Growth differentiation factor 9 activates the TGF-β pathway in follicle atresia of Muscovy ducks

Abstract

Muscovy ducks' high broodiness hinders industry growth. Studying broodiness regulation contributes to the theoretical foundation for enhancing reproductive performance in Muscovy ducks. Experiment 1, a total of 18 Muscovy ducks were divided into 2 groups: Laying group (LO) and Broody group (BO). To collect ovaries for morphological and transcriptome analysis. Experiment 2, Primary Muscovy ducks granulosa cells (GC) were isolated and treated with or without GDF9 at appropriate concentrations as indicated. Experiment 3, GC were treated with or without GDF9 in the presence or absence of a receptor inhibitor. The cell viability, cell apoptosis rate and levels of TGF-β pathway were determined. In vivo, there was a gradual disappearance of follicles in the ovaries and accompanied by follicle atrophy and a concentration of cytoplasm in BO group. The transcriptome expression profile revealed a total of 1,185 up-regulated differentially expressed transcripts (DEs) and 1,258 down-regulated DEs in the BO group compared to the LO group. The up-regulated differentially expressed GDF9 is involved in regulating the TGF-β pathway, which is among the top 10 pathways identified through the KEGG pathway analysis (P < 0.05). Additionally, the fluorescence intensity of apoptosis is primarily observed in the granulosa layers of the ovary. Compared to the LO group, the mRNA level of TGF-β pathway and the protein of GDF9 and p-Smad2/3 were increased in ovary of the BO group (P < 0.05). In vitro, GDF9 supplementation demonstrated does-related promotion of GC (P < 0.01). Compared to CTRL group, 12 ng/mL GDF9 supplementation to GC increased the rate of cell apoptosis, the mRNA and protein expression of TGF-β pathway and the apoptosis-related genes. Pretreatment of GC with GDF9-receptor inhibitor largely abrogated the negative function of GDF9 treatment (P < 0.05). In summary, granulosa cell apoptosis leading to follicle atresia in broodiness of Muscovy ducks is associated with GDF9 activation of the TGF-β pathway. This discovery lays a solid foundation for understanding duck follicular development and enhancing egg production in Muscovy ducks.

Keywords: Muscovy duck; SMDA; apoptosis; broodiness; granulosa cell.

Figure 1

The ovarian morphology and histological characteristics in Muscovy ducks. (A and B) Macroscopic structure of the ovary. (C and D) HE staining for morphological characteristics of the ovary. The scale bar in the HE staining image is 200 μm. Green arrow, Granulosa cell layer. LO, laying Muscovy ducks group; BO, Broody Muscovy ducks group, n = 9.

Figure 2

Quantitative analysis and validation of transcriptome data. (A) Scatter plot, red dots indicate up-regulated mRNAs, blue dots indicate down-regulated mRNAs; gray dots indicate mRNAs with no significant difference. (B) Heat map, red and blue indicate high and low expression of mRNAs. (C) the differentially expression mRNA verified by Real-time quantitative PCR. LO, laying Muscovy ducks group; BO, Broody Muscovy ducks group. n = 3, Ovaries from 9 ducks in each group were randomly divided into 3 sets, with the ovarian tissues from each set homogenized in liquid nitrogen to form 3 biological replicates.

Figure 3

Top 10 significantly clustered GO biological processes and total significantly enriched KEGG pathways in ovary. (A) GO biological processes, the Y-axis represents second-level GO terms for the 3 significant terms, including reproductive structure development, developmental processes involved in reproduction, and gonad development. The upper X-axis indicates the percentage of mRNA included in that second-level category out of the total, and the lower X-axis represents the number of mRNA aligned to that second-level category. The ovarian mRNA samples were obtained from complete ovarian tissues (excluding both connective tissue and yolk) from the laying and broody groups. The 3 colors represent 3 major classifications: BP (biological process), CC (cellular component), and MF (molecular function). (B) KEGG pathways, the Y-axis represents the names of KEGG pathways, while the X-axis represents the rich factor, defined as the ratio of the number of enriched mRNA in the KEGG pathway to the number of annotated mRNA (Sample number/Background number). A larger rich factor indicates a higher level of enrichment. The size of the points indicates the number of mRNA in the KEGG pathway, and the color of the points corresponds to different values of P adjust. LO, laying Muscovy ducks group; BO, Broody Muscovy ducks group. n = 3, Ovaries from 9 ducks in each group were randomly divided into 3 sets, with the ovarian tissues from each set homogenized in liquid nitrogen to form 3 biological replicates.

Figure 4

The protein level of GDF9 on follicles at different stages. (A) the complete ovary during the laying period can be classified based on follicle diameter. (B) The GDF9 protein levels were measured in follicles at various developmental stages after removing connective tissue and yolk. SWF, small white follicles. LWF, large white follicles. SYF, small yellow follicles. LYF, large yellow follicles. F6, F5, F4, F3, F2 and F1, different grades of preovulation follicles. n = 3, Ovaries from 9 ducks in each group were randomly divided into 3 sets, with the ovarian tissues from each set homogenized in liquid nitrogen to form 3 biological replicates.

Figure 5

The level of cell apoptosis rate and TGF-β signaling expression in ovary of Muscovy ducks. (A and B) The HE staining for the granular layer in small yellow follicle, the white arrows indicate the distribution area of granulosa cells, while the black lines represent the thickness of the granulosa cell layer, the scale bar represents 100 μm. C_1-3-D_1-3, TUNEL fluorescence staining for apoptosis positive cells in ovary. DAPI staining of ovarian tissue cell nuclei (blue); Apoptosis positive cells detection in the ovary (red), scale bar represents 50 μm. E, Statistical quantification of data in Figures (C and D). (F) The protein level of GDF9 detected by ELISA in ovary. (G) the mRNA expression of TGF-β signaling in ovary. (H) the protein of Smad2/3 and p-Smad2/3 normalized to β-actin expression. (I) Statistical quantification of the data in picture H. LO, laying Muscovy ducks group; BO, Broody Muscovy ducks group. The marker was FSHR. * represents 0.01 < P ≤ 0.05 and ** represents P ≤ 0.01. n = 6.

Figure 6

Effects of GDF9 on primary Muscovy ducks Granulosa cells viability, cell apoptosis and TGF-β signaling expression. (A) Identification of granulosa cells: DAPI staining for granulosa cell nuclei (blue); granulosa cell marker (red); scale bar represents 100 μm. (B) Granulosa cells were incubated with GDF9 for 24 h at concentrations of 0, 3, 6, 12, 25, 50, and 100 ng/mL. Cell viability was determined with a CCK-8 assay. (C) The relative mRNA level of TGF-β signaling was evaluated in cells incubated with GDF9 for 24 h at a concentration of 12 ng/mL. (D) Western blot analysis of Smad2/3 and p-Smad2/3 with densitometric values normalized to β-actin. (E) Statistical analysis of the data in D. (F) Cell apoptosis was assessed using flow cytometry. (G) Statistical analysis of F. CTRL, cells cultured in basal medium; GDF9, cells cultured with 12 ng/mL GDF9 for 24 h. * represents 0.01 < P ≤ 0.05 and ** represents P ≤ 0.01. n = 3, each set of experiments was independently performed 3 times, with each experiment consisting of 3 replicates per treatment.

Figure 7

Effects of GDF9 on cell apoptosis and TGF-β signaling expression in cells treated with inhibitors. (A) Cells were incubated with inhibitors (LY) for 2, 6, 12 and 24 h at concentrations of 0, 1 μmol/L, 2 μmol/L, 5 μmol/L, 10 mmol/L. Cell viability were determined with a CCK-8 assay. (B-C) BMPR2-TGFBR1 receptor inhibitors knockdown efficiency in cells transfected with LY. (D) the mRNA expression levels of TGF-β signaling in Granulosa cells treated with or without LY. (E) the protein levels of Smad 2/3 and p-Smad2/3 in Granulosa cells treated with or without LY. (F) Cell apoptosis was determined with flow cytometry in Granulosa cells treated with or without LY. (G) Statistical analysis of F. CTRL, cells cultured in basal medium; GDF9, cells cultured with 12 ng/mL GDF9 for 24 h. LY, cells cultured with 2 μmol/L of LY for 6h; GDF9 + LY, cells cultured with 2 μmol/L of LY for 6h followed by treatment with 12 ng/mL GDF9 for 24 h. * represents 0.01 < P ≤ 0.05 and ** represents P ≤ 0.01. n = 3, each set of experiments was independently performed 3 times, with each experiment consisting of 3 replicates per treatment.

Figure 8

The mechanism of GDF9-induced granulosa cell apoptosis associated with follicle atresia. Muscovy ducks exhibit strong broody behavior, which significantly impacts their ovarian physiology. Compared to laying ducks (LO), the ovaries of broody Muscovy ducks (BO) undergo frequent follicular atresia, leading to ovarian regression and the cessation of ovulation. Transcriptome sequencing data revealed that Growth Differentiation Factor 9 (GDF9) is significantly upregulated in the ovarian tissue during the broody phase. GDF9, secreted by oocytes, binds to its receptors, TGFBR1 (ALK5) and BMPR2, on granulosa cells. This activates the canonical Smad-dependent signaling cascade, where Smad2/3 are phosphorylated and form a complex with Smad4. The Smads complex translocates to the nucleus and regulates the transcription of apoptosis-related genes, such as Bcl-2, Bax, and Caspase-3, ultimately leading to the induction of granulosa cell apoptosis.