Progesterone-regulated B4galnt2 expression is a requirement for embryo implantation in mice  Pei-Tzu Li, M.S., Chi-Jr Liao, M.S., Wen-Guey Wu, Ph.D., Lung-Chi Yu, Ph.D., Sin Tak Chu, Ph.D.  Fertility and Sterility  Volume 95, Issue 7, Pages 2404-2409.e3 (June 2011) DOI: 10.1016/j.fertnstert.2011.03.043 Copyright © 2011 Terms and Conditions

Figure 1 B4galnt2 expression in the uterus. (A) Quantitative PCR of B4galnt2 in the mouse uterus during the estrous cycle at proestrous (Pro), estrous (Est), metestrous (Met), and diestrous (Die) stages. Amplification of B4galnt2 was set to monitor the template cDNA. (B) Reverse transcription–PCR (RT-PCR) demonstrated the regulation of B4galnt2 expression by sex hormones in the uterus of immature mice. The mice were treated with vehicle (C), LH (2.75 IU per mouse), FSH (2.5 IU per mouse), E2 (30 ng/g), or P (150 μg/g) for 3 days, and RNA was then extracted from the tissues. The RT-PCR products are shown at left; densitometric analysis is shown at right. The relative amount of B4galnt2 expression was normalized to the value of β-actin, and statistical analysis was performed using ANOVA, according to the data from four independent experiments. ∗∗P<.01 vs. control. (C) Ovariectomized mice were treated with vehicle (Con), P (P; 150 μg/g body weight), E2 (E2; 30 ng/g body weight), or a combination of P and E2 (P + E2; 150 μg/g body weight + 30 ng/g body weight) for 3 days, and RNA was extracted from the oviduct and uterus. Statistical analysis was performed using ANOVA according to the data from four independent experiments. ∗∗∗P<.001 vs. control; +++P<.001 vs. P + E2. Fertility and Sterility 2011 95, 2404-2409.e3DOI: (10.1016/j.fertnstert.2011.03.043) Copyright © 2011 Terms and Conditions

Figure 2 Effect of P on B4galnt2 promoter–driven CAT reporter gene expression. The 2,000-bp B4galnt2 promoter construct was transiently transfected into the TM4 cell line. (A) We divided seven putative GRE regions into group I (including A–D) and group II (including E–G). The gray blocks indicate the GRE sequence in each region. (B) The GRE regions in group I or group II were separately assayed after stimulation with different hormones. The transfected cells were treated with hormones for 24 h, and CAT reporter gene expression was determined. P = 4.0 μM P; E2 = 20 nM E2; Dex = 3.0 μM dexamethasone. (C) Each GRE in group I was used separately in the CAT promoter assay after stimulation with different hormones as described above. Gray bars indicate the control for supplementation using <0.5% DMSO. Black bars indicate hormonal supplements. (D) Quantitative PCR analysis shows the regulation of B4galnt2 expression by sex hormones in primary cultured endometrial cells (5 × 104 cells/mL). The cultured cells were treated with 4.0 μM P (P), 20 nM E2 (E2), or a combination of P and E2 (P + E2; 4.0 μM + 20 nM) for 24 h. Control cells were incubated with DMSO (<0.5%). The results represent the mean ± SEM for each group. ∗P<.05; ∗∗P<.01; ∗∗∗P<.001. Fertility and Sterility 2011 95, 2404-2409.e3DOI: (10.1016/j.fertnstert.2011.03.043) Copyright © 2011 Terms and Conditions

Figure 3 B4galnt2 expression in pregnancy in mice. Ribonucleic acid was extracted from the uteri of mice at different stages of pregnancy (from E0.5 to E18.5 and including day 1 of parturition). (A) Quantitative PCR was performed, and the values were compared with the value of β-actin. Each value represents the mean ± SEM of four different samples. The data are representative of four experiments carried out at different times with different pregnant mice. ∗P<.05 vs. E0.5. (B) Protein (20 μg) expression during pregnancy was detected by Western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Localization of B4GALNT2 expression in the pregnant uterus at E10.5 was assessed using an immunohistochemical assay. The brown color indicates B4GALNT2. Without the first antibody incubation was as the negative control. E = embryo; Pl = placenta; D = endometrial decidua; M = myometrium. Statistical analysis was performed using ANOVA according to data from four independent experiments. The results represent the mean ± SEM for each group. Fertility and Sterility 2011 95, 2404-2409.e3DOI: (10.1016/j.fertnstert.2011.03.043) Copyright © 2011 Terms and Conditions

Figure 4 In vivo implantation analysis. (A) The RNAi containing vector was transfected into TM4 cells using TurboFect in vitro Transfection Reagent. One day later, B4galnt2 gene expression was performed by PCR (left) and Western analysis (right). (B) Using pKLO.1/B4galnt2 siRNA60 (RNAi60) transfection is for in vivo implantation assay. Embryonic day 3.5 mice were transfected with siRNA and harvested at E10.5 as a representative experiment. The right uterine horn was injected with 8 μg Mock vector, and the left uterine horn was injected with 8 μg RNAi60 containing vector. (C) On days 3.5 (E3.5) and 10.5 (E10.5), the pregnant mice were injected with vectors, respectively. The implanted embryos were counted in the groups of Mock vector–injected horn as control (black bars) and RNAi60 containing vector–injected horn (gray bars). ∗P<.05 vs. control. Fertility and Sterility 2011 95, 2404-2409.e3DOI: (10.1016/j.fertnstert.2011.03.043) Copyright © 2011 Terms and Conditions