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-

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

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

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

) ⁄ )
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

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

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

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

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)

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

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

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

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

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

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

(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

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

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.

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-

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-

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.