitro fertilization for infertile couples, someoocytes do not initiate cleavage even after intracytoplasmic sperm injection, thus remaining unfertilized.
Morphological studies have shown that some of theunfertilized oocytes terminate the fertilization process
at the stage of chromatin decondensation of sperm,
suggesting that inadequate replacement of protamineby osH1 is a cause of fertilization failure [29]. Accordingly, further investigation of osH1 may provide
new understanding of human fertilization and embryogenesis.
In conclusion, cDNA encoding a human oocytespeciﬁc linker histone H1, osH1, was identiﬁed for theﬁrst time in human oocytes using a novel method forsingle-cell analysis of gene expression that was developed in the present work.

Acknowledgments
We thank Dr. Toshio Fukazawa for critical reading, Atsuko
Soeda for technical assistance, and Kazuyo Nakamura for proofreading.

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