Fig 2: Follicle-stimulating hormone (FSH) reduces the amount of SHP2/ERa complex in human granulosa cells (COV434). (A) To obtain the effective concentration of phenylhydrazonopyrazolone sulfonate 1 (PHPS1), an MTT assay was performed in COV434 cells, and 3 µM of PHPS1 was identified as the least effective concentration. (B) Immunofluorescence experiment of FoxL2 (green) and SHP2 (red) identified that SHP2 shows nuclear localization in COV434 cells, and PHPS1 had no effect on SHP2 nuclear localization. (C) FSH- and E2-stimulated COV434 cells showed the differential localization of SHP2 and ERa after 24 h. Estradiol (E2) significantly enhanced the nuclear localization of SHP2, while FSH significantly reduced its nuclear localization, compared to that in the control group. (D) Co-immunoprecipitation identified that SHP2 and ERa form a complex in COV434 cells, and E2 or FSH treatments had a significant effect on this complex. (E) FSH augmented extracellular signal-regulated kinase (ERK) and AKT signaling in the COV434 cell line compared to that in the control and E2-treated groups, wherein signaling was significantly reduced. The experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Src-homology-2-containing phosphotyrosine phosphatase (SHP2)/estrogen receptor alpha (ERa) complex and ERa-dependent gene expression in cumulus cells. (A) SHP2 and ERa proteins analyzed in germinal vesicle (GV)- and MII-stage cumulus–oocyte complexes (COCs) via immunofluorescence. Both proteins showed nuclear localization and exited the nucleus with the progression of meiotic maturation. Data are expressed as means ± SEM. GV and MII COCs were immune-labeled with SHP2 (green) and ERa (red) and counterstained with 10 µg/ml 4',6'-diamidino-2-phenylindole to visualize DNA (mean ± SEM). (B) Co-immunoprecipitation of SHP2 and ERa showed that both proteins form a complex at the GV COC stage, and the complex is significantly reduced with oocyte maturation. (C) Natriuretic peptide C (NPPC) and natriuretic peptide receptor 2 (NPR2) genes analyzed via RT-qPCR in GV, germinal vesicle breakdown (GVBD), and MII-stage COCs and compared with phenylhydrazonopyrazolone sulfonate 1 (PHPS1) treatment of GVBD- and MII-stage oocytes. (D) CX37 and CX43 genes analyzed via RT-qPCR in GVBD- and MII-stage COCs and compared with the PHPS1 treatment of GVBD- and MII-stage oocytes. (E) Cultured bovine granulosa cells were stimulated with follicle-stimulating hormone (FSH) and FSH plus PHPS1, and the p-AKT protein was analyzed along with total AKT. The experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Cumulus-cell cytoplasmic Src-homology-2-containing phosphotyrosine phosphatase (SHP2) inhibition deteriorates oocyte maturation and embryo development. (A) SHP2/ERa (GFP-labeled SHP2 and red fluorescent protein-labeled ERa) proteins analyzed via immunofluorescence in cumulus cells during germinal vesicle breakdown (GVBD) and MII stages of control cumulus–oocyte complexes (COCs) and phenylhydrazonopyrazolone sulfonate 1 (PHPS1)-treated COCs. (B) Representative images of p-ERK1/2 and p-AKT in GVBD- and MII-stage oocytes in the control and PHPS1-exposed groups. 4',6'-Diamidino-2-phenylindole was used to stain the nucleus. (C) Immunofluorescence images of Caspase-3 and phosphorylated NF-Kb in control and PHPS1-treated bovine day 8 blastocysts. Qualitative analysis showed that PHPS1 treatment enhances p-NF-kB nuclear localization and enhances Caspase-3 expression. All experiments were repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Src-homology-2-containing phosphotyrosine phosphatase (SHP2) expression in bovine ovary and upregulation with follicle-stimulating hormone (FSH) or epidermal growth factor (EGF) treatment. (A) Representative images of SHP2 protein expression (green, fluorescein isothiocyanate) in bovine pre-ovulatory oocyte granulosa cells. (B) SHP2 protein expression in bovine ovarian medulla. The lower images are zoom-ins of the upper images. (C) SHP2 expression in the cumulus cells of bovine oocyte (germinal vesicle, GV). (D) SHP2 expression in cultured cumulus cells obtained from GV-stage oocytes. (E) Cultured cumulus cells treated with FSH or EGF and PTPN11 (SHP2), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B (AKT3) genes analyzed via RT-qPCR (the experiment was repeated thrice, and data are shown as mean ± SEM. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.