Current Biology 16, 166–172, January 24, 2006 ª2006 Elsevier Ltd All rights reservedDOI 10.1016/j.cub.2005.11.071
Comparison of Gene Expression in Male and Female
Mouse Blastocysts Revealed Imprinting of the
Shin Kobayashi,1Ayako Isotani,1,2Nathan Mise,3
Masamichi Yamamoto,4Yoshitaka Fujihara,1,5
Kazuhiro Kaseda,1Tomoko Nakanishi,1,6
Masahito Ikawa,1Hiroshi Hamada,4Kuniya Abe,3
and Masaru Okabe1,2,5,*
1Research Institute for Microbial Diseases
Suita, Osaka 565-0871
2Graduate School of Pharmaceutical Sciences
Suita, Osaka 565-0871
3Technology and Development Team for Mammalian
RIKEN Tsukuba Institute
Tsukuba Ibaraki 305-0074
4Developmental Genetics Group
Graduate School of Frontier Biosciences
Suita, Osaka 565-0871
5Graduate School of Medicine
Suita, Osaka 565-0871
Mammalian male preimplantation embryos develop
more quickly than females [1, 2]. Using enhanced
green fluorescent protein (EGFP)-tagged X chromo-
gene expression patterns between male and female
mouse blastocysts by DNA microarray. We detected
nearly 600 genes with statistically significant sex-
linked expression; most differed by 2-fold or less. Of
11 genes showing greater than 2.5-fold differences,
four were expressed exclusively or nearly exclusively
sex dependently. Two genes (Dby and Eif2s3y) were
mapped to the Y chromosome and were expressed in
male blastocysts. The remaining two (Rhox5/Pem and
Xist) were mapped to the X chromosome and were
predominantly expressed in female blastocysts. More-
over, Rhox5/Pem was expressed predominantly from
the paternally inherited X chromosome, indicating
sex differences in early epigenetic gene regulation.
Sex-Linked Differential Gene Expression
In mammals, phenotypic gender is normally determined
at the time of gonadal differentiation . However, this
may not be the sole determinant. For example, male
blastocysts develop more quickly than female blasto-
cysts [1, 2], and expression of several genes, such as
murine Xist [4, 5], bovine G6PD [6, 7], ZFX , HPRT 
and INF–t , and murine Zfy and Sry , is different in
each sex. However, there is no report on global differ-
ences in gene expression in male and female blasto-
cysts, largely because of the technical difficulty in sex-
ing many blastocysts quickly and accurately. We
overcame this by using a transgenic mouse line in which
the X chromosome is tagged with a ubiquitously-
expressed EGFP transgene (XGFP) [10–12]. The system
were mated with wild-type females (XX). Only the female
embryos (XXGFP) fluoresce green because of the pater-
nally inherited XGFP.
More than 1000 sexed blastocysts were studied. The
global gene-expression patterns of male and female
blastocysts were compared with the Agilent Mouse De-
velopment DNA microarray, which contains 20,371 tran-
Three independent experiments were carried out, and
Figure 1C shows the normalized results. We identified
591 differentially expressed genes (blue spots); three
hundred and eleven were expressed to a higher degree
to a higher degree in males (p < 0.01). Most of the differ-
ences were 2-fold (200%) or less (Figure 1C).
Of seven genes previously reported to be expressed
differentially in male and female blastocysts of the cow
and mouse [4–9], three were not represented in this mi-
croarray and one was not expressed in the mouse, leav-
ing three candidate genes (Xist, G6pd, and Hprt) avail-
able for analysis. These were all significantly differently
expressed by sex and are listed in Table 1.
with blastocysts sexed by XGFP. Of these, Xist and
Rhox5/Pem on the X chromosome were predominantly
expressed in females, whereas Dby and Eif2s3y on the
Y chromosome were exclusively expressed in males
(Figure 2). The same results were obtained with wild-
type blastocysts not expressing EGFP (Figure S1 in the
Supplemental Data available with this article online).
Verification of Imprinting of the Rhox5/Pem Gene
Because Rhox5/Pem is located on the X chromo-
some and is predominantly expressed in females, we
6Present address: Institute of Applied Biochemistry, University of
Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan.
To distinguish paternal from maternal expression, we
produced inter-subspecific F1 mice by crossing M. m.
musculus (C57BL/6;B6) with M. m. molossinus (JF1/
Ms;JF1). A DNA polymorphism in the 30-UTR of the
Rhox5/Pem gene (Figure 3A) is detectable by restriction
fragment length polymorphism (RFLP) analysis with
BsrBI. This was used to analyze the active allele in F1
blastocysts derived from mating between C57BL/6 fe-
males and JF1/Ms males (B6 3 JF1) and the reciprocal
cross of JF1/Ms females with C57BL/6 males (JF1 3
B6). The RT-PCR products (Figure 3B) clearly show
that the gene was predominantly expressed from the
paternally inherited X chromosome.
Because early preimplantation-stage embryos al-
ready show dosage compensation via inactivation of
the paternal X chromosome (Xp) [13–15], we examined
whether there was expression of the paternal allele of
bryos from the M. m. musculus 3 M. m. molossinus
crosses. Figure 3C shows that the gene was expressed
predominantly from Xp after the 8 cell stage to the blas-
tocyst stage. This indicated that Rhox5/Pem escaped
Rhox5/Pem expression levels in male and female
post-implantation stages were also examined in em-
bryos at 5.5, 6.5, and 7.5 days post-coitum (dpc) by
whole-mount in situ hybridization (Figure 4). Despite the
predominant expression in female blastocysts, Rhox5/
Pem was expressed in both male and female later-stage
embryos: not in the epiblasts, but in extraembryonic
ectoderm (ExE) and visceral endoderm (VE). When the
expressed allele was examined in inter-subspecific
crosses, Rhox5/Pem was expressed predominantly as
the maternal allele in the 7.5 dpc embryos (Figure 3C,
In eutherian mammals, male preimplantation embryos
develop more rapidly than females in a number of spe-
cies, such as the mouse [1, 2], cow [16, 17], human
, sheep , and pig . These differences precede
gonadal sex commitment. Here we found minor but sta-
tistically significant sex differences inthe expressions of
nearly 600 genes. These may have arisen from slight dif-
ferencesindevelopmental stagesbetween themaleand
female blastocysts, so we cannot conclude that all are
associated with sex differentiation. However, we have
demonstrated here that at least four genes are certainly
expressed differently, not by stage but by sex. We pre-
sume that the actual number of genes involved in such
sex differences is likely to exceed the small number of
previously reported genes [4–9].
Figure 5indicates the chromosomal distribution ofthe
no obvious tendency in the distribution of male upregu-
ted 212 of these X-linked genes on the X chromosome,
most of the variance in expression remained within the
range of 1 % FC % 2 (Figure S2). These included previ-
ously reported female upregulated genes such as G6pd
and Hprt (Table 1, lower row). This slight differential
Figure 1. Screening of Differentially Expressed Genes between
Sexes at the Preimplantation Stage
(A) The scheme of a XGFPsexing system.
(B) Blastocysts at 3.5 dpc. Only female embryos become green.
(Left: bright field. Right: dark field).
(C) Expression profiling of 20K genes between nonfluorescent male
and fluorescent female blastocysts. For each gene, average signal
intensity is displayed on a scatter plot. Genes that showed signifi-
cantly different expression levels between females and males at the
1% significance level (p < 0.01; the complete description of the sta-
tistical methods is available in the technology section of the Rosetta
Biosoftware website, http://www.rosettabio.com/tech/default.htm)
are displayed as blue spots, and the others are displayed as gray
spots. The numbered genes were re-examined for their expression
by RT–PCR. Dotted lines indicate 2.0-fold (200%) expression
X-Linked Imprinted Gene Rhox5/Pem
expression of many X-linked genes may be attributable
to incomplete X inactivation or to the reactivation of X-
linked genes in epiblast cells.
Many of the genes showing differential expression
between sexes were linked to the X chromosome, but
only two genes were linked to the Y chromosome.
More are likely to be present because some Y-linked
genes (Sry and Zfy) that are expressed in blastocysts
on the array are listed in Table S1. However, we clearly
detected differential expression of Dby and Eif2s3y.
Because the Y chromosome may accelerate growth of
preimplantation mouse embryos, whereas paternally
derived X chromosomes or double X chromosomes re-
tard it [21–24], our findings may help elucidate these
As with the Xist gene, Rhox5/Pem was predominantly
expressed from the paternally derived X chromosome in
the blastocyst. This is a member of a homeobox gene
cluster that is mainly expressed in reproductive tissues
such as those of the testis, ovary, and placenta, which
may play a role in controlling the development of these
organs . At present we have no information on the
mechanism that enables preferential expression of the
paternal Rhox5/Pem allele from the 8 cell to the blasto-
cyst stage, when the paternal X is thought to be inacti-
vated, or how the subsequent switch to expression of
the maternal allele is brought about. Further experi-
ments are needed to address these issues. However it
is known that some imprinted genes show complicated
expression patterns. One example is mouse Grb10,
nal expression in other tissues [26, 27]. It is not clear if
this imprinting of Rhox5/Pem disappeared after implan-
tation, but the expression pattern shown in Figure 3C
suggests stage-specific imprinting of this gene.
Using gene targeting, MacLean et al. showed that
Rhox5/Pem null males were subfertile with reduced
sperm count and decreased sperm motility . How-
ever, they did not report the effect of gene disruption
on embryonic development. It would be worthwhile to
examine the early embryonic development between
Table 1. Upregulated Genes in Female Blastocysts
Gene Name Accession NumberMap PositionN-Fold Changep ValueIntensity (Male) Intensity (Female)
4.9 3 1026
6.3 3 10228
4.4 3 1023
4.6 3 10212
1.5 3 1023
2.0 3 10233
All genes showing more than a 2.5-fold (250%) change in expression level are listed in the upper part of the table.
aNumbers correspond to those indicated in Figure 1C.
bGenes that were previously reported to be differently expressed.
Table 2. Upregulated Genes in Male Blastocysts
Gene NameAccession Number Map PositionN-Fold Changep ValueIntensity (Male)Intensity (Female)
6.5 3 1023
1.1 3 10213
4.2 3 1023
All genes for which the change in expression level is less than 22.5-fold are listed.
aNumbers correspond to those indicated in Figure 1C.
Figure 2. RT-PCR Analysis of Differentially Expressed Genes
Genes showing expression differences of more than 2.5-fold (250%)
(see Tables 1 and 2) were chosen, and RT–PCR allowed the differen-
tial expression of the genes to be re-examined. Among these genes,
four (Xist, Rhox5/Pem, Dby, and Eif2s3y) showed obvious differ-
ences. It should be noted that very weak expression of Xist and
Rhox5/Pem was detected in male blastocysts when additional
PCR cycles were carried out.
of a family (Rhox1-12) whose members might compen-
sate for loss of the Rhox5/Pem function. All 12 Rhox
genes are contained within an approximately 0.7 Mb
segment of the X chromosome, and expression analysis
of the 12 Rhox genes in male and female blastocysts re-
vealed that Rhox9/Psx2 was also predominantly ex-
pressed in females, as with Rhox5/Pem. This suggests
an imprinted cluster near the Rhox5/Pem gene.
Other X-linked imprinted genes (Xlr3b, Xlr4b, and
Xlr4c) have been reported [28, 29], and their expression
levels are 5–6 times higher in the developing brain of
males than in females. However, Rhox5/Pem reported
here is paternally expressed, whereas Xlr genes are ma-
ternally expressed. Thisfindingofimprinted Rhox5/Pem
indicates epigenetic differences between male and
female embryos as early as the preimplantation stage.
X-linked imprinted genes in general appear to affect the
ated with later cognitive dysfunction, if they are mutated
[24, 30, 31]. Thus, studies on the potential imprinted re-
gions of Xlr genes and Rhox5/Pem may provide new in-
sights into imprinting-related disorders.
Nearly 600 genes were differentially expressed between
male and female blastocysts. Among these, Xist and
Rhox5/Pem were expressed predominantly in females,
and Dby and Eif2s3y were exclusively expressed in
males. Moreover, Rhox5/Pem is an imprinted gene ex-
pressed from the paternally derived X chromosome, in-
dicating epigenetic sex differences as early as the pre-
Figure 3. Verification of the Rhox5/Pem Gene Imprinting
(A) The single-nucleotide polymorphism detected in C57BL/6;B6 (M. m. musculus) and JF1/Ms;JF1 (M. m. molossinus) is shown. The BsrBI site
(GAGCAG)inthe B6 allelewas changed toGAGCGG inthe JF1allele.These alleles could bedistinguished bydigestion with BsrBI;40bp and216
bp digested PCR fragments appeared in the B6 sample, whereas a 256 bp undigested PCR product was seen in the JF1/Ms samples.
(B) Predominant expression of Rhox5/Pem from paternal X chromosome was determined by RT–PCR and RFLP analysis with inter-subspecific
hybrid mice F1 progenies.
(C)ImprintedexpressionofRhox5/Pem atearlyembryonicstages. TheexpressionofRhox5/Pemwas examined inunfertilized eggs(B6)and at2
cell, 8 cell, morula, and blastocyst stages (B63JF1) (left panel). Allelic expression of Rhox5/Pem was analyzed in B6 3 J F1 embryos (8 cell, mor-
ula, blastocyst, and 7.5 dpc embryonic stages; right panel).
X-Linked Imprinted Gene Rhox5/Pem
The handling and surgical manipulation of all experimental animals
were carried out according to the guidelines of the Committee on
the Use of Live Animals in Teaching and Research of Osaka Univer-
sity. The strain B6C3F1 TgN (act EGFP) Osb CX-38 (G38) described
previously  was used as an EGFP-expressing transgenic mouse
line to distinguish between male and female embryos.
Blastocyst Collection and RNA Extraction
Eight-week-old B6C3F1femalemice weresuperovulatedwith 5IUof
pregnant mare serum gonadotropin (PMSG) followed by 5 IU of hu-
man chorionic gonadotropin (hCG) 48 hr later and were mated with
XGFPY male mice. Four-cell-stage embryos were collected from the
oviducts 55 hr after the hCG injection, placed in kSOM, and incu-
bated in a humidified atmosphere of 5% CO2in air at 37ºC for an ad-
ditional 38 hr. We separated male (EGFP-negative) and female
(EGFP-positive) embryos atthe blastocyst stage byobserving green
fluorescence under a dissection microscope. RNA samples were
prepared from sexed blastocysts with ISOGEN (NIPPON GENE
Inc., Japan), which is based on acid guanidine thiocyanate-phenol-
chloroform extraction. To verify differential gene expression in non-
transgenic embryos in vivo, we obtained wild-type C57BL/6 blasto-
cysts from the uterus of superovulated C57BL/6 females mated
with wild-type C57BL/6 males 92 hr after hCG injection. Genomic
DNA and RNA were extracted from individual blastocysts. Male
and female blastocysts were pooled separately after sex determina-
tion PCR with the following Ube1x primers: 50-TGGTCTGGACCC
GATG-30. For each independent experiment, 30–40 wild-type
blastocysts were sexed and pooled samples were used for expres-
Comparative Expression Analysis with DNA Microarray
nih.gov/projects/geo/). The GEO accession number is GSE2934.
RT-PCR of Candidate Genes for Sexually Dimorphic Expression
Reverse transcription was carried out with the pooled total RNA ex-
tracted from 100 blastocysts via Superscript RT (Invitrogen). One
hundredth of the resulting cDNA samples was amplified by PCR
withExTaq DNApolymerase (TaKaRa).Reactionmixtures contained
13 ExTaq buffer, 2.5 mMdNTP, 40 pmolof primers,and 1.25 units of
were 1 min at 96ºC, followed by 30–33cycles for Xist, Rhox5/Pem,
Dby, and Eif2s3y and 24 cycles for b-actin. Cycles were 96ºC for
15 s, 65ºC for 30 s, and 72ºC for 30 s, with a final 1 min extension at
72ºC. Primer sets were as follows: 50-AAGTGTGACGTTGACATC
CG-30and 50-GATCCACATCTGCTGGAAGG-30for b-actin, 50-CT
GG-30for Xist, 50-AGAGATGAGCAAGGTGCACA-30and 50-CGAAC
CTAGAGCCCTGGAG-30for Rhox5/Pem, 50-CGACCATATCTTCCAT
TTTCC-30and 50-GCCTGGACCAGCAATTTGTTG-30for Dby, 50-GC
Figure4. Expression ofRhox5/Pemin5.5,6.5,and7.5dpc Maleand
No obvious differences were detected. Rhox5/Pem expression was
detected in extraembryonic ectoderm (ExE) and visceral endoderm
detected in extraembryonic ectoderm (ExE), but not in epiblast, at
7.5 dpc. The scale bar represents 50 mm.
Figure 5. The Chromosomal Distribution of Differentially Expressed
The upper and lower panels show upregulated genes in males and
females, respectively. Open and closed bars correspond to the
value of n-fold changes as shown in the figures. p < 0.01. The calcu-
lation methods were the same as those described in the legend for
CGTC-30for Eif2s3y. PCR primers for Dby and Eif2s3y were from
. All PCR reactions were repeated at least once. The same PCR
conditions were used for examining the wild-type blastocysts, except
the amount of starting material (30–40 blastocysts) was different.
Verification of Imprinting
TworeciprocalF1hybrid blastocysts, (C57BL/63JF1)F1and(JF13
C57BL/6) F1, were produced by an in vitro fertilization (IVF) method.
In each experiment at least 30 blastocysts were sexed with a PCR-
cordingtotheirsex.Onethirdoftheresulting cDNAsamples derived
from pooled RNAs were amplified by PCR with the above primers.
To verify the imprinting status of Rhox5/Pem, we digested amplified
by using an Agilent Bio Analyzer 2100 (Agilent). The allelic expres-
sion of Rhox5/Pem in 2 cell, 8 cell, and morula stages were analyzed
with non-sexed C57BL/6 3 JF1 pooled samples (20–50 pooled em-
bryos) by RFLP analysis. Embryos (7.5 dpc) were sexed by PCR as
described above; six females were pooled, and expression was an-
alyzed. We carried out duplicate RT–PCR and RFLP analyses.
In Situ Hybridization and Histology
Mouse embryos were staged on the basis of their morphology .
Whole-mount in situ hybridization was performed as described in
our previous paper . The RNA probes encompass nucleotides
1–431 of Rhox5/Pem (NM_008818), and this sequence is specific
Supplemental Data are available with this article online at http://
This work was supported by a Grant-in-Aid for Scientific Research
from The Ministry of Education, Culture, Sports, Science, and Tech-
nology and by the 21st Century COE program from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan.
Received: July 13, 2005
Revised: November 7, 2005
Accepted: November 25, 2005
Published: January 23, 2006
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