Báo cáo khoa học: Targeted disruption of one of the importin a family members leads to female functional incompetence in delivery docx

Targeted disruption of one of the importin a family
members leads to female functional incompetence in
delivery
Tetsuji Moriyama
1
, Masahiro Nagai
2
, Masahiro Oka
1,2,3
, Masahito Ikawa
4
, Masaru Okabe
4
and
Yoshihiro Yoneda
1,2,3
1 Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Japan
2 Department of Biochemistry, Graduate School of Medicine, Osaka University, Japan
3 JST, CREST, Graduate School of Frontier Biosciences, Osaka University, Japan
4 Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Japan
Introduction
In eukaryotic cells, the nuclear and cytoplasmic com-
partments are separated by the nuclear envelope. The
nuclear envelope contains nuclear pore complexes that
allow macromolecules to be exchanged between the
two compartments. The nucleocytoplasmic transport
system functions as a key mediator of signal transduc-
tion by regulating protein localization. The nuclear
import of proteins generally depends on the presence
of specific signal sequences called nuclear localization
signals (NLSs), and the basic type of NLS is recog-

nized by an importin a⁄ b heterodimer and targeted to
nuclear pores. Importin b possesses affinity for nucleo-
porins, which are components of the nuclear pore
complex that mediate nuclear import. On the other
hand, the importin a family generally binds to both
the nuclear import cargo and importin b, indicating
that importin a functions as an adaptor between the
cargo proteins and importin b. In the nucleus,
the import complex encounters the GTP-bound form
of Ran (RanGTP), which is a member of the Ras
Keywords
estrogen; gene knockout; importin a;
nuclear transport; reproduction
Correspondence
Y. Yoneda, Department of Frontier
Biosciences, Osaka University, Graduate
School of Frontier Biosciences, Osaka
University, 1-3 Yamada-oka, Suita, Osaka
565-0871, Japan
Fax: +81 6 6879 4609
Tel: +81 6 6879 4606
E-mail:
(Received 30 October 2010, revised 10
February 2011, accepted 22 February 2011)
doi:10.1111/j.1742-4658.2011.08079.x
Importin a mediates the nuclear import of proteins through nuclear pore
complexes in eukaryotic cells, and is common to all eukaryotes. Previous
reports identified at least six importin a family genes in mice. Although
these isoforms show differential binding to various import cargoes in vitro,
the in vivo physiological roles of these mammalian importin a isoforms

remain unknown. Here, we generated and examined importin a5 knockout
(impa5
) ⁄ )
) mice. These mice developed normally, and showed no gross his-
tological abnormalities in most major organs. However, the ovary and
uterus of impa5
) ⁄ )
female mice exhibited hypoplasia. Furthermore, we
found that impa5
) ⁄ )
female mice had a 50% decrease in serum progester-
one levels and a 57% decrease in progesterone receptor mRNA levels in
the ovary. Additionally, impa5
) ⁄ )
uteruses that were treated with exoge-
nous gonadotropins displayed hypertrophy, similarly to progesterone recep-
tor-deficient mice. Although these mutant female mice could become
pregnant, the total number of pups was significantly decreased, and some
of the pups were dead at birth. These results suggest that importin a5 has
essential roles in the mammalian female reproductive organs.
Abbreviations
EBAG9, estrogen receptor-binding fragment-associated antigen 9; EFP, estrogen-responsive finger protein; ER, estrogen receptor; FRT, FLP
recombinase target; FSHR, follicle-stimulating hormone receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hCG, human
chorionic gonadotropin; impa5
) ⁄ )
, importin a5 knockout; LHR, luteinizing hormone receptor; Ltf, lactotransferrin; NLS, nuclear localization
signal; PMSG, pregnant mare serum gonadotropin; PR, progesterone receptor; SEM, standard error of the mean.
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1561
superfamily that localizes to the nucleus, and this inter-
action causes the cargo protein to dissociate from the

complex [1,2]. It has been reported that there is only a
single importin a gene in budding yeast, whereas at
least six importin a family genes have been found in
mice and humans. These importin a molecules are clas-
sified into three subtypes on the basis of their sequence
homology. The importin a1 subfamily in mice consists
of importin a1 (karyopherin a2, PTAC58, Rch1); the
importin a3 subfamily includes importin a3 (karyoph-
erin a4, Qip1) and importin a4 (karyopherin a3,
Qip2); and the importin a5 subfamily includes impor-
tin a5 (karyopherin a1, NPI-1) and importin a7 (kary-
opherin a6) [3]. Additionally, very recently, a novel
importin a isoform (importin a8, karyopherin a7) was
identified that is expressed during oocyte maturation
and early embryonic development [4].
These importin a isoforms have distinct binding
characteristics for various NLS-containing proteins
in vitro [5–7]. Furthermore, previous studies indicated
that the importin a isoforms are differentially expressed
in adult mouse and human tissues [8–10]. More
recently, it was reported that these transport factors
function as major players in determining cell fate [11].
Thus, these results suggest that each importin a iso-
form may contribute to a variety of physiological func-
tions in multicellular organisms. Indeed, in the fruit fly
Drosophila melanogaster, which expresses three classes
of importin a homologs in unique temporal and spatial
patterns, it has been shown that mutants lacking any
single importin a isoform have defects in gametogenesis
[12–16], indicating that all of the importin a isoforms

are required for germline development. In addition,
importin a1 (mammalian importin a5 subfamily homo-
log) is required for normal wing development [12], and
importin a2 (mammalian importin a1 subfamily homo-
log) is involved in synapse, axon and muscle develop-
ment [17,18]. Importin a3 (mammalian importin a3
subfamily homolog) is required for flies to mature into
adults and for tiling of photoreceptor axons in the
visual system [14,19]. Moreover, it was very recently
reported that destruction of mouse importin a8 causes
a significant reduction in fertility and fecundity [20].
The mammalian importin a5 subfamily has higher
homology with plant and fungal importin a than the
other mammalian importin a isoforms, suggesting that
the other importin a isoform genes in mammals arose
from an importin a5-like progenitor [1]. Although
Shmidt et al. reported that importin a5 mutant mice
did not exhibit any obvious morphological or behav-
ioral abnormalities [21], these mice have not been ana-
lyzed in detail. To gain further insights into the in vivo
physiological significance of importin a5 in mammals,
we generated importin a5 knockout (impa5
) ⁄ )
) mice
using the Cre–lox system, which differs from the
method used by Shmidt et al., and analyzed them in
detail. Here, we report that an importin a5 deficiency
affects the female reproductive organs and causes func-
tional deterioration of the female reproductive tract.
Results

Targeted disruption of the mouse importin a5
gene
To study the physiological significance of importin a5
in mammals, we used gene targeting to generate
impa5
) ⁄ )
mice (Fig. 1A). Because exons 2 and 3 of the
importin a5 gene encode the translation start site and
importin b-binding site, we disrupted these areas with
a Cre–loxP system. Targeted ES cell clones were identi-
fied by PCR (Fig. 1B) and Southern blotting (Fig. 1C),
and were used to generate impa5
) ⁄ )
mice as described
in Experimental procedures. The absence of the impor-
tin a5 protein was confirmed by western blot analysis
with tissue lysates from impa5
) ⁄ )
mice (Fig. 1D).
Intercrossing between the heterozygous parents pro-
duced homozygous knockout animals in the expected
Mendelian ratio (wild-type ⁄ heterozygote ⁄ homozygous
knockout = 19 : 35 : 13). Both male and female
mutant mice developed normally and showed no appar-
ent gross developmental abnormalities (Fig. 1E,F). A
previous study with impa5
) ⁄ )
mice demonstrated that
importin a4 was markedly upregulated in the brain,
suggesting that the counter-regulation of another im-

portin a isoform may compensate for the lack of a
single isoform in vivo in mammals [21]. Therefore, to
determine whether the lack of importin a5 affects the
expression of other importin a isoforms in our
impa5
) ⁄ )
mice, we compared the protein expression
levels of each importin a isoform in various tissues
from impa5
) ⁄ )
and wild-type mice by western blotting.
There were no obvious differences in the expression of
other importin a isoforms between impa5
) ⁄ )
and wild-
type mice (Fig. S1).
Genital hypoplasia in impa5
) ⁄ )
female mice
Tissue sections from impa5
) ⁄ )
and wild-type mice were
compared for three pairs of male and female animals.
Histological analyses showed that impa5
) ⁄ )
mice had
no gross abnormalities in the brain (Fig. 2A), spinal
cord, sciatic nerve, thymus, lung, heart, liver (Fig. 2B),
pancreas, mammary gland, testis, vagina (Fig. 2C), etc.
(Fig. S2). Analyses of hematological and biochemical

parameters showed mild increases in aspartate
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1562 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS
aminotransferase and alanine aminotransferase levels,
and decreases in total cholesterol levels and platelet
counts (Tables S1 and S2), suggesting a slight deteriora-
tion in liver function. However, a detailed histological
analysis and apoptosis test with the terminal deoxynu-
cleotidyl transferase dUTP nick end labeling method
did not show any abnormalities in the liver. These
results indicate that loss of importin a5 does not obvi-
ously affect the organization and function of most
organs. However, we noticed that the reproductive
tracts in all impa5
) ⁄ )
females had crucial differences
from wild-type female mice. The impa5
) ⁄ )
ovary had a
reduced number of growing follicles at the maturation
stage (Fig. 2D). The uteruses of impa5
) ⁄ )
mice had thin
myometrial, stromal and epithelium layers, and imma-
ture endometrial glands, as compared with the uterine
morphology of wild-type female mice (Fig. 2E). In
order to elucidate the cause of the abnormalities
observed in the reproductive tracts of impa5

) ⁄ )
females,
we examined the pattern of importin a5 protein expres-
sion in wild-type ovary and uterus by immunohisto-
chemistry (Fig. 3). Abundant importin a5 signals were
observed in both the ovary and uterus of wild-type
female mice, but not in sections prepared from impa5
) ⁄ )
female mice. Interestingly, importin a5 was strongly
expressed in granulosa cells of ovaries (Fig. 3A), and in
the luminal and glandular epithelium of the uterus
(Fig. 3B). These expression patterns suggest that impor-
tin a5 may have especially important functions in the
maturation of the ovum and uterine epithelial layers.
A
B
D
C
EF
Fig. 1. Generation of importin a5-deficient mice. (A) Schematic representation of homologous recombination of the targeting vector and
recombination steps. The numbered closed boxes denote the translated exons of the gene. (B) PCR analysis for the confirmation of homolo-
gous recombination of the short arm side. Genomic DNA isolated from ES clones was used as a template. A 2.9-kb band was detected in
the targeted allele but not in the wild-type allele. (C) Southern blot analysis for the confirmation of homologous recombination of the long
arm side. PvuII–PacI-restricted DNA yielded 12-kb and 9.2-kb bands for wild-type and recombinant alleles, respectively. The small box in (A)
represents the DNA probe used to screen for homologous recombination of the long arm side. (D) Immunoblotting analysis of importin a5
protein expression. Importin a5 and GAPDH protein expression was detected by immunoblotting with 15 lg of various tissue lysates from
impa5
) ⁄ )
and wild-type mice. Arrowhead: importin a5 protein band. *Nonspecific band. (E, F) Growth curves for male (E) and female (F)
impa5

) ⁄ )
, impa5
+ ⁄ )
and wild-type mice. Each mouse was weighed 1–8 weeks after birth. Error bars indicate the standard deviation. KO,
knockout; TK, thymidine kinase; WT, wild-type.
T. Moriyama et al. Reproductive organ abnormalities in impa5
) ⁄ )
mice
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1563
To determine the effects of importin a5 disruption
on reproduction, the fertility of impa5
) ⁄ )
mice was
examined. For 28 days, impa5
) ⁄ )
and impa5
+ ⁄ )
females were mated with impa5
) ⁄ )
or impa5
+ ⁄ )
males,
and the numbers of pregnant female mice, pups and
live pups were counted. Mating of impa5
) ⁄ )
females
with wild-type males resulted in significantly smaller
litter sizes (Table 1). Furthermore, we found that
impa5
) ⁄ )

female mice had significantly increased num-
bers of dead pups in their cages after delivery. The
mean number of live pups born to impa5
+ ⁄ )
females
was 6.8 ± 1.5, whereas impa5
) ⁄ )
females had an aver-
age litter size of 1.3 ± 2.7 (P < 0.001). In addition,
most of the dead pups had twisted bodies and ⁄ or bite
marks (Fig. 4A).
To determine when these pups died, we analyzed
embryonic day 18.5 embryos. However, they appeared
to develop normally, and we did not observe dead
embryos at this embryonic stage. On the other hand,
impa5
) ⁄ )
females had vaginal bleeding near the time of
delivery (Fig. 4B), and five of 17 impa5
) ⁄ )
females died
as a result of severe bleeding. In particular, one female
died while a pup remained trapped within her vagina
(Fig. 4D). In addition, some females appeared to take
a significant amount of time to deliver their pups
(Fig. 4C). These results indicate that impa5
) ⁄ )
females
had severe difficulty in delivering their pups, suggesting
that the depressed reproductive organ functions of

impa5
) ⁄ )
females damaged the pups during delivery,
and led to a decreased litter size and reduced pup sur-
vival. In contrast, impa5
) ⁄ )
male, imp a5
+ ⁄ )
male and
impa5
+ ⁄ )
female mice were as fertile as wild-type mice,
and they had comparable litter survival rates. These
results indicate that loss of the importin a5 gene causes
not only morphological but also functional deteriora-
tion of the female reproductive tract.
Reduced serum progesterone levels in impa5
) ⁄ )
female mice
The female ovaries mature in response to cycling sex
hormones. In particular, 17-b-estradiol stimulates the
proliferation of uterine layer cells, suggesting that
impa5
) ⁄ )
female mice may have imbalanced 17-b-estra-
diol levels. However, steroid hormone measurements
with sensitive enzyme immunoassays revealed that the
serum 17-b-estradiol levels were comparable between
impa5
) ⁄ )

and wild-type females (Fig. 5A). In contrast,
we found that impa5
) ⁄ )
mice had significantly reduced
progesterone levels, by  50%, as compared with wild-
type mice (Fig. 5B). The reduction in serum progester-
one is consistent with the decrease in the number of
mature follicles in the ovaries of impa5
) ⁄ )
mice,
because progesterone is produced specifically after ovu-
lation from the corpus luteum in the ovary.
Abnormal uterine development in impa5
) ⁄ )
females after treatment with exogenous
gonadotropin
To gain insights into the defective reproductive organs
of impa5
) ⁄ )
females and determine whether these mice
A
B
C
D
E
Fig. 2. Histological analysis of impa5
) ⁄ )
(left panel) and wild-type
(right panel) mice. (A) Brain. (B) Liver. (C) Vagina. (D) Ovary (two
impa5

) ⁄ )
female ovaries). (E) Uterus. CER, cerebral cortex; ep, epi-
thelial layer; HPC, hippocampus; o, oriens layer; pr, pyramidal cell
layer; r, stratum radiatum; st, stromal layer; ug, uterine gland.
Tissue sections of impa5
) ⁄ )
and wild-type mice were stained with
hematoxylin and eosin.
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1564 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS
ovulate normally, 4-week-old mice were given exoge-
nous gonadotropins, including pregnant mare serum
gonadotropin (PMSG) and human chorionic gonado-
tropin (hCG). After the hormone treatments, impa5
) ⁄ )
mice produced almost the same number of mature
oocytes as wild-type mice (9.9 ± 3.3 for impa5
) ⁄ )
,
n = 7; 10.3 ± 3.8 for wild type, n = 6). Additionally,
the volumes of impa5
) ⁄ )
ovaries were significantly
enlarged after hormone treatment, and the numbers of
growing follicles increased to levels that were compara-
ble to those in control ovaries (Fig. 6A). These results
imply that disruption of importin a5 does not lead to
defects in oogenesis, but results in decreased respon-

siveness of ovary cells to sex hormones. Furthermore,
we found that the uteruses of gonadotropin-treated
impa5
) ⁄ )
mice had abnormal uterine structures, and
that the luminal epithelium and endometrial stroma
appeared hyperplastic, as compared with wild-type
controls (Fig. 6B). It is of note that these histological
changes were similar to the previously reported pheno-
types of uteruses from progesterone receptor (PR)-
deficient mice that were treated with estrogen and
progesterone [22], raising the possibility that PR
expression is particularly suppressed in impa5
) ⁄ )
mice.
Decreased expression of genes downstream of
the estrogen receptor (ER)
Next, to further examine the possibility that PR
expression is reduced in impa5
) ⁄ )
mice, we examined
the mRNA expression levels of not only PR but also
ERa,ERb, follicle-stimulating hormone receptor
(FSHR) and luteinizing hormone receptor (LHR) in
the ovary by quantitative real-time PCR (Fig. 7A).
The gene expression levels for ERa,ERb, FSHR and
LHR were not different between impa5
) ⁄ )
and wild-
type mice, whereas PR expression was significantly

downregulated, by 57%, in impa5
) ⁄ )
mice as com-
pared with wild-type mice, indicating that importin a5
plays an essential role in regulating expression of the
PR gene. Because estrogen plays a crucial role in regu-
lating PR in target tissues, and the proximal promoter
of the PR gene possesses several estrogen-responsive
elements [23], our data suggest that loss of importin a5
leads to the downregulation of ER signaling in
PR-expressing cells and subsequent suppression of PR.
To examine this possibility, we examined the mRNA
expression levels of genes that are downstream of ER,
such as those encoding ER-binding fragment-associ-
ated antigen 9 (EBAG9) [24], estrogen-responsive fin-
ger protein (EFP) [25], and lactotransferrin (Ltf) [26],
by quantitative real-time PCR (Fig. 7B). Although the
expression levels of the follicle-stimulating hormone-
responsive gene encoding cyclin D2 [27,28] were not
significantly different between impa5
) ⁄ )
and wild-type
mice, EBAG9 and EFP expression was significantly
downregulated in imp a5
) ⁄ )
mice, by  20%. These
findings indicate that importin a5 is prominently
involved in gene regulation by ER and its cofactors.
On the other hand, the protein levels in the uterus
AC

D
B
Fig. 3. Immunohistochemistry for impor-
tin a5 expression in ovarian and uterine sec-
tions. Ovarian and uterine sections prepared
from wild-type (A, B) and impa5
) ⁄ )
(C, D)
female mice were stained for importin a5.
ep, epithelial layer; G, granulosa cell layer;
O, oocyte; st, stromal layer; ug, uterine
gland.
Table 1. Fertility data of wild-type, heterozygous and homozygous
male and female impa5
) ⁄ )
mice. Each pair (male ⁄ female = 1 : 1)
was transferred to a mating cage for 28 days. The cages were
monitored daily and for an additional 28 days, and the numbers
of pregnant female mice, pups and live pups were counted.
*P < 0.05.
Genotype
(importin a5)
Pregnancy
rate
Litter size
(mean ± SEM)
Litter survival
rate, % (no.)Male Female
+ ⁄ ++⁄ +9⁄ 9 6.8 ± 1.5 100 (61 ⁄ 61)
+ ⁄ ) + ⁄ ) 12 ⁄ 12 7.0 ± 0.9 100 (84 ⁄ 84)

) ⁄ ) + ⁄ +9⁄ 10 7.0 ± 1.7 97 (61 ⁄ 63)
+ ⁄ + ) ⁄ ) 13 ⁄ 15 4.5 ± 2.5* 29 (17 ⁄ 59)
T. Moriyama et al. Reproductive organ abnormalities in impa5
) ⁄ )
mice
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1565
(Fig. 7C) and ovary and the subcellular localization of
ERa in the uterus (Fig. 7D) were not different between
impa5
) ⁄ )
and wild-type mice, suggesting that importin
a5 does not affect the nuclear import of ER.
Discussion
The impa5
) ⁄ )
females showed depressed reproductive
organ functions, such as a reduced number of growing
follicles at the maturation stage in the ovary and
immature layer construction in the uterus, and
decreased levels of serum progesterone. Furthermore,
administration of exogenous gonadotropin restored
follicle growth in the ovary and the release of oocytes
in impa5
) ⁄ )
females, although their uteruses showed
hypertrophy (see discussion below). In addition, analy-
sis of the mRNA expression levels of estrogen-depen-
dent genes in impa5
) ⁄ )
ovaries revealed that the

transcriptional activity of ER was downregulated. It is
A
C
B
D
Fig. 4. Photographs of impa5
) ⁄ )
mice after
the delivery date. A series of photographs
show the cage (A) and impa5
) ⁄ )
mice after
(B) and during (C) delivery, and a dead
impa5
) ⁄ )
female mouse with pups trapped
within the birth canal (D). (C) This impa5
) ⁄ )
female took at least 2 days to give birth,
and all of her pups were dead. (D) This dead
impa5
) ⁄ )
mother still had two undelivered
pups in her uterus. The open arrowheads
indicate the dead pups, and the filled arrow-
heads indicate the bleeding point.
AB
Fig. 5. Serum 17-b-estradiol and progesterone levels in female
mice. Serum samples were collected, and the 17-b-estradiol and
progesterone levels were measured. (A) Serum 17- b-estradiol levels

in impa5
) ⁄ )
and wild-type mice. The 17-b-estradiol values were 9.4
and 10.4 pgÆmL
)1
for female impa5
) ⁄ )
and wild-type mice, respec-
tively, with P = 0.653. (B) Serum progesterone levels in impa5
) ⁄ )
and wild-type mice. The progesterone values were 1.25 ngÆmL
)1
and 2.51 ngÆmL
)1
for impa5
) ⁄ )
and wild-type females, respectively,
with P = 0.015. *P < 0.05; impa5
) ⁄ )
mice, n = 8; wild-type mice,
n = 8. Error bars indicate the SEM.
A
B
Fig. 6. Histological analysis of reproductive organs from 4-week-old
impa5
) ⁄ )
mice that were induced to superovulate. (A, B) Histologi-
cal analysis of the (A) ovary and (B) uterus from 4-week-old
impa5
) ⁄ )

mice that were treated with PMSG and hCG. Tissue sec-
tions from impa5
) ⁄ )
and wild-type mice were stained with hema-
toxylin and eosin.
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1566 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS
generally accepted that ER-mediated transcriptional
and biological activation requires the recruitment of a
number of cofactors, including SRC-1, CBP ⁄ p300,
TRAP220, ASC-1, SRA, and p68 [29], which facilitate
a functional interaction between the receptor and the
general transcription machinery. Our results showed
that there are no differences between impa5
) ⁄ )
and
wild-type mice in the amount and localization of the
ERa protein, suggesting that importin a5 may specifi-
cally mediate the nuclear import of at least some of
these cofactors, although we cannot completely exclude
the possibility that disruption of importin a5 reduces
the import efficiency of ER. On the other hand, mice
knocked out for over 200 genes have shown reproduc-
tive defects as a major phenotype; the genes include
encoding those encoding transcription factors and
nuclear proteins, such as C ⁄ EBPb, p27
kip1
, and

cyclin D2 [30]. Accordingly, the defects observed in the
reproductive organs of impa5
) ⁄ )
mice could result
from the combined effects of the inefficient nuclear
import of such factors.
The number of pups born to impa5
) ⁄ )
females was
clearly reduced. This phenotype could result from vari-
ous causes, including the dysfunction of the ovary
and ⁄ or uterus. The impa5
) ⁄ )
ovaries had a reduced
number of growing follicles. Several studies have
reported that estrogen augments the effects of follicle-
stimulating hormone on granulosa cells [31], granulosa
cell growth, and the number of granulosa cells in the
ovary [32,33]. Our data showed that an importin a5
deficiency resulted in decreased ER signaling, suggest-
ing that the abnormalities in impa5
) ⁄ )
females may be
caused by defects in the known functions of estrogen
in the ovary. Furthermore, we found that not only the
serum progesterone levels but also the mRNA expres-
sion levels of PR in the ovaries were reduced in
impa5
) ⁄ )
mice. Because progesterone and its receptor

are thought to play important roles in ovulation
[22,34], it is likely that this phenotype in impa5
) ⁄ )
female mice is at least partly attributable to the
reduced serum progesterone levels and decreased PR
expression in the ovary. Alternatively, importin a5is
highly expressed in granulosa cells of the ovarian folli-
cle (Fig. 3), which secrete progesterone, suggesting that
importin a5 may be involved in progesterone synthesis
and corpus luteum development. In addition, when
impa5
) ⁄ )
females were subjected to superovulation
with exogenous gonadotropins, the uterus showed
hypertrophy, suggesting that impa5
) ⁄ )
mice have uter-
ine abnormalities, which may harm the implanting
embryos. Furthermore, the number of live pups born
to impa5
) ⁄ )
females was decreased, probably because
of incomplete delivery of some pups. On the other
hand, as all of the embryonic day 18.5 embryos from
impa5
) ⁄ )
females appeared to have developed nor-
mally, it is likely that the impa5
) ⁄ )
uterus does not

Fig. 7. Decreased activation of estrogen signaling in impa5
) ⁄ )
mice. (A, B) Expression of ERa,ERb, PR, FSHR and LHR genes, as
well as ER and FSHR downstream genes, in impa5
) ⁄ )
and wild-
type ovaries. Real-time PCR was performed with ERa,ERb, PR,
FSHR, LHR, EFP, EBAG9, Ltf and cyclin D2 gene-specific primers,
and impa5
) ⁄ )
and wild-type ovaries. The graphs represent the
impa5
) ⁄ )
⁄ wild-type ratio for the amount of each mRNA. The data
are expressed as the mean copies of each mRNA per the mRNA
levels of the housekeeping gene, hypoxanthine-guanine phosphori-
bosyl transferase. Impa5
) ⁄ )
mice, n = 6; wild-type mice, n =5.
*P < 0.05. Error bars indicate the SEM. (C) ERa protein expression
in impa5
) ⁄ )
mice. The protein expression levels for importin a5,
ERa and actin were detected by immunoblotting with 10 lgof
uterus lysates from four animals of each genotype. (D) Localization
of the ERa protein in impa5
) ⁄ )
mice. Immunofluorescence staining
for ERa was performed with impa5
) ⁄ )

and wild-type uteruses.
Nuclei within the same field were counterstained with 4¢,6-diamidino-
2-phenylindole (DAPI) (right panel).
T. Moriyama et al. Reproductive organ abnormalities in impa5
) ⁄ )
mice
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1567
affect embryonic development after implantation. Fur-
ther studies are necessary to fully understand why
impa5
) ⁄ )
females have reduced litter sizes.
As described above, we found that impa5
) ⁄ )
females
were unable to effectively deliver their pups, and had
abnormal parturition concomitant with vaginal bleeding
or pups being trapped within the birth canal. Progester-
one and estrogen are key regulators of uterine develop-
ment, myometrial growth, and contractility [35]. It has
been reported that progesterone prepares the uterine
wall for implantation of the fertilized egg, maintains
the pregnant state by promoting myometrial relaxation,
remodels the stromal extracellular matrix cervix, and
contracts the uterus in parturition [36,37]. Estrogen also
promotes uterine growth and augments myometrial con-
tractility. Collectively, it is likely that the abnormal
delivery observed in impa5
) ⁄ )
mice results from defects

in progesterone and ⁄ or estrogen signaling.
Our previous study on mouse embryonic stem cells
demonstrated that switching of importin a subtype
expression, i.e. downregulation of importin a1 followed
by upregulation of importin a5, is critical for neural dif-
ferentiation [11]. However, impa5
) ⁄ )
mice had normal
development and were born at the expected Mendelian
ratio, with no obvious morphological abnormalities in
the brain (Fig. 2A) and spinal cord, consistent with a
previous study [21]. As importin a7, which belongs to
the importin a5 subfamily, is expressed in many mouse
tissues (Fig. S1) [8] and has 81% identity with impor-
tin a5 and close to 90% identity in the NLS-binding
regions, it is possible that these two importin a isoforms
have overlapping roles in nuclear transport.
A previous study found that impa5
) ⁄ )
mice had no
morphological abnormalities and that the importin a4
protein was remarkably upregulated in the brains of
impa5
) ⁄ )
mice [21]. On the other hand, as compared
with wild-type mice, our impa5
) ⁄ )
mice did not exhibit
any apparent differences in the expression levels of im-
portin a4 or other isoforms in any tissues, including

the brain. Furthermore, we found that loss of impor-
tin a5 caused morphological defects and functional
deterioration of the female reproductive tract, although
our impa5
) ⁄ )
mice, like the previously reported
impa5
) ⁄ )
mice, were born at the expected Mendelian
ratio, and were viable and fertile. The mouse line in
the previous study was generated with a gene trap tar-
geting method, which may lead to incomplete disrup-
tion of protein expression and potentially influence the
expression of other genes, including the importin a4
gene. Alternatively, different genetic backgrounds
could affect the results of importin a5 disruption.
Although impa5
) ⁄ )
females had defective reproduc-
tive organs, impa5
) ⁄ )
males were fertile and showed no
gross morphological or functional defects. Notably, we
found that importin a7 was strongly expressed in the
testis, especially in round spermatids; this is similar to
importin a5 expression in the adult mouse testis
(Fig. S3). Therefore, it is likely that a large amount of
importin a7 compensates for the lack of importin a5in
the testis. Furthermore, importin a6, which also belongs
to the importin a5 subfamily in humans, is expressed

only in the testis [38], suggesting that the importin a5
subfamily members have overlapping roles in the testis.
These findings also led us to hypothesize that the impor-
tin a5 subfamily expanded throughout evolution to effi-
ciently generate and ⁄ or protect male germ cells. Further
analyses with impa5
) ⁄ )
⁄ impa7
) ⁄ )
double-deficient mice
will be required to further investigate this hypothesis.
In summary, we used a knockout mouse model of
importin a5, one of six importin a family genes in
mice, to demonstrate that importin a5 plays an essen-
tial role in female reproduction that is not compen-
sated for by other members of the importin a family.
Primates, particularly humans, have evolved ingenious
and complicated birthing mechanisms to ensure sur-
vival of the next generation, and studies have identified
a variety of risk factors associated with stillbirths. Our
studies on impa5
) ⁄ )
mice identified a novel risk factor
that causes female infertility and ⁄ or the difficulty in
parturition, i.e. abonormality of the nucleocytoplasmic
transport system in the reproductive organs.
Experimental procedures
Generation of impa5
) ⁄ )
mice

The targeting vector was constructed to target exons 2 and
3, which encode the start codon of mouse importin a5, by
flanking these exons with a loxP site and a loxP and FLP
recombinase target (FRT) site-flanked Neo cassette. A 2.1-
kb PstI–XhoI fragment or 3.3-kb SpeI–AscI and 5.4-kb
PacI–NheI fragments, which were cloned from 129 ⁄ Sv (D3)
ES cell genomic DNA by PCR, were inserted as the short
and long arms into the NsiI–XhoIorNheI–AscI and PacI–
AvrII sites in the pNT1.1 vector, respectively. The targeting
vector was linearized by NotI digestion and introduced into
ES cells of line D3. The colonies that had undergone
homologous recombination were detected by Southern blot
analysis with a probe (Fig. 1A, Probe) and PCR analysis
with specific primers [Fig. 1A, Fw(1), Re(1)]. Correctly tar-
geted ES clones were used to generate germline chimeras
that transmitted the floxed allele of importin a5 and the
phosphoglycerate kinase–Neo cassette (the allele was named
impa5
floxN
), in which the phosphoglycerate kinase promoter
drives expression of the neomycin (Neo) resistance gene.
The impa5
floxN ⁄ +
mice were mated with CAG-Flpe trans-
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1568 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS
genic mice [39] that express the Flp recombinase to remove
the intronic neomycin expression cassette, and then with

CAG-Cre transgenic mice that ubiquitously express Cre re-
combinase [40]. As the Flp and Cre recombinases could
potentially affect the phenotype of the knockout mice, the
heterozygous animals were mated with C57BL ⁄ 6 mice to
remove these recombinases. The matings between these
impa5
+ ⁄ )
mice were performed to generate impa5
) ⁄ )
mice.
The wild-type, loxed and floxed alleles were confirmed by
PCR analysis with three primers [Fw(2), Re(2), and Re(3)]
and mouse tail genomic DNA as a template in order to
genotype the littermates. The 623-bp, 420-bp or 777-bp
PCR products indicate the wild-type, mutant and floxed
alleles, respectively (the primers used to confirm the genera-
tion of impa5
) ⁄ )
mice are shown in Table S3). Animals
were housed in a temperature-controlled room with a 12-h
light ⁄ dark cycle in a specific pathogen-free environment.
Food and water were available ad libitum. Animal proce-
dures were conducted in compliance with the ethical guide-
lines of the Graduate School of Frontier Bioscience, Osaka
University.
Antibodies
The following antibodies were used for immunoblotting and
immunohistochemistry: a rat monoclonal antibody against
importin a1 (Yasuhara et al., submitted) (1 : 500), anti-
KPNA4 IgG (ab6039; Abcam, Cambridge, MA, USA)

(1 : 2000), goat anti-importin a4 IgG (IMGENEX, San
Diego, CA, USA) (1 : 2000), mouse anti-KPNA1 IgG
(Abnova, Teipeh, Taiwan) (immunoblotting, 1 : 500), poly-
clonal rabbit anti-KPNA1 IgG (ProteinTech, Chicago, Il,
USA) (immunohistochemistry, 1 : 300), anti-importin a5
(NPI-1) ⁄ a7 IgG (MBL, Nagoya, Japan) (immunoblotting,
1 : 500), a rat monoclonal antibody against importin a7
(Mizuguchi et al., in submitted) (immunohistochemistry,
1 : 100), mouse anti-karyopherin b IgG (BD Transduction
Laboratories, San Jose, CA, USA) (1 : 1000), anti-glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH) IgG (Ambi-
on, Austin, TX, USA) (1 : 5000), and anti-ERa IgG;
(MC-20) (Santa Cruz, CA, USA) (immunoblotting, 1 : 500;
immunohistochemistry, 1 : 300).
Immunoblotting
Eight-week-old impa5
) ⁄ )
and wild-type mice were perfused
with 0.01 m NaCl ⁄ P
i
under pentobarbital sodium anesthesia
(50 mgÆkg
)1
body weight, intraperitoneal; Dainippon Sumi-
tomo Pharma, Osaka, Japan). Their organs were removed
and homogenized with RIPA buffer [10 mm Tris ⁄ HCl
(pH 7.2), 150 mm NaCl, 0.1% SDS, 1.0% Triton X-100,
1.0% sodium deoxycholate, 5 mm EDTA, 10 lgÆmL
)1
each

of leupeptin, pepstatin, and aprotinin, and 1 mm phen-
ylmethanesulfonyl fluoride). These lysates were centrifuged
at 20 400 g for 30 min, and the supernatants were then
collected as the cytosolic fractions. The protein concentra-
tions of the fractions were determined with a bicinchoninic
acid kit (Pierce, Rockford, IL, USA), and 10 or 15 lgof
total tissue lysate was loaded in each lane for SDS ⁄ PAGE
and then transferred onto poly(vinylidene difluoride) mem-
branes (Millipore, Schwalbach, Germany) with a semidry-
type blotting apparatus (Horizblot; ATTO, Tokyo, Japan).
Molecular mass markers (Precision Plus Protein Standards;
Bio-Rad Laboratories, Hercules, CA, USA; Magic
Mark XP, Invitrogen, Carlsbad, CA, USA) were used to
estimate the molecular masses of the bands. The mem-
branes were immunoblotted with the indicated antibodies
and horseradish peroxidase-conjugated secondary antibod-
ies (Jackson ImmunoResearch Laboratories, West Grove,
PA, USA) (1 : 2000).
Histological analysis and immunohistochemistry
Tissues were fixed in 10% formalin (Mildform 10 N; Wako
Pure Chemical Industries, Osaka, Japan) and embedded in
paraffin. After dehydration of the tissues with increasing
concentrations of ethanol, the specimens were sectioned at
3-lm thickness. The sections were dealcoholized, stained
with hematoxylin and eosin, dehydrated, mounted in Ente-
llan New (Merck, Darmstadt, Germany), and then photo-
graphed with a Provis AX-80 microscope (Olympus,
Tokyo, Japan). For immunohistochemistry, sections of the
uterus and testis were subjected to the antigen retrieval
heating method with an autoclave (120 °C, 20 min,

216 kPa) and TE buffer (10 mm Tris ⁄ 1mm EDTA,
pH 9.0). The sections were treated with a goat serum block-
ing buffer (2% goat serum, 1% BSA, 0.1% gelatin, 0.1%
Triton X-100, and 0.05% Tween-20), and incubated with
the indicated antibodies. After washing, the sections were
incubated with EnVision+ Rabbit ⁄ horseradish peroxidase
(Dako, Carlsbad, CA, USA) or an Alexa Fluor 488-conju-
gated secondary antibody (Invitrogen) (1 : 500).
Hormone measurements
Blood from a mouse in estrus was collected via the vena
cava under inhalation anesthesia (isoflurane), and centri-
fuged at 800 g for 10 min at 4 ° C. The serum supernatant
samples were collected and stored at )80 °C until further
use. 17-b-Estradiol and progesterone were measured with an
enzyme immunoassay kit from Cayman Chemical Company
(Ann Arbor, MI, USA). Briefly, the serum samples were
incubated with rabbit antiserum specific for 17-b-estra-
diol ⁄ progesterone and tracer (17-b-estradiol ⁄ progesterone
acetylcholinesterase conjugate) in plates precoated with an
anti-rabbit IgG. The plates were washed, and Ellman’s
Reagent (which contained the substrate for acetylcholinester-
ase) was then added to each well. The plates were read at
405 nm with a Microplate Reader (Dainippon Sumitomo
Pharma).
T. Moriyama et al. Reproductive organ abnormalities in impa5
) ⁄ )
mice
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1569
Hormone treatment
For female reproductive organ histology, 4-week-old virgin

mice were intraperitoneally injected with 5 IU of PMSG
(Serotropin; ASKA Pharmaceutical, Tokyo, Japan), and
induced to ovulate after 48 h with 5 IU of hCG (Gonatro-
pin; ASKA Pharmaceutical). Thirteen hours after hCG
treatment, the tissues were excised under pentobarbital
sodium anesthesia.
Quantitative analysis of the expression of
ERa, PR, FSHR, and their downstream target
genes
The real-time PCR reactions were carried out with an ABI
Prism 7900 (Applied Biosystems, Foster City, CA, USA).
The amplicons were designed to amplify > 150-bp frag-
ments (primers used for real-time PCR assay are shown in
Table S4). A One Step SYBR PrimeScript RT-PCR Kit II
(Takara Bio, Shiga, Japan) was used for the one-step RT-
PCR reactions containing total RNA from impa5
) ⁄ )
and
wild-type ovaries as a template. According to the manufac-
turer’s protocol, reverse transcription was conducted at
42 °C for 5 min and then at 95 °C for 10 s, and this was
followed by an initial activation at 95 °C for 5 s and 60 °C
for 30 s for a total of 40 cycles. Briefly, standard curves
were generated for all target genes with prepared serial dilu-
tions of total RNA from a control wild-type mouse at con-
centrations of 25 ng per well, 5 ng per well, 1 ng per well,
200 pg per well, and 40 pg per well. We examined the
amplification efficiency of the quantitative RT-PCR curve,
and confirmed that it was a single, sharp peak, indicating
that only one specific PCR product was amplified with

these primer sets. RNA from wild-type and impa5
) ⁄ )
mice
was diluted to 1 ng per well, and then used as a template to
amplify and quantify the target genes. The amount of tar-
get gene was determined from the standard curve, and nor-
malized to the housekeeping gene, hypoxanthine-guanine
phosphoribosyltransferase.
Statistical analysis
All data are expressed as the means ± standard deviations
or standard errors of the mean (SEMs), and P < 0.05 and
P < 0.001 were considered to be statistically significant,
based on Student’s t-test.
Acknowledgements
We thank A. F. Stewart for kindly providing the
CAG-Flpe transgenic mice, and J. Miyazaki for provid-
ing the CAG-Cre transgenic mice. We also thank
A. Kawai and Y. Esaki for technical assistance. This
work was supported, in part, by the Ministry of Edu-
cation, Culture, Sports, Science and Technology of
Japan, the CREST program of the Japan Science and
Technology Agency (JST), and the Takeda Science
Foundation.
References
1 Goldfarb DS, Corbett AH, Mason DA, Harreman MT
& Adam SA (2004) Importin alpha: a multipurpose
nuclear-transport receptor. Trends Cell Biol 14, 505–
514.
2 Yasuhara N, Oka M & Yoneda Y (2009) The role of
the nuclear transport system in cell differentiation.

Semin Cell Dev Biol 20, 590–599.
3 Yoneda Y (2000) Nucleocytoplasmic protein traffic
and its significance to cell function. Genes Cells 5,
777–787.
4 Tejomurtula J, Lee KB, Tripurani SK, Smith GW &
Yao J (2009) Role of importin alpha8, a new member
of the importin alpha family of nuclear transport pro-
teins, in early embryonic development in cattle. Biol Re-
prod 81, 333–342.
5 Sekimoto T, Imamoto N, Nakajima K, Hirano T &
Yoneda Y (1997) Extracellular signal-dependent nuclear
import of Stat1 is mediated by nuclear pore-targeting
complex formation with NPI-1, but not Rch1. EMBO J
16, 7067–7077.
6 Welch K, Franke J, Kohler M & Macara IG (1999)
RanBP3 contains an unusual nuclear localization signal
that is imported preferentially by importin-alpha3. Mol
Cell Biol 19, 8400–8411.
7 Tseng SF, Chang CY, Wu KJ & Teng SC (2005)
Importin KPNA2 is required for proper nuclear
localization and multiple functions of NBS1. J Biol
Chem 280, 39594–39600.
8 Tsuji L, Takumi T, Imamoto N & Yoneda Y (1997)
Identification of novel homologues of mouse importin
alpha, the alpha subunit of the nuclear pore-targeting
complex, and their tissue-specific expression. FEBS Lett
416, 30–34.
9 Nachury MV, Ryder UW, Lamond AI & Weis K
(1998) Cloning and characterization of hSRP1 gamma,
a tissue-specific nuclear transport factor. Proc Natl

Acad Sci USA 95, 582–587.
10 Kamei Y, Yuba S, Nakayama T & Yoneda Y (1999)
Three distinct classes of the alpha-subunit of the nuclear
pore-targeting complex (importin-alpha) are differen-
tially expressed in adult mouse tissues. J Histochem
Cytochem 47, 363–372.
11 Yasuhara N, Shibazaki N, Tanaka S, Nagai M, Kamik-
awa Y, Oe S, Asally M, Kamachi Y, Kondoh H &
Yoneda Y (2007) Triggering neural differentiation of
ES cells by subtype switching of importin-alpha. Nat
Cell Biol 9, 72–79.
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1570 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS
12 Ratan R, Mason DA, Sinnot B, Goldfarb DS &
Fleming RJ (2008) Drosophila importin alpha1
performs paralog-specific functions essential for gameto-
genesis. Genetics 178, 839–850.
13 Gorjanacz M, Adam G, Torok I, Mechler BM,
Szlanka T & Kiss I (2002) Importin-alpha 2 is critically
required for the assembly of ring canals during
Drosophila oogenesis. Dev Biol 251, 271–282.
14 Mason DA, Fleming RJ & Goldfarb DS (2002)
Drosophila melanogaster importin alpha1 and alpha3
can replace importin alpha2 during spermatogenesis but
not oogenesis. Genetics 161, 157–170.
15 Mason DA, Mathe E, Fleming RJ & Goldfarb DS
(2003) The Drosophila melanogaster importin alpha3
locus encodes an essential gene required for the devel-

opment of both larval and adult tissues. Genetics 165,
1943–1958.
16 Mathe E, Bates H, Huikeshoven H, Deak P, Glover
DM & Cotterill S (2000) Importin-alpha3 is required at
multiple stages of Drosophila development and has a
role in the completion of oogenesis. Dev Biol 223, 307–
322.
17 Mosca TJ & Schwarz TL (2010) The nuclear import
of Frizzled2-C by Importins-beta11 and alpha2 pro-
motes postsynaptic development. Nat Neurosci 13,
935–943.
18 Mosca TJ & Schwarz TL (2010) Drosophila Importin-
alpha2 is involved in synapse, axon and muscle develop-
ment. PLoS ONE 5, e15223.
19 Ting CY, Herman T, Yonekura S, Gao S, Wang J,
Serpe M, O’Connor MB, Zipursky SL & Lee CH
(2007) Tiling of r7 axons in the Drosophila visual
system is mediated both by transduction of an activin
signal to the nucleus and by mutual repulsion. Neuron
56, 793–806.
20 Hu J, Wang F, Yuan Y, Zhu X, Wang Y, Zhang Y,
Kou Z, Wang S & Gao S (2010) The novel importin-
alpha family member KPNA7, is required for normal
fertility and fecundity in the mouse. J Biol Chem 43,
33113–33122.
21 Shmidt T, Hampich F, Ridders M, Schultrich S, Hans
VH, Tenner K, Vilianovich L, Qadri F, Alenina N,
Hartmann E et al. (2007) Normal brain development in
importin-alpha5 deficient-mice. Nat Cell Biol 9, 1337–
1338.

22 Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes
AR, Montgomery CA Jr, Shyamala G, Conneely OM
& O’Malley BW (1995) Mice lacking progesterone
receptor exhibit pleiotropic reproductive abnormalities.
Genes Dev 9, 2266–2278.
23 Kastner P, Krust A, Turcotte B, Stropp U, Tora L,
Gronemeyer H & Chambon P (1990) Two distinct
estrogen-regulated promoters generate transcripts
encoding the two functionally different human proges-
terone receptor forms A and B. EMBO J 9, 1603–1614.
24 Watanabe T, Inoue S, Hiroi H, Orimo A, Kawashima
H & Muramatsu M (1998) Isolation of estrogen-respon-
sive genes with a CpG island library. Mol Cell Biol 18,
442–449.
25 Inoue S, Orimo A, Hosoi T, Kondo S, Toyoshima H,
Kondo T, Ikegami A, Ouchi Y, Orimo H & Muramatsu
M (1993) Genomic binding-site cloning reveals an estro-
gen-responsive gene that encodes a RING finger pro-
tein. Proc Natl Acad Sci USA 90, 11117–11121.
26 Pentecost BT & Teng CT (1987) Lactotransferrin is the
major estrogen inducible protein of mouse uterine secre-
tions. J Biol Chem 262, 10134–10139.
27 Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner
H, Park MY, Robker RL, Richards JS, McGinnis LK,
Biggers JD et al. (1996) Cyclin D2 is an FSH-responsive
gene involved in gonadal cell proliferation and oncogen-
esis. Nature 384, 470–474.
28 Robker RL & Richards JS (1998) Hormone-induced
proliferation and differentiation of granulosa cells: a
coordinated balance of the cell cycle regulators cy-

clin D2 and p27Kip1. Mol Endocrinol 12
, 924–940.
29 Leo C & Chen JD (2000) The SRC family of nuclear
receptor coactivators. Gene 245, 1–11.
30 Matzuk MM & Lamb DJ (2002) Genetic dissection of
mammalian fertility pathways. Nat Cell Biol 4(Suppl),
s41–s49.
31 Tonetta SA & diZerega GS (1989) Intragonadal
regulation of follicular maturation. Endocr Rev 10,
205–229.
32 Goldenberg RL, Vaitukaitis JL & Ross GT (1972)
Estrogen and follicle stimulation hormone interactions
on follicle growth in rats. Endocrinology 90, 1492–1498.
33 Richards JS (1980) Maturation of ovarian follicles:
actions and interactions of pituitary and ovarian hor-
mones on follicular cell differentiation. Physiol Rev 60,
51–89.
34 Tanaka N, Espey LL, Stacy S & Okamura H (1992)
Epostane and indomethacin actions on ovarian kallik-
rein and plasminogen activator activities during ovula-
tion in the gonadotropin-primed immature rat. Biol
Reprod 46, 665–670.
35 Mesiano S & Welsh TN (2007) Steroid hormone control
of myometrial contractility and parturition. Semin Cell
Dev Biol 18, 321–331.
36 Garibay-Tupas JL, Okazaki KJ, Tashima LS, Yamam-
oto S & Bryant-Greenwood GD (2004) Regulation of
the human relaxin genes H1 and H2 by steroid hor-
mones. Mol Cell Endocrinol 219, 115–125.
37 Kamat AA, Feng S, Bogatcheva NV, Truong A, Bishop

CE & Agoulnik AI (2004) Genetic targeting of relaxin
and insulin-like factor 3 receptors in mice. Endocrinol-
ogy 145, 4712–4720.
38 Kohler M, Ansieau S, Prehn S, Leutz A, Haller H &
Hartmann E (1997) Cloning of two novel human im-
portin-alpha subunits and analysis of the expression
T. Moriyama et al. Reproductive organ abnormalities in impa5
) ⁄ )
mice
FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS 1571
pattern of the importin-alpha protein family. FEBS Lett
417, 104–108.
39 Rodriguez CI, Buchholz F, Galloway J, Sequerra R,
Kasper J, Ayala R, Stewart AF & Dymecki SM (2000)
High-efficiency deleter mice show that FLPe is an alter-
native to Cre-loxP. Nat Genet 25, 139–140.
40 Sakai K & Miyazaki J (1997) A transgenic mouse line
that retains Cre recombinase activity in mature oocytes
irrespective of the cre transgene transmission. Biochem
Biophys Res Commun 237, 318–324.
Supporting information
The following supplementary material is available:
Fig. S1. Comparisons of importin a isoform expression
in impa5
) ⁄ )
and wild-type mice.
Fig. S2. Histological analysis of impa5
) ⁄ )
(left panel)
and wild-type (right panel) mice.

Fig. S3. Immunofluorescence staining for importin a5
and a7 expression in testis sections.
Table S1. Overview of hematological parameters in
impa5
) ⁄ )
and wild-type mice.
Table S2. Overview of biochemical parameters in
impa5
) ⁄ )
and wild-type mice.
Table S3. Primers used to confirm the generation of
impa5
) ⁄ )
mice.
Table S4. Primers used for real-time PCR assay.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
Reproductive organ abnormalities in impa5
) ⁄ )
mice T. Moriyama et al.
1572 FEBS Journal 278 (2011) 1561–1572 ª 2011 The Authors Journal compilation ª 2011 FEBS