Gene expression pattern and subcellular localization of mouse Kpna7. A and B, gene expression pattern of Kpna7 in tissues (A) and oocytes and preimplantation embryos (B). Mouse Kpna7 gene was specifically expressed in oocytes, zygotes, and two-cell stage embryos. Its expression was dramatically down-regulated after the two-cell stage. C–J, Kpna7 protein expression and subcellular localization in preimplantation stages. Mouse Kpna7 protein was highly expressed in GV oocytes (C), MII oocytes (D, localizes to the spindle (solid arrow) but not the chromatin (open arrow)), and zygotes (E–H, from early to late zygote stage). Its expression was dramatically down-regulated in two-cell stage embryos (I and J). K–N, negative control. No significant and specific staining signal had been observed in GV and MII oocytes with the preimmune serum. O–R, specificity analysis of Kpna7 antibody in mouse pancreatic epithelial cells. Kpna7 antibody only recognizes the ectopically expressed mouse Kpna7 protein (arrows) but not other importins (open arrows). (bars, C–J and O–R, 20 μm; K–N, 10 μm). Br, brain; He, heart; Ki, kidney; Li, liver; Pa, pancreas; Sk, skeleton; Ov, ovary; Te, testis; GV, germinal vesicle stage oocyte; MII, metaphase-II stage oocyte; Zy, zygote; 2c, two-cell; 4c, four-cell; 8c, eight cell; Mo, morula; Bl, blastocyst.

Kpna7 targeting design and sequence analysis. A, original design of the Kpna7 gene targeting. Exons 5 and 6 were designed to be deleted, and finally the truncated transcript should be destroyed because it caused incorrect splicing and wrong encoding. B, existence of a previously unexpected new exon after exon 6 caused in-frame encoding between exon 4 and the new exon 7, which generated one 1215-bp long truncated Kpna7 mRNA. The truncated Kpna7 mRNA encodes a protein that lacks the exon 5- and exon 6-encoded polypeptide fragment but with a new exon 7-encoded polypeptide fragment. C and D, genomic PCR (C) and RT-PCR (D) analysis of the wild type (wt1-3), Kpna7 heterozygous (F1-162, F2-14, and F2-101), and homozygous (F2-42, F2-46, F3-163, and F3-164) mutation mice.

Kpna7 gene mutation caused loss of KPNA7 protein expression in the mouse. A–D, KPNA7-specific antibody staining in wild type GV (A), MII (B) oocytes, zygotes (Zy, C), and two-cell embryos (D). E–H, KPNA7-specific antibody staining in Kpna7 mutation GV (E), MII (F) oocytes, zygotes (G), and two-cell embryos (H). Significant and specific positive signal had been observed in oocytes and early embryos that isolated from wild type mice but not Kpna7 mutation mice (bars, 20 μm).

Mutation of Kpna7 caused reproductivity reduction by inducing partial fetal lethality. A–D, uteri were isolated from Kpna7 mutation mice. A, one uterus isolated from a Kpna7 mutation mouse contained embryonic 7.5 day (E7.5) embryos. All the embryos were alive on E7.5. B, one uterus containing E12.5 embryos. Three embryos were alive but others had died and been absorbed. C and D, typical littermates observed in Kpna7 mutation mice. C shows E13.5 littermates isolated from one female. Six of 13 of the fetuses were alive. The other embryos had died and been absorbed. Arrows indicate one typical absorbed embryo and its placenta. D shows E16.5 littermates isolated from one female. Five fetuses were alive. Two residues were found.

Mutation of Kpna7 caused reproductivity reduction and sex imbalance. A, statistical analysis of reproductive ability of wild type, Kpna7^+/mut, and Kpna7^mut/mut mice. The litter size is reduced in Kpna7 heterozygous and homozygous mutation mice than the control group. * and ** representing the average litter size of Kpna7^+/mut and Kpna7^mut/mut group are smaller than the wild type group (p < 0.01 and p < 0.001, respectively). # representing the litter size of Kpna7^mut/mut group is smaller than Kpna7^+/mut group (p < 0.05). B, genetic analysis of the heterozygous/homozygous and female/male mice that generated from heterozygous parents. The mice of wild type (+/+), heterozygous (+/mut), and homozygous (mut/mut) were counted. The sex ratio of each gene type was also calculated. C, sex determination of the dead embryos by genomic PCR. Six males (xist and zfy double-positive) and 18 females (zfy-negative) were found in 24 dead embryos collected from six homozygous females of middle or late gestation.

Mutation of Kpna7-induced preimplantation developmental abnormalities in vitro. A–C, developmental abnormalities of naturally fertilized zygotes are induced in Kpna7 mutation mice. Many of the zygotes of Kpna7 group failed to develop into blastocyst stage (A). C, statistical analysis of preimplantation development 120 and 144 h after hCG injection. 48.4% of the Kpna7 mutation zygotes fail or are delayed from developing into blastocyst stage. D–F, parthenogenesis developmental abnormalities of naturally fertilized zygotes are induced in Kpna7 mutation mice. D and E, development of parthenogenesis one-cell embryos that collected from Kpna7 mutation and wild type mice. F, statistics analysis of parthenogenesis preimplantation development 120 and 144 h after hCG injection. Most (88.6) of the parthenogenesis one-cell embryos from Kpna7 mutation mice failed to develop into blastocyst stage. G–N, cell cycle is loss of control in parthenogenesis embryos of mutation mice after activation. G and H show embryos 48 h after hCG injection in Kpna7 mutation and wild type group (solid arrow, embryo containing four cells; open arrows, embryos containing two cells). I and J show statistics analysis of two-cell embryos and non-two-cell embryos 48 h after hCG injection in Kpna7 mutation group (I) and wild type group (J), respectively. K–N show four-cell embryos from Kpna7 mice with different morphological styles that observed 22 h after activation (48 h after hCG injection). The embryos are stained with CDC25 (solid arrows, abnormal morphology; open arrows, polar body DNA; bars, A, B, G, and H, 100 μm; D and E, 200 μm; K–N, 20 μm).

Mutation of Kpna7 caused down-regulation of a set of chromatin remodeling factors. A and B, qualitative RT-PCR analysis shows that Kpna7 mutation induces down-regulation of dppa2, dppa4, and piwil2 in late two-cell embryos in Kpna7 mutation mice. C, real time RT-PCR analysis also shows that dppa2, dppa4, and piwil2 but not dppa3 are dramatically down-regulated in Kpna7 mutation group. hdac3 mRNA level is up-regulated. D–G, immunostaining analysis of DPPA2 protein levels in late two-cell stage embryos. Arrows indicate the DPPA2 signal (red). D and E show embryos injected with enhanced EGFP and NLS-EGFP, respectively. The DPPA2 signal is high in both wild type (D, solid arrow) and NLS-EGFP-injected late two-cell embryos (E, solid arrow). F and G, typical results of embryos derived from Kpna7 mutation mice. The DPPA2 signal is dramatically down-regulated (F, open arrow) or even hardly detected (G, open arrow) in late two-cell stage embryos. H–J, statistical analysis of DPPA2 staining intensities in late two-cell embryos. H–J show the results of wild type (H), Kpna7 heterozygous (I), and homozygous mutation mice (J), respectively. (Bars, 20 μm.)

Epigenetic analysis of oocytes, zygotes, and two-cell embryos isolated from Kpna7 mutation mice. A–F, histone acetylation analysis. H4K8ace (A and B), H4K12ace (C and D), and H3K14ace (E and F) levels in zygotes of Kpna7 mutation mice and wild type mice are similar. G–L, H3K4me1, H3K9me3, and HP1B staining. H3K4me1 (G and H), H3K9me3 (I and J), and HP1B (K and L) staining levels in late two-cell embryos of Kpna7 mutation mice and wild type mice are similar. M–R, H3K27me3 staining in MII oocytes, zygotes, and parthenogenesis two-cell embryos. H3K27me3 levels are down-regulated in MII oocytes (M, N, S, and T) and zygotes (O, P, U, and V) of Kpna7 mutation mice. DAPI and H3K27me3 double staining are shown in top right of M and N to show their colocalization. H3K27me3 levels are down-regulated in parthenogenesis two-cell embryos (Q, R, W, and X) but not in normally fertilized two-cell embryos (data not shown) of Kpna7 mutation mice. (Solid arrows indicate the high levels of staining in wild type group, and open arrows indicate the low levels of staining in Kpna7 mutation group; bars, 20 μm).

Biochemical analysis of the interaction between KPNA7 and KPNB1 and proposed models. A and B, mouse KPNA7 and KPNB1 interaction analysis. Western blotting analysis of samples obtained by immunoprecipitation. Expression vectors were cotransfected into 293T cells, respectively. Kb1 and Ka7 represent KPNB1 and KPNA7, respectively. A, KPNB1 protein (Kb1-FG) is used as the bait protein and FLAG antibody is used to perform immunoprecipitation. KPNA2 (Ka2-his) is used as a positive control. KPNA7 protein with deleted IBB domain is used as a negative control. B, KPNA7 and KPNB1 are used as bait proteins, respectively, to further confirm the interaction between KPNA7 and KPNB1. C, proposed model in cell level for how the KPNA7 mutation caused the phenotypes/abnormalities.