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: yyoneda@anat3.med.osaka-u.ac.jp (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. 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Drosophila Importinalpha2 is involved in synapse, axon and muscle development 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, . 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 ,. 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. 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 ,