The Hippo/MST Pathway Member SAV1 Plays a Suppressive Role in Development of the Prehierarchical Follicles in Hen Ovary

<div><p>The Hippo/MST signaling pathway is a critical player in controlling cell proliferation, self-renewal, differentiation, and apoptosis of most tissues and organs in diverse species. Previous studies have shown that Salvador homolog 1 (SAV1), a scaffolding protein which functions in the signaling system is expressed in mammalian ovaries and play a vital role in governing the follicle development. But the exact biological effects of chicken SAV1 in prehierarchical follicle development remain poorly understood. In the present study, we demonstrated that the SAV1 protein is predominantly expressed in the oocytes and undifferentiated granulosa cells in the various sized prehierarchical follicles of hen ovary, and the endogenous expression level of <i>SAV1</i> mRNA appears down-regulated from the primordial follicles to the largest preovulatory follicles (F2-F1) by immunohistochemistry and real-time RT-PCR, respectively. Moreover, we found the intracellular SAV1 physically interacts with each of the pathway members, including STK4/MST1, STK3/MST2, LATS1 and MOB2 using western blotting. And SAV1 significantly promotes the phosphorylation of LATS1 induced by the kinase of STK4 or STK3 in vitro. Furthermore, SAV1 knockdown by small interfering RNA (siRNA) significantly increased proliferation of granulosa cells from the prehierarchical follicles (6–8 mm in diameter) by BrdU-incorporation assay, in which the expression levels of <i>GDF9</i>, <i>StAR</i> and <i>FSHR</i> mRNA was notably enhanced. Meanwhile, these findings were consolidated by the data of SAV1 overexpression. Taken together, the present results revealed that SAV1 can inhibit proliferation of the granulosa cells whereby the expression levels of <i>GDF9</i>, <i>StAR</i> and <i>FSHR</i> mRNA were negatively regulated. Accordingly, SAV1, as a member of the hippo/MST signaling pathway plays a suppressive role in ovarian follicle development by promoting phosphorylation and activity of the downstream LATS1, may consequently lead to prevention of the follicle selection during ovary development.</p></div

Effects of overexpressing <i>SAV1</i> on expression of <i>FSHR</i>, <i>StAR</i>, <i>CCND2</i> and <i>GDF9</i> mRNA examined and cell proliferation.

<p>Granulosa cells were transfected with reconstructed pFLAG-SAV1 plasmids, pFLAG CMV-2 empty vector (negative control) and no plasmid (blank control). (A) The expression of <i>SAV1</i> gene before and after the GCs transfected with pFLAG-SAV1expression vector for 24 h was examined by qRT-PCR. Quantification of mRNA expression as normalized to the <i>18S rRNA</i> gene, the values on the bar graphs are the mean ± SEM of 10 hens (n = 10) from a representative experiment. (B) Expression levels of SAV1 protein in the GCs before and after the transfection with with pFLAG-SAV1 vector was detected by western blotting. The β-actin was used as the loading control. (C) The influence of <i>SAV1</i> overexpression on <i>FSHR</i>, <i>StAR</i>, <i>CCND2</i> and <i>GDF9</i> mRNA abundances in the granulosa cells from prehierarchichal follicles (6 to 8 mm in diameter) was examined. (D) The effects of SAV1 overexpression on the GC proliferation were detected by BrdU incorporation assay (original magnification × 200). Of which panel a, blank control; panel b, negative control; panel c-d, RNAi group. (E) The number of BrdU⁺ cells was expressed as percentage and calculated relative to the total number of cells counted in the microscope fields observed in the negative control. For each group, bars with superscript symbol indicates that difference was significant compared to the control group ** P<0.01,* P<0.05.</p

Quantification of expression levels of the <i>SAV1</i> mRNA in variously sized follicles from freshly collected ovaries.

<p>The data are the mean ± SEM from 10 hens (n = 10), and bars with different letters above them differ significantly in the amount of mRNA expression as normalized to the <i>18S rRNA</i> gene (<i>P</i> < 0.05). Horizontal ordinate represents the examined follicle sizes as indicated: 0-1mm, small follicles (<1 mm in diameter); 1–3.9 mm, the prehierarchical follicles were from 1 to 3.9 mm in diameter; 4–4.9 mm, from 4 to 4.9 mm in diameter; and the others followed similarly.</p

Phosphorylation of LATS1 by SAV1 in vitro.

<p>(A) CHO cells (which do not express endogenous chicken LATS1) was transfected with the pCMV-HA-LATS1 expression construct or an empty pCMV-HA vector for 24 h and lysed, cell lysates were immunoprecipitated using an antibody to HA. The immunoprecipitates were analyzed by western blotting with the LATS1 antibody, and the purified LATS1 protein was then used as a substrate to be incubated with the lysates from the cells transfected with the pFLAG-SAV1 construct or an empty pFLAG-CMV-2 vector alone, or co-transfected with the the pcDNA3-STK4 or STK3 expression vector aforementioned for an in vitro phosphorylation assay. The β-actin was used as the loading control. (B) Blotting signal intensity was quantified densitometrically after phosphorimaging (shown in A), and normalized for loading by comparison to the signal for β-actin. The signal intensity of LATS1 or phosphorylated LATS1 was expressed as the ratio β-actin signal in arbitrary units shown in B (n = 5 per mean ± SEM). The Five independent experiments were carried out in triplicate. The results are representative of at least three independent experiments. Statistical significance was marked with different superscript symbols ** P<0.01,* P<0.05.</p

Primer pairs designed for quantitative real-time PCR.

<p>Primer pairs designed for quantitative real-time PCR.</p

Phosphorylation of SAV1 by SKT3 and SKT4 in vitro.

<p>CHO cells (which do not express endogenous chicken SAV1, STK4 and STK3) was co-transfected with the pFLAG-SAV1 expression construct or an empty pFLAG-CMV-2 vector, and the pcDNA3-STK4 or STK3 expression vector or a vacant pcDNA3 vector for 24 h. The cells were lysated and analysed by immunoblotting procedure using an antibody to SAV1 and phosphorylated SAV1 (p-SAV1). The bands were observed corresponding to the antibody of p-SAV1 in the presence of STK4 or STK3 in the cell lysates, but no band was detected for the same lysates in the absence of STK4 or STK3.</p

Interaction of SAV1 and STK4, STK3, LATS1, MOB2 and YAP1 by an coimmunoprecipitation experiment.

<p>Chicken GC cells were transfected with an empty pFLAG-CMV-2 expression vector (−) or pFLAG-SAV1 expression construct (+). After transfection for 24 h, the cells were lysed, and the lysates were immunoprecipitated with a control chicken IgG or an antibody to FLAG. The lysates (Lysate) and immunoprecipitates (IP) were analyzed by immunoblotting (IB) with SAV1, STK4, STK3, LATS1, MOB2 and YAP1 antibodies. (A) When the empty pFLAG-CMV-2 vector was used as a template (Lysate, −), endogenous SAV1 was detected with a faint band observed, when the pFLAG-CMV-2-SAV1 construct was used as a template (Lysate, +), SAV1 was synthesized. The endogenous STK4, STK3, LATS1, MOB2 and YAP1 expression was also detected in the lysates prior to immunoprecipitation. The β-actin was used as the loading control (B) When these lysates were immunoprecipitated with chicken IgG, no band was obtained for FLAG-SAV1in immunoprecipitates from cells expressing FLAG-SAV1, and no band was obtained for all of STK4, STK3, LATS1, MOB2 and YAP1(IP: chocken IgG). When the lysates were immunoprecipitated with an antibody to FLAG, endogenous STK4, STK3, LATS1 and MOB2 was coimmunoprecipitated in cells expressing FLAG-FOXL2 (IP: FLAG, +), but YAP1 was not coimmunoprecipitated in the cells. No one of the proteins was examined in the cells expressing the empty expression vector (IP: FLAG, −).</p

Immunohistochemical analysis of SAV1 protein expression in the ovarian follicles.

<p>Paraformaldehyde-fixed tissue sections were immunostained using anti-chicken SAV1 as described above. Panel A, strong staining is observed in all oocytes (OC) and granulosa cells (GCs) within the variously sized prehierarchical follicles (×10). Panel B, the amplified primordial follicle and primary follicle (×100), paralleling the one marked in the read box of panel A; Panel C, the amplified developing PF follicles with two or three layers of GCs (×20), paralleling the one marked in the yellow box of panel A. Panel D, the amplified small PF follicles with one or more layers of GCs (×40). Panel E, the amplified large PF follicles with more layers of GCs (×20). Panel F, one of the negative controls for SAV1, the sections were immunostained with pre-immune serum, no significant expression was detected. Negative control sections were prepared with the primary antibody pre-incubated with a blocking peptide for 1 h at room temperature and for 2 h at 4°C. Five birds were used for immunohistochemical analysis and representative microscopic fields were selected. Scale bar = 100 μm.</p

Knockdown of CDH17 in AGS cells inhibited cell proliferation, migration, adhesion, colony formation and induced a cell-cycle arrest and apoptosis.

<p>Different assay were described in materials and methods. (<b>A</b>) Real-time PCR analysis showed the knock down of CDH17 at the mRNA level in AGS cells (C), AGS cells transiently transfected with pcDNA^TM6.2-GW/EmGFP-scramble miR plasmid (NC) and pcDNA^TM6.2-GW/EmGFP-CDH17 miR plasmids (90-1, 90-2, 90-3, and 90-4); (<b>B</b>) Western blotting for the effect of CDH17 knockdown using different concentrations of Tet for 48 h in TR-AGS-CDH17_KD cells; (<b>C</b>) Cell proliferation, migration (6 days post treatment), adhesion (6 days post treatment. **<i>P</i><0.01) and colony formation in soft agar (7 days post treatment. **<i>P</i><0.01); (<b>D</b>) Proliferation rescue assay. TR-inducible AGS-CDH17_KD stable cells were seeded in 60 mm ×15 mm dish and cultured for 10 days with or without 5.0 µg/ml Tet. For rescue group, the culture medium containing Tet was replaced by fresh medium on day 4, and cells were continued to be cultured to day 10. Cells in each group were harvested on day 2, 4, 6, 8 and 10 for cell number counting (left) and Western blotting analysis (right); (<b>E</b>) Cell cycle analysis after cells were treated with 5.0 µg/ml Tet for 6 days; and (<b>F</b>) Cell apoptosis analysis after cells were treated with 5.0 µg/ml Tet for 6 days by flow cytometry.</p

Schematic diagram of the regulatory and signaling network of CDH17 in GC.

<p>This schematic diagram demonstrates the inducing effect of CDH17 on Ras/Raf/MEK/ERK signaling pathway and illustrates the hypothetic involvement of integrins in GC. CDH17 indirectly affects integrins to stabilize their structure and activity. The up-regulation of cadherin-integrin signaling activates the Ras/Raf/MEK/ERK pathway. The activation of ERK regulates various nuclear and cytoplasmic substrates, including p53 and p21, which involve in diverse cellular responses, such as cell proliferation, migration, adhesion, colony formation, cell-cycle and apoptosis.</p