Sorcin is involved during embryo implantation via activating VEGF/PI3K/Akt pathway in mice

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   Figure 1

   Expression of sorcin during the period of early pregnancy (A) and during estrous cycle (B) in mouse uterus. Protein expression was measured in uterine tissue lysates by Western blotting. Images of representative blots (upper panel) and densitometric quantitation of band intensity (lower panel) have been shown. n = 3. Results are expressed as mean ± s.e.m., P values are ^a P < 0.001 vs non-pregnant, ^# P < 0.001 vs D5 IS and ^a P<0.001 vs Proestrus. IS, implantation site; IIS, inter-implantation site; NP, non-pregnant. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 2

   Immunohistochemistry of sorcin expression in mouse uteri during early pregnancy. Sorcin expression was measured in endometrium i.e. glandular epithelium (GE), luminal epithelium (LE), and stroma (S) at different days of pregnancy (D1, D4, D5, D6 and D7). Graph in lower panel shows image score analysis. IS, implantation site; NC, Negative control; NP, non-pregnant. P values are ^a P < 0.001 vs NP group. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 3

   Effect of sorcin inhibition on the number of implantation sites. The right horn was injected with anti-sorcin or sorcin siRNA and the left horn was injected with control IgG/scrambled siRNA. (A and B) Representative images of uterus on D5 and D10 from the antibody- and siRNA- treated groups. (C) Left panel shows representative images of Western blotting for receptivity markers in scrambled control- and siRNA- treated uterine horn collected on D5 of pregnancy; Right panel shows densitometric quantitation of band intensity. n = 3. P values are ^a P < 0.001, ^b P < 0.01 vs control. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 4

   Attachment and expansion of BeWo trophoblast spheroids on RL95-2 cells. The RL95-2 monolayer grown in the 96-well plate were pretreated with sorcin siRNA for 6 h and allowed for 24 h of incubation, followed by the addition of spheroids stained with CMFDA cell tracker dye to each well and co-cultured for 1 h. (A) Graph showing the percent attachment of spheroid on sorcin-silenced RL95-2 cells. (B) Representative images of BeWo spheroids outgrowth on RL95-2 monolayer. The spheroids were photographed at 1 h and 24 h after co-culture. Data are presented as spheroid expansion relative to their original size at 1 h. P values are ^a P < 0.001 vs control. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 5

   Role of ovarian hormones in regulation of sorcin expression. (A) Ovariectomized mice treated with E₂ (100 ng) or P₄ (1 mg) were used to detect the expression pattern of sorcin in uterus. Representative Western blots for sorcin expression in vehicle- and hormone-treated groups have been shown. (B) For delayed implantation, pregnant mice were ovariectomized on D3. To maintain delay in implantation, P₄ (1 mg/30 g body weight) was given and to initiate implantation, estrogen surge (25 ng/30 g body weight) was given to progesterone-primed delayed implantation mice on D7. The expression of sorcin in response delayed and after activation by E₂ surge was measured by Western blotting. (C) Immunohistochemical analysis of sorcin expression in LE, GE and stromal cells of uterine tissue in delayed and active groups. (D and E) Mouse primary EECs were treated with E₂ (10 nM), P₄ (1 µM) and E₂ + P₄ and Western blotting and immunocytochemistry was done to demonstrate the expression of sorcin protein. Representative images have been shown. Densitometry results are expressed as mean ± s.e.m., n = 3. P values are ^a P < 0.001 vs control. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 6

   Effect of sorcin silencing on cytosolic free Ca⁺² , VEGF level, VEGFR-2 expression in mouse primary EECs in vitro and on signaling molecules (PI3K, Akt and NOS) in D5 uterus in vivo. (A) Fluorescence was measured from cells loaded with 2 µM Fluo-3-AM dye. Exposure of cells to 10 µM ionomycin induced a Ca⁺² transient. (B) VEGF level in culture supernatants of mouse primary EECs measured by enzyme-linked immunosorbent assay. (C) Representative images of immunocytochemical analysis showing VEGFR-2 expression in EECs. (D) Representative images showing Western blot analysis of VEGF, VEGFR-2, PI3K, Akt and NOS in uterine horn collected on D5 pregnancy. Details have been given in ‘material and methods’ section. Results are expressed as mean ± s.e.m., P values are ^a P < 0.001 and ^b P < 0.01 vs scrambled control. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 7

   The proliferation, migration and invasion assay of HUVECs. (A) The proliferation of HUVECs was detected in various groups; HUVECs were cultured with supernatants (20% V/V) from mouse primary EECs transfected with sorcin siRNA or scrambled siRNA. In additional group, a blocking anti-VEGF antibody was added in the supernatant from scrambled siRNA-treated EECs. Details have been given in ‘materials and methods’ section. (B) Analysis of migration of HUVECs was performed by scratch-wound healing assay. Representative images showing migration of HUVECs cultured for 24 h in the supernatant of control EECs and in the supernatant of EECs with sorcin blockade (×200 magnification). Cell migration distance of the cells between the scratch edges was observed after 24 h. (C) Transwell migration and matrigel invasion assays were done to analyse migration and invasion of HUVECs. The graph in the lower panel shows the relative percentage of migrated and invaded cells over control in three wells in each independent experiment (random fields were photographed and number of cells from three fields per well were counted). Results are expressed as mean ± s.e.m., n = 3. P values are ^a P < 0.001 vs scram control. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.
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   Figure 8

   Schematic hypothetical representation of the molecular mechanism of sorcin in endometrium during embryo implantation. Sorcin maintains calcium homeostasis (Colotti et al. 2014) and induces angiogenic factor VEGF in endometrium and activates its downstream PI3K/Akt signaling cascade. Akt activates nitric oxide synthase (NOS) which in turn enhances the local nitric oxide (NO) level which regulates cellular proliferation, migration and invasion of endothelial cells and leads to the process of angiogenesis at implantation sites in the endometrium. A full color version of this figure is available at https://doi.org/10.1530/JME-17-0153.

• © 2018 Society for Endocrinology