Fig 1.
Rac1 is induced in the uterus during early pregnancy.
(A) Induction of Rac1 mRNA in the uterus during experimentally-induced decidualization. Uterine RNA was purified from mice at different times after decidual stimulation and analyzed by qPCR. Relative levels of Rac1 mRNA expression in uteri after decidual stimulation are compared to those in unstimulated control uteri. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Letters indicate statistically significant differences (P < 0.0001). (B) Expression of Rac1 during early pregnancy overlaps with the decidual phase of gestation. qPCR was performed to monitor the expression of Rac1 mRNA in uteri on days 1 to 8 of gestation. The relative levels of gene expression on different days of pregnancy were determined by setting the expression level of Rac1 mRNA on day 1 of pregnancy at 1.0. Rplp0, encoding a ribosomal protein, was used to normalize the level of RNA. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Letters indicate statistically significant differences (P < 0.0001). (C) Localization of active RAC1 protein in uterine stromal cells during early pregnancy. Uterine sections on day 7 of pregnancy were subjected to immunofluorescence (IF) histochemistry using anti-RAC1-GTP antibody. Panels a, b, and c show immunostaining of RAC1-GTP; panels d, e, and f show staining with non-immune IgG. AMD, MD and E indicate antimesometrial decidua, mesometrial decidua and embryo, respectively.
Fig 2.
Loss of Rac1 expression in the uterus of Rac1d/d mice.
(A) Efficient ablation of Rac1 in the uterus during decidual phase of pregnancy. Uterine RNA was purified from Rac1f/f and Rac1d/d mice on days 6, 8, or 10 of pregnancy and analyzed by qPCR. Relative levels of Rac1 mRNA expression in uteri of Rac1d/d mice are compared to those in Rac1f/f control mice. Data represent mean ± SEM from four separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (***P < 0.001) (B) A marked decline in the level of active RAC1 protein in the stromal cells of Rac1d/d uteri. Uterine sections obtained from day 8 pregnant Rac1f/f (left panel) and Rac1d/d (right panel) mice were subjected to IF using anti-RAC1-GTP antibody. Note the lack of RAC1 immunostaining in uteri of the mutant mice. (C) Expression of various members of the Rho family of GTPases was unaffected in Rac1d/d uteri. Real-time PCR was performed to monitor the expression of Rac1, Rac2, Rhoa, and Cdc42 in the uteri of day 8 pregnant Rac1f/f and Rac1d/d mice. Data represent mean ± SEM from four separate samples and were analyzed by t-test. Asterisks indicate statistically significant differences (****P < 0.0001).
Table 1.
Ablation of uterine Rac1 leads to severe female infertility.
Fig 3.
Early implantation is unaffected in Rac1 conditional knockout mouse.
(A) Gross morphology of Rac1f/f and Rac1d/d uteri at days 6 and 8 of gestation. (B) Rac1f/f and Rac1d/d mice were subjected to artificial decidual stimulation for 96 hours as described in the Materials and methods. For each mouse, one uterine horn was stimulated, while the other horn was left undisturbed. Gross morphology of Rac1f/f and Rac1d/d uteri following the application of the decidual stimulus is shown. (C) Comparative wet weight gains in uteri of Rac1f/f and Rac1d/d mice. Following artificial decidualization, stimulated and unstimulated horns were assessed for wet weight gain. The histogram shows the ratios of average weights of stimulated over unstimulated horns from Rac1f/f and Rac1d/d mice. Data represent mean ± SEM from four separate samples and were analyzed by t-test, P > 0.05). (D) Uterine sections from Rac1f/f and Rac1d/d mice on day 8 of pregnancy were subjected to alkaline phosphatase activity (ALPL, upper) and IF staining using an antibody specific for the prolactin-related protein (PRL82A, lower). AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively. (E) Comparable expressions of various markers of decidualization in Rac1f/f and Rac1d/d uteri. Total RNA was isolated from uteri on day 8 of pregnancy and qPCR analysis was performed using primers specific for Pgr, Bmp2, and Gja1. Data represent mean ± SEM from four separate samples and were analyzed by t-test, P > 0.05).
Fig 4.
Pregnancy failure in Rac1d/d mice in mid gestation is associated with lack of angiogenesis.
(A) Gross morphology of Rac1f/f and Rac1d/d uteri at days 10 and 15 of gestation. Hemorrhagic sites are indicated by arrowheads. (B) Angiogenesis is impaired in Rac1 conditional-knockout mouse. qPCR was performed to analyze the expression of angiogenic factors, Epas1/Hif2α, Nrp1, Angpt2, Sphk1, Vegfa, and Angpt1 in uteri of Rac1f/f and Rac1d/d mice on day 8 of pregnancy. Data represent mean ± SEM from four separate samples and were analyzed by t-test. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01). (C) Uterine sections of Rac1f/f and Rac1d/d mice on day 8 were subjected to IF staining with PECAM1 antibody. AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively
Fig 5.
Enhanced trophoblast proliferation in Rac1 conditional-knockout mouse.
(A) Expanded trophoblast cells in Rac1d/d uteri on day 8 of gestation. a: Hematoxylin and Eosin (H & E) staining of uterine sections from Rac1f/f and Rac1d/d mice on day 8 of pregnancy. Three representative images of Rac1d/d uterine sections are shown. b: Quantitation of embryonic areas in H & E stained uterine sections of Rac1f/f and Rac1d/d mice on day 8 of pregnancy. Data represent mean ± SEM from six separate samples and were analyzed by non-parametric t-test. Asterisks indicate statistically significant differences (*P < 0.05). (B) Increased proliferation of trophoblast cells in the ectoplacental cone (EPC) of Rac1d/d uteri. Uterine sections from Rac1f/f and Rac1d/d mice on days 7 (panels a and b) and 8 of pregnancy (panels c and d) were subjected to IF using PCNA and cytokeratin 8 (KRT8) antibodies. AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively.
Fig 6.
Abnormal trophoblast proliferation, differentiation and disorganized placentation in Rac1 conditional-knockout mouse.
(A & B) Increased population of trophoblast giant cells (TGCs) in the placenta of Rac1d/d uteri. (A) H and E staining of uterine sections from Rac1f/f and Rac1d/d mice on day 10 of pregnancy. TGC and MD indicate trophoblast giant cells and mesometrial decidua, respectively. TGCs are demarcated by dashed lines. Arrows indicate chorionic plate and arrow-heads indicate TGCs. (B) Uterine sections from Rac1f/f and Rac1d/d mice on day 10 of pregnancy were subjected to IF using PL1. (C) Uterine sections from Rac1f/f and Rac1d/d mice on day 10 of pregnancy were subjected to IF using TPBPA and KRT8 antibodies. (D) H and E staining of uterine sections from Rac1f/f and Rac1d/d mice on day 12 of pregnancy. TGC, MD and E indicate trophoblast giant cells, mesometrial decidua, and embryo, respectively.
Fig 7.
Rac1 regulates vesicular exocytosis in decidual cells by controlling Rab27b.
(A & B) Expression of Rab27b mRNA and protein is downregulated in Rac1-null stromal cells. (A) qPCR was performed to monitor the expression of Rab27a and Rab27b in the uteri of Rac1f/f and Rac1d/d mice on day 8 of pregnancy. Data represent mean ± SEM from four separate samples and were analyzed by t-test. Asterisks indicate statistically significant differences (***P < 0.001). (B) IF of RAB27B in Rac1f/f and Rac1d/d uteri on day 8 of pregnancy. AMD, MD, and E denote antimesometrial decidua, mesometrial decidua, and embryo respectively. (C & D) Secretions by decidual cells are reduced in the conditioned media of Rac1-null stromal cells. Stromal cells isolated from Rac1f/f and Rac1d/d uteri on day 4 of pregnancy were cultured for 96 hours, fixed and subjected to IF using VEGFA (C, Left) and IGFBP4 (D, Left) antibodies. Conditioned media from cultured stromal cells isolated from Rac1f/f and Rac1d/d uteri were analyzed for VEGFA (C, Right) and IGFBP4 (D, Right) by ELISA. Data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 8.
RAC1 regulates secretory function of human endometrial stromal cells.
(A) RAC1 is induced in primary human endometrial stromal cells undergoing in vitro decidualization. Human endometrial stromal cells (HESCs) were subjected to differentiation in response to estrogen, progesterone, and 8-Br-cAMP as described in the Materials and Methods. The expression of RAC1 mRNA was assessed by qPCR. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05). (B & C) Inhibition of biological activity of RAC1 in HESCs inhibits RAB27B expression and VEGFA secretion in the conditioned media. HESCs were subjected to differentiation in the absence or presence of 25μM InSolution Rac1 Inhibitor II (Z62954982) for eight days. (B) qPCR was performed to monitor the expression of RAC1, RAC2, RHOA, CDC42, VEGFA, and RAB27B, N = 2–3, P< 0.001. (C & D) ELISA was performed to measure VEGFA and IGFBP1 secretion in the conditioned media. VEGFA ELISA data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05 and ***P < 0.001). IGFBP1 ELISA data represent mean ± SEM from two separate samples and were analyzed by t-test, P > 0.05.
Fig 9.
A model depicting the mechanism of Rac1 action in mouse uterus during early pregnancy.