Effects of Shh Signal on the Proliferation and Differentiation of Follicular Granulosa Cells in Dairy Cows

doi:10.13560/j.cnki.biotech.bull.1985.2025-0333

Abstract

Abstract:

Objective To investigate the effect of sonic hedgehog (Shh) signal on the proliferation and differentiation of bovine follicular granulosa cells. Method Ovaries from estrous dairy cows were collected and the expression characteristics of Shh in ovarian tissues were detected by IHC. Bovine follicular granulosa cells were isolated and cultured, and divided into: control group, Ad-Shh group (transfected with Shh overexpression adenovirus, MOI = 50 for 48 h), and Ad-Shh + Cyc group (treated with Shh pathway inhibitor cyclopamine (Cyc, 5 μmol/L) for 24 h on the basis of overexpression of Shh). The proliferation activities of cells was analyzed by immunofluorescence, MTS, and EdU methods; the cell cycle variations were analyzed by flow cytometry; and the expressions of Shh, Ki67, and proliferation and differentiation-related proteins were detected by Western blot. Result IHC results showed that Shh was highly expressed in the bovine ovarian tissues; immunofluorescence results indicated that the overexpression of Shh promoted the expression of Ki67 in granulosa cells, and Cyc reversed this change (P<0.01); MTS and EdU results demonstrated that Cyc reversed the enhancing effect of overexpression of Shh on cell proliferation activity (P<0.01). Flow cytometry results showed that the overexpression of Shh promoted the transition of granulosa cells from G1 phase to S phase (the proportion of G1 phase cells decreased from 91.42% to 71.58%, a decrease of 19.84%, and the proportion of S phase cells increased from 4.88% to 22.22%, an increase of 17.34%), and Cyc slowed down the cell cycle process (P<0.01). Western blot results showed that the overexpression of Shh promoted the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.01), and Cyc reversed the promoting effect of overexpression of Shh on the expression of Ki67, cell proliferation, and cell differentiation-related proteins (P<0.05). Conclusion Shh signal positively regulates the proliferation and differentiation of bovine follicular granulosa cells.

Key words: Shh signal, follicular development, follicular granulosa cells, proliferation, differentiation

SUN Han-bing, XIANG Nian, FANG Xin-xin, YUE Jun-xiu, XU Qiu-shi, MA Li. Effects of Shh Signal on the Proliferation and Differentiation of Follicular Granulosa Cells in Dairy Cows[J]. Biotechnology Bulletin, 2025, 41(9): 326-334.

Figures/Tables 6

Fig. 1 Expression characteristics of the Shh signal in the ovarian tissue of dairy cowsA:Expression characteristics of the Shh signaling pathway in the ovarian tissue of dairy cows. The brown color indicates Shh protein, and the blue color indicates the cell nucleus. B: Magnified results of the part enclosed in figure A

Fig. 2 Immunofluorescence expression of Ki67 in the GCs of dairy cow folliclesA: The immunofluorescence results of Ki67 expression in GCs. B: Statistical analysis of Ki67 positive cell rate; *P<0.05, **P<0.01, the same below

Fig. 3 Influence of Shh signal on the proliferation activity of GCs in dairy cow folliclesA: Cell proliferation rate was detected by MTS. B: EdU detects the results of cell proliferation. C: Statistical analysis of EdU positive cell rate. D: Grayscale images of protein expression of Shh and proliferation marker protein Ki67. E: The relative expression of Shh protein. F: The relative expression of the proliferation marker protein Ki67

Fig. 4 Influences of Shh signal on the proliferation process of GCs in dairy cow folliclesA-C: Variation in the cell cycle process of GCs in dairy cow follicles. D: Variation in the cell cycle progression

Fig. 5 Influence of Shh signal on the cell cycle of GCs in dairy cow folliclesA: Grayscale images of the protein expressions of cyclins (CCND1, CCND2, CCND3, and CCNE1). B: Relative protein expression of cyclins. C: Grayscale images of the protein expressions of cyclin-dependent kinases (CDK2、CDK4、CDK6). D: Relative protein expression of cyclin-dependent kinases

Fig. 6 Influence of Shh signal on the expression of differentiation proteins in GCs of dairy cow folliclesA: Grayscale images of the protein expressions of cell differentiation marker molecules (StAR, CYP19A1, and LHCGR). B-D:Relative protein expression of cell differentiation marker molecules (StAR, CYP19A1, and LHCGR)

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