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. 2023 Jan 9:9:1081643.
doi: 10.3389/fvets.2022.1081643. eCollection 2022.

Epidermal growth factor regulates autophagy activity and endocytosis of yak cumulus cells in a concentration-dependent manner

Rui Ma  1   2 , Sijiu Yu  1   2 , Yan Cui  1   2 , Yangyang Pan  1   2 , Meng Wang  1   2 , Libin Wang  1   2 , Jinglei Wang  1   2 , Ling Zhao  1   2 , Hui Zhang  1   2
Affiliations

Epidermal growth factor regulates autophagy activity and endocytosis of yak cumulus cells in a concentration-dependent manner

Rui Ma et al. Front Vet Sci. .

Abstract

Introduction: Autophagy and endocytosis are crucial biological activities in mammalian follicle development and oocyte maturation, which are easily affected by external environmental factors. Epidermal growth factor (EGF), as an important component of follicular fluid, regulates the growth and apoptosis of follicular cells. However, its regulatory mechanism of autophagy and endocytosis in mammals, especially in large domestic animals such as plateau yak, remains unclear. This study aimed to investigate the regulatory mechanism of EGF on autophagy and endocytosis in yak cumulus cells.

Methods: Yak cumulus cells were treated with different concentrations of EGF and appropriate concentrations of EGFR inhibitor gefitinib (10 μM). The dynamic expression levels of Atg5, Beclin1, LC3, Cav1 and Cav2 were detected by immunofluorescence staining, qRT-PCR and Western-blot.

Results: EGF inhibited autophagy in yak cumulus cells by down-regulating the expression of Atg5, Beclin1, and LC3. The level of autophagy varied with the concentration of ligands, and the inhibition was most significant at 100 ng/mL. Noteworthy, EGF can promote endocytosis by regulating the expression of Cav1 and Cav2, but the EGFR-mediated signaling pathway is not the main way to regulate the expression of these proteins.

Discussion: These results provide a reference for further exploring the effects of growth factors on livestock germ cells and the regulatory role of autophagy-endocytosis crosstalk mechanism in follicle development and oocyte maturation, to improve the fecundity of yaks.

Keywords: EGF; autophagy; cumulus cells; endocytosis; yak.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Yak cumulus cells culture and distribution of Atg5, Beclin1, LC3, Cav1, Cav2 protein in CCs. (A) The growth pattern of the second generation yak cumulus cells (10×) (a–d showed cell growth at 6, 24, 48, and 72 h, respectively). (B) Localization of target proteins in yak cumulus cells. Red fluorescence, Atg5, Beclin1, LC3, Cav1, and Cav2 protein, respectively; Green fluorescence, β-tubulin protein; Blue fluorescence: cell nucleus.
Figure 2
Figure 2
Relative expression levels of Atg5, Beclin1 and LC3 gene in yak CCs treated with different concentrations of EGF and the optimal concentration of gefitinib (EGFR inhibitor). (A–C) Relative mRNA expression levels of Atg5, Beclin1, and LC3 in yak CCs (EGF group). (D) Relative mRNA expression levels of Atg5, Beclin1, and LC3 in yak CCs (Gefitinib group). *on the bars indicates values that differ significantly (*P < 0.05).
Figure 3
Figure 3
Western-blot detection of Atg5, Atg5-Atg12, Beclin1, LC3I, LC3II, Cav1, Cav2, and β-actin proteins under different EGF concentrations and EGFR inhibitor action group. From left to right: 0, 50, 100, 150, 200 ng/mL EGF group and EGFR inhibitor gefitinib group.
Figure 4
Figure 4
Relative expression levels of Atg5, Beclin1, and LC3 protein in yak CCs treated with different concentrations of EGF and the optimal concentration of gefitinib (EGFR inhibitor). (A–F) Relative protein expression levels of Atg5, Atg5-Atg12, Beclin1, LC3I, LC3II, and LC3II/LC3I in yak CCs (EGF group). (G, H) Relative protein expression levels of Atg5, Atg5-Atg12, Beclin1, LC3I, LC3II, and LC3II/LC3I in yak CCs (Gefitinib group). *on the bars indicates values that differ significantly (*P < 0.05).
Figure 5
Figure 5
Relative expression levels of Cav1 and Cav2 gene in yak CCs treated with different concentrations of EGF and the optimal concentration of gefitinib (EGFR inhibitor). (A, B) Relative mRNA expression levels of Cav1 and Cav2 in yak CCs (EGF group). (C) Relative mRNA expression levels of Cav1 and Cav2 in yak CCs (Gefitinib group). *on the bars indicates values that differ significantly (*P < 0.05).
Figure 6
Figure 6
Relative expression levels of Cav1 and Cav2 protein in yak CCs treated with different concentrations of EGF and the optimal concentration of gefitinib (EGFR inhibitor). (A, B) Relative protein expression levels of Cav1 and Cav2 in yak CCs (EGF group). (C) Relative protein expression levels of Cav1 and Cav2 in yak CCs (Gefitinib group). *on the bars indicates values that differ significantly (*P < 0.05).
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