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2.
Figure 4

Figure 4. Uterine deletion of Trp53 confers decidual polyploidy, terminal differentiation, and senescence. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A) Uterine loss of p53 increased Ki67-positive decidual cells with large nuclei. Higher-magnification views of the boxed regions are shown below. Arrowheads indicate Ki67-positive cells with large, irregularly shaped nuclei. Scale bars: 200 μm. (B) Representative histogram of flow cytometry analyses showing DNA content (propidium iodide). Ploidy is denoted by -N (i.e., 2N, diploid; 4N–10N, polyploid). (C) In Trp53loxP/loxPPgrCre/+ decidua, the number of diploid cells decreased, whereas the number of polyploid cells increased (mean ± SEM; *P < 0.05, Student’s t tests). (D) Uterine Trp53 deletion enhanced SA–β-gal activity in decidua. Scale bar: 200 μm.

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.
3.
Figure 1

Figure 1. Efficient deletion of uterine Trp53. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A and B) Trp53 mRNA was undetectable in 2-month-old Trp53loxP/loxPPgrCre/+ uteri analyzed by RT-PCR (A) and Northern hybridization (B). Actb and Rpl7 were used as internal controls. (C) p53 protein levels were very low to undetectable in irradiation-induced 2-month-old Trp53loxP/loxPPgrCre/+ uteri assessed by Western blotting. Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ dams were killed 6 hours after 9.5 Gy whole-body γ irradiation. Protein extracts from mouse lymphoma cell line WR19L were used as a positive control. (D) Immunostaining of p53 in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ decidua on day 8 of pregnancy. Immunoreactive p53 was undetectable in Trp53loxP/loxPPgrCre/+ decidua. Fast Green solution was used to counterstain the cytoplasm. Dark green-black staining shows nuclear p53 localization. Scale bar: 200 μm.

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.
4.
Figure 2

Figure 2. Mice with uterine deletion of Trp53 show normal implantation. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A) Representative photographs of uteri with implantation sites (blue bands) on day 5 of pregnancy in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ females, mated with fertile Trp53loxP/loxPPgr+/+ males. Arrows and arrowheads indicate implantation sites and ovaries, respectively. (B) Number of implantation sites on day 5 of pregnancy (mean ± SEM; P > 0.05; Student’s t test). (C) Representative photomicrographs of H&E-stained histology, and in situ hybridization of Ptgs2, in implantation sites on day 5 of pregnancy in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ dams. Arrowheads indicate the location of implanting blastocysts. Scale bars: 200 μm. (D) In situ hybridization of Lif in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ uterine sections on days 1 and 4 of pregnancy. Scale bars: 200 μm. (E and F) Lif mRNA levels in day 1 uteri of Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ dams (E) and in day 4, 0900 h, uteri of Trp53loxP/loxPPgr+/+, Trp53loxP/loxPPgrCre/+, WT, and Lif+/– dams (F), as determined by Northern hybridization. Values are mean ± SEM of 2–4 independent samples. *P < 0.05, Student’s t tests.

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.
5.
Figure 6

Figure 6. Uterine deletion of Trp53 promotes preterm birth through COX2/PGFS/PGF2α pathway. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A) Preterm birth (deliveries earlier than day 19) occurred in 52% of Trp53loxP/loxPPgrCre/+ dams (*P < 0.05). Numbers in parentheses denote the ratio of dams with preterm birth/total dams. (B) All pups born before day 19 were dead around the time of delivery or died immediately after birth (*P < 0.05 versus Trp53loxP/loxPPgr+/+). Numbers in parentheses denote the ratio of dead pups/total pups. (C and D) Levels of COX2 and PGFS were upregulated in day 16 uteri lacking p53. (C) Uterine samples from which placentas and fetuses had been removed were used for Western blotting, and (D) quantitative analysis of band intensities were all normalized against actin (mean ± SEM of 3 independent samples from different mice; *P < 0.05). (E) Conditional loss of uterine p53 upregulated PGF levels in day 16 uteri. A total of 15 independent samples from 3 different mice were evaluated (mean ± SEM; *P < 0.05). (F and G) Preterm birth and neonatal deaths in Trp53loxP/loxPPgrCre/+ mice were reversed by oral gavage of celecoxib (10 mg/kg/dose, administered twice) on day 16. In G, numbers in parentheses denote the ratio of dead pups/total pups. *P < 0.05 versus vehicle-treated Trp53loxP/loxPPgr+/+; **P < 0.05 versus vehicle-treated Trp53loxP/loxPPgrCre/+.

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.
6.
Figure 3

Figure 3. Uterine deletion of Trp53 restricts normal decidual growth. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A) Representative photographs of day 8 pregnant implantation sites in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ mice. (B and C) Number and weight of implantation sites in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ mice on day 8 of pregnancy (mean ± SEM; *P < 0.05). (D and E) Trp53loxP/loxPPgr+/+ blastocysts were transferred into uteri of Trp53loxP/loxPPgr+/+ or Trp53loxP/loxPPgrCre/+ recipients on day 4 of pseudopregnancy. The number and weight of implantation sites were evaluated on day 8, 4 days after embryo transfer (mean ± SEM; *P < 0.05). Numbers in parentheses denote the ratio of implantation sites/total blastocysts transferred. (F) In situ hybridization of Hoxa10 and Bmp2 in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ uteri on day 8 of pregnancy. Em, embryo; M, mesometrial pole; AM, antimesometrial pole. Scale bar: 200 μm. (G) Northern hybridization of Hoxa10 and Bmp2 in day 8 uteri of Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ dams. In each group, 2–3 independent samples were examined (mean ± SEM; *P < 0.05). (H and I) Loss of p53 upregulated pAkt, p110α, and p21 levels in day 8 implantation sites. As assessed by Western blotting (H), quantitatively analyzed band intensities of pAkt were normalized against total Akt, and those of p110α and p21 were normalized against actin (I). In each group, 3 independent samples from different mice were examined (mean ± SEM; *P < 0.05).

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.
7.
Figure 5

Figure 5. Decidual senescence persists in uteri deleted of Trp53. . From: Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

(A and B) Number and weight of implantation sites in Trp53loxP/loxPPgr+/+ and Trp53loxP/loxPPgrCre/+ mice on day 16 of pregnancy (mean ± SEM; *P < 0.05). (C) Uterine loss of p53 impeded decidual growth. Placental labyrinth and spongiotrophoblast layers were demarcated by cytokeratin-8 immunostaining. (D) Uterine Trp53 deletion enhanced SA–β-gal activity primarily in the decidual layer on days 12 and 16. (C and D) Decidual boundaries are demarcated by double-sided arrows. Myo, myometrium; Dec, decidua; Sp, spongiotrophoblast; Lb, labyrinth; BV, blood vessel. Scale bars: 500 μm. (E and F) Levels of p21, pAkt, and p110α were upregulated in day 16 uteri lacking p53. (E) Uterine samples from which placentas and fetuses had been removed were used for Western blotting. (F) Quantitative analysis of band intensities of pAkt were normalized against total Akt, and those of p21 and p110α were normalized against actin (mean ± SEM; *P < 0.05). In each group, 3 independent samples from different mice were examined. (G) Serum levels of E2 and P4 (mean ± SEM; P > 0.05). At least 4 independent samples were examined in each group. (H) Representative photomicrographs of H&E-stained histological sections of ovaries on day 16 of pregnancy. CL, corpus luteum; F, follicle. Scale bar: 200 μm. (I) Western blotting of ovarian 20α-HSD levels on day 16 of pregnancy. Day-20 WT mice were used as a positive control. (J) Quantitative analysis of 20α-HSD band intensities from I were normalized against actin (mean ± SEM; P > 0.05).

Yasushi Hirota, et al. J Clin Invest. 2010 March 1;120(3):803-815.

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