Huan Yang, Caixia Lei, Wei Zhang Reproductive BioMedicine Online

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Expression of galectin-3 in mouse endometrium and its effect during embryo implantation Huan Yang, Caixia Lei, Wei Zhang Reproductive BioMedicine Online Volume 24, Issue 1, Pages 116-122 (January 2012) DOI: 10.1016/j.rbmo.2011.09.003 Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 mRNA expression of Gal-3 in mice endometria. Day 0 corresponds to no pregnancy. Eight mice were used in each group and embryos of days 6 and 8 were separated from the endometria. Each sample was analysed in triplicate. The Gal-3 mRNA concentrations were normalized relative to β-actin. The data were analysed by one-way ANOVA and least significant difference analysis (∗P<0.05, versus day 0). Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Protein expression of Gal-3 in mice endometria. (A) Western blot analysis of Gal-3 and β-actin proteins. (B) Gal-3 protein expression normalized relative to β-actin. Day 0 corresponds to no pregnancy. Total protein (30μg) was extracted from endometria of non-pregnant and pregnant mice from different groups. There were eight mice in each group and embryos were separated from the uteri of day-6 and day-8 pregnant mice. Each sample was analysed in triplicate. The data were analysed by one-way ANOVA and least significant difference analysis. (∗P<0.05, versus day 0). Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Immunohistochemical analysis of mouse uteri by staining with Gal-3 on day 0, day 2, day 4, day 6 and day 8. Control=each day, three samples were used for control, which used normal rat immunoglobulin to replace anti-mouse Gal-3 antibody. There were 20 mice in each group. G=gland; L=lumen of uterus; SC=stromal cells. Bar=100μm. Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Analysis of transfection efficiency by fluorescent microscopy of mice uterus. (A) Fluorescent image. (B) Optical image. Fluorescent dsRNA oligomer (10μmol/l) was injected into mouse uteri 16–40h after vaginal-plug detection and the fluorescent distribution was analysed 6h later. Experiments were performed in duplicate. Bars=150μm. Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Knock-down Gal-3 expression in mouse uteri. (A) At 16–40h of pregnancy, silencing RNA (siRNA)/liposome were injected into horns of mice and Gal-3 protein was analysed by Western blot on day 4 of pregnancy. (B) Gal-3 protein expression normalized relative to β-actin. Control=negative siRNA/liposome injection; siGal-3=Gal-3 siRNA/liposome injection. Samples from eight mice were analysed in triplicate. Data were analysed by two-tailed paired t-test (∗P<0.05). Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Effect of Gal-3 expression on mouse litter size. The number of implanted embryos was determined by counting the number of implantation sites per uterus examined on day 9 of pregnancy. Eight mice were transfected 16–40h after detection of vaginal plug with Gal-3 siRNA (right horn) and control siRNA (left horn). Litter size was significantly lower in the right uterine horns (siGal-3; 3.0±1.1) compared with the left uterine horns (control; 8.0±0.9) when compared by two-tailed paired t-test (∗P<0.05). Reproductive BioMedicine Online 2012 24, 116-122DOI: (10.1016/j.rbmo.2011.09.003) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions