Notch2 improves granulosa cell functions in premature ovarian failure by activating the Wnt2/β-catenin pathway

Abstract

Background: Notch2 and Wnt2/β-catenin pathway improve granulosa cell (GC) functions, and there are interactions between Notch and Wnt/β-catenin in some cells. We aimed to investigate whether Notch2 improves GC functions in premature ovarian failure (POF) by activating the Wnt2/β-catenin pathway.

Methods: Notch2 expression was interfered in mice or KGN cells, then, mice were treated with cyclophosphamide and busulfan intraperitoneally, and KGN cells were exposed to cyclophosphamide to establish POF models. In vivo, the number of follicles at different stages was counted, and interactions between Notch2 and Wnt2 were detected. In vitro, cell viability and cycle were measured. Additionally, hormone levels, oxidative stress (OS) degrees, cell apoptosis, Notch2 and Wnt2/β-catenin pathway-related genes were detected in vivo and in vitro. Finally, Wnt/β-catenin pathway inhibitor (IWR-1), agonist (SKL2001) and β-catenin knockdown were used.

Results: Notch2 overexpression not only improved hormone levels, follicular development, OS degree and ovarian cell apoptosis, but also activated Wnt2/β-catenin pathway for POF mice. Moreover, Notch2 interacted with Wnt2 in POF mice. In vitro, Notch2 knockdown decreased cell viability, disrupted cell cycle, increased cell apoptosis, worsened hormone levels, promoted OS degree and inhibited Wnt2/β-catenin pathway for POF. Importantly, the protective effects of Notch2 overexpression and the worsening impacts of Notch2 knockdown on POF were reversed by IWR-1 and SKL2001. β-Catenin knockdown further impaired GC functions in POF models that underwent Notch2 and β-catenin knockdown.

Conclusion: Notch2 may improve GC functions in POF by activating the Wnt2/β-catenin pathway, suggesting that the Notch2-mediated Wnt2/β-catenin pathway is a novel therapeutic target for POF.

Keywords: Cyclophosphamide; Granulosa cell functions; Notch2; Premature ovarian failure; Wnt2/β-catenin pathway.

Fig. 1

Notch2 overexpression enhanced ovarian functions in premature ovarian failure (POF) mice by activating the Wnt/β-catenin pathway. (A) Quantitative real-time PCR (qPCR) for the validation of oe-Notch2 overexpression efficiency. (B) Representative images of ovarian size at the end of the study. (C) Quantification of ovarian weight at the end of the study. (D) Enzyme-linked immunosorbent assay (ELISA) for follicle-stimulating hormone (FSH), estradiol (E2) and anti-mullerian hormone (AMH) levels in serum. (E) qPCR for the expressions of steroidogenic enzyme-related genes (StARD1, Cyp11a1 and 3-β-HSD) in ovarian tissues. (F) Representative images of hematoxylin-eosin (HE) staining and quantification of primordial follicles, primary follicles, secondary follicles, antral follicles, atretic follicles and mature follicles for ovarian tissues. Magnification×200, scale bar = 100 μm; Magnification×40, scale bar = 500 μm. PF: primitive follicle; PrF: primary follicle; SF: secondary follicle; AnF: antral follicle; AF: atretic follicle; MF: mature follicle. ^△P < 0.05 and ^△△P < 0.01 vs. Normal; P < 0.05 and P < 0.01 vs. Control; ^#P < 0.05 and ^##P < 0.01 vs. oe-NC; ^@P < 0.05 and ^@@P < 0.01 vs. oe-Notch2. Results were presented as mean ± standard deviation (SD)

Fig. 2

Notch2 overexpression suppressed oxidative stress and ovarian cell apoptosis in POF mice by activating the Wnt/β-catenin pathway. (A) Levels of oxidative stress markers (malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)) in ovarian tissues. (B) Flow cytometry for cell apoptosis in ovarian tissues. (C) Western blot analysis was applied to measure the cleaved caspase-3/caspase-3 ratio in ovarian tissues. (D) qPCR for the expressions of cell cycle-related genes (CDK4, CCND2 and P21) and apoptosis-related genes (Bax and Bcl2) in ovarian tissues. P < 0.05 and P < 0.01 vs. Control; ^#P < 0.05 and ^##P < 0.01 vs. oe-NC; ^@P < 0.05 and ^@@P < 0.01 vs. oe-Notch2. Results were presented as mean ± SD

Fig. 3

Notch2 interacted with Wnt2 and β-catenin in POF mice. (A) qPCR was used to detect Wnt-2 and β-catenin mRNA expressions in the ovarian tissues. (B) Immunohistochemistry was applied to measure Notch2 protein expression in the ovarian tissues. Magnification×200, scale bar = 100 μm. (C) Western blot analysis was utilized to test Notch2 pathway-related protein expressions (Notch2, Jag1 and Hes2) and Wnt2/β-catenin pathway-related protein expressions (Wnt2, β-catenin, Axin2 and LEF1) in the ovaries. (D) Co-immunoprecipitation (Co-IP) was conducted to further verify the protein-protein interactions between Notch2 and Wnt2. P < 0.05 and P < 0.01 vs. Control; ^#P < 0.05 and ^##P < 0.01 vs. oe-NC; ^@P < 0.05 and ^@@P < 0.01 vs. oe-Notch2. Results were presented as mean ± SD

Fig. 4

Notch2 knockdown worsened GC functions in POF models by suppressed Wnt/β-catenin pathway (A) qPCR revealed the knockdown efficiency of sh-Notch2 for KNG cells. (B) Cell counting kit-8 (CCK-8) assay revealed cell viability for KNG cells. (C) Flow cytometry displayed cell cycle distribution for KNG cells. (D) Flow cytometry displayed cell apoptosis for KNG cells. (E) Western blot analysis displayed the cleaved caspase-3/caspase-3 ratio for KNG cells. (F) qPCR for the expressions of CDK4, CCND2, P21, Bax and Bcl2 mRNAs for KNG cells. (G) ELISA exhibited FSH, E2 and AMH levels for KNG cells. (H) qPCR for the expressions of StARD1, Cyp11a1 and 3-β-HSD mRNAs for KNG cells. (I) MDA, GSH and SOD levels were detected for KNG cells. P < 0.05 and P < 0.01 vs. Normal; P < 0.05 and P < 0.01 vs. Control; ^*P < 0.05 and ^**P < 0.01 vs. Model; ^#P < 0.05 and ^##P < 0.01 vs. sh-NC; ^@P < 0.05 and ^@@P < 0.01 vs. sh-Notch2. Results were presented as mean ± SD

Fig. 5

Notch2 acted on the Wnt2/β-catenin pathway in POF cells. (A) qPCR analysis for the expressions Wnt2 and β-catenin mRNAs in KNG cells. (B) Western blot analysis for the expressions of Notch2, Jag1, Hes2 Wnt2, β-catenin, Axin2 and LEF1 proteins in KNG cells. P < 0.05 and P < 0.01 vs. Control; ^*P < 0.05 and ^**P < 0.01 vs. Model; ^#P < 0.05 and ^##P < 0.01 vs. sh-NC; ^@P < 0.05 and ^@@P < 0.01 vs. sh-Notch2. Results were presented as mean ± SD

Fig. 6

βcatenin knockdown worsened GC functions in POF cells treated with sh-Notch2 and SKL2001 (A) CCK-8 assay was used to measure cell viability for KNG cells among groups. (B) Flow cytometry was conducted to test cell cycle distribution for KNG cells among groups. (C) Flow cytometry was carried out to evaluate cell apoptosis for KNG cells among groups. (D) qPCR was performed to detect CDK4, CCND2, P21, Bax and Bcl2 mRNA expressions in KNG cells among groups. (E) ELISA was utilized to measure FSH, E2 and AMH levels for KNG cells among groups. (F) qPCR was performed to detect StARD1, Cyp11a1 and 3-β-HSD in KNG cells among groups. (G) MDA, GSH and SOD levels among groups were detected for KNG cells. P < 0.05 and P < 0.01 vs. Model; ^#P < 0.05 and ^##P < 0.01 vs. sh-Notch2 + SKL2001 + sh-NC. Results were presented as mean ± SD

Fig. 7

βcatenin knockdown inhibited Wnt2/β-catenin pathway in POF cells treated with sh-Notch2 and SKL2001 (A) qPCR analysis for the expressions Wnt2 and β-catenin mRNAs in KNG cells. (B) Western blot analysis for the expressions of Notch2, Jag1, Hes2 Wnt2, β-catenin, Axin2 and LEF1 proteins in KNG cells. ^#P < 0.05 and ^##P < 0.01 vs. sh-Notch2 + SKL2001 + sh-NC. Results were presented as mean ± SD