Apolipoprotein A1 and heterogeneous nuclear ribonucleoprotein E1 implicated in the regulation of embryo implantation by inhibiting lipid peroxidation  Jia Jia, Jinhai Gou, Xia Zhao, Tao Yi, Zhengyu Li  Reproductive BioMedicine Online  Volume 33, Issue 5, Pages 635-645 (November 2016) DOI: 10.1016/j.rbmo.2016.07.011 Copyright © 2016 Terms and Conditions

Figure 1 The expression of hnRNP-E1 (A, B) and apoA1 (C, D) in mouse uterine horns during the peri-implantation period (D1 to D8) in established models of pregnancy, pseudopregnancy, artificial decidualization and delayed implantation. In the ovariectomy model (E, F, G, and H), the mice were killed following injection of oestradiol-17β, or progesterone, or both for three days, with the treatment beginning two weeks after ovariectomy, and ‘normal’ in panel means ‘normal pregnancy’. Specific up-regulation of apoA1 in the implantation period was previously reported (Gou et al., 2015). Protein concentrations were examined by western blotting (examples in panels A, C, E, G) and quantified using Quantity One v4.62 software. Values were normalized to β-actin and the protein concentration on D1/Normal is arbitrarily set at 1, and the values of other groups are expressed as a ratio of it (panels B, D, F, H). Values are recorded as mean ± SD and comparison among multiple groups is performed by one-way ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01. There were three mice in each group. apoA1 = apolipoprotein A1; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 2 Uterine horns were collected from pregnant mice on day 1, 5 and 8 of pregnancy. In mice killed on day 5 the implantation sites are visualized as blue bands (A); the expressions of apoA1 and hnRNP-E1 in implantation and inter-implantation sites are examined by western blotting (B) and quantified using Quantity One v4.62 software (C, D). Values were normalized to β-actin and the value of implantation sites is arbitrarily set at 1, and the value of inter-implantation sites is expressed as aratio to it. There were five mice in each group. Values are recorded as mean ± SD. Comparison between two groups is performed by t-test. **P < 0.01. Examples of light micrographs showing the histological location of apoA1 (E) and hnRNP-E1 (F) during the peri-implantation period identified by immunohistochemistry. Black arrow: blastocyst in uterine horn Lower panels show the sections outlined in the upper panel at higher magnification. apoA1 = apolipoprotein A1; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 3 (A) and (B): On day 3 of pregnancy mice were injected with 20 µl/horn (1 nmol) of the specific siRNA suspension into one uterine horn and scrambled siRNA (negative control) into the other horn. Mice were killed on day 5 and the uteri collected. Knockdown of apoA1 and hnRNP-E1 in endometrium was determined by western blotting and quantified by Quantity One v4.62 software. Values were normalized to β-actin and the value of normal group (untreated normal pregnant mice) is arbitrarily set at 1, and the values of other groups are expressed as a ratio to it; (C): The mRNA concentrations of apoA1 and hnRNP-E1 relative to GAPDH in endometrium on day 5, determined by qRT-PCR. Values in Y-axis are expressed as the natural logarithm. Values are recorded as mean ± SD and comparison among multiple groups is performed by one-way ANOVA followed by Dunnett's test. **P < 0.01. There were three mice in each group. apoA1 = apolipoprotein A1; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1; qRT-PCR = reverse transcription PCR. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 4 In-vivo effect of apoA1 and hnRNP-E1 knockdown on embryo implantation. On day 3 of pregnancy three mice were injected with 20 µl/horn (1 nmol) of the specific siRNA suspension into one uterine horn and scrambled siRNA (negative control) into the other horn (A, B). Mice were killed on day 5 and the number of implantation sites counted. In-vivo effect of apoA1 and hnRNP-E1 knockdown on pregnancy outcome. On day 3 of pregnancy one group of mice (n = 5) were injected with specific siRNA into both uterine horns and the other group were injected with negative control (n = 5). The mice were allowed to maintain pregnancy until delivery and the number of pups was recorded (C, D). Values are recorded as mean ± SD. Comparison between two groups is performed by t-test. **P < 0.01. apoA1 = apolipoprotein A1; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 5 Lipid peroxidation during the peri-implantation period in the pregnancy, pseudopregnancy and delayed implantation models. (A): The expression of eNOS in the normal pregnancy group was examined by western blotting and quantified by Quantity One v4.62 software. (B) and (C): Quantities of MDA and SOD in the normal pregnancy (B, C), pseudopregnancy (D, E) and delayed implantation (F, G) models. Values were normalized to β-actin and the value of D1 is arbitrarily set at 1, and other values are expressed as ratios to it. Values are recorded as mean ± SD, and comparison among multiple groups is performed by one-way ANOVA followed by Dunnett's test. *P < 0.05. There were three mice in each group. eNOS = endothelial nitric oxide synthase; MDA = malondialdehyde; SOD = superoxidase dismutase. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 6 In-vivo effect of apoA1 and hnRNP-E1 knockdown on lipid peroxidation. On day 3 of pregnancy mice were injected with 20 µl/horn (1 nmol) of the specific siRNA suspension into one uterine horn and scrambled siRNA (negative control) into the other horn. Mice were killed on day 5 and uteri were collected. The expression of eNOS is examined by western blotting (A, C) and quantified using Quantity One v4.62 software (B, D). Values were normalized to β-actin and the value of the normal group (untreated pregnant mice) is arbitrarily set at 1, and the values of other groups are expressed as ratios to the normal group. Values are recorded as mean ± SD, and comparison among multiple groups is performed by one-way ANOVA followed by Dunnett's test. **P < 0.01. There were three mice in each group. apoA1 = apolipoprotein A1; eNOS = endothelial nitric oxide synthase; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions

Figure 7 In-vivo effect of apoA1 and hnRNP-E1 knockdown on lipid peroxidation. On day 3 of pregnancy mice were injected with 20 µl/horn (1 nmol) of the specific siRNA suspension into one uterine horn and scrambled siRNA (negative control) into the other horn. Mice were killed on day 5 and uteri were collected. The quantities of MDA (A, B) and SOD (C, D) after apoA1 or hnRNP-E1 knockdown were determined by western blotting and quantified using Quantity One v4.62 software. Values were normalized to β-actin and the value of the blank group is arbitrarily set at 1, and the values of other groups are expressed as ratios to the blank group. Values are recorded as mean ± SD, and comparison among multiple groups is performed by one-way ANOVA followed by Dunnett's test. *P < 0.05, **P < 0.01. There were three mice in each group. apoA1 = apolipoprotein A1; hnRNP-E1 = heterogeneous nuclear ribonucleoprotein E1; MDA = malondialdehyde; SOD = superoxidase dismutase. Reproductive BioMedicine Online 2016 33, 635-645DOI: (10.1016/j.rbmo.2016.07.011) Copyright © 2016 Terms and Conditions