Proteomic characterization of bovine granulosa cells in dominant and subordinate follicles

Abstract

Background: Characterization of molecular factors regulating ovarian follicular development is critical to understanding its functional mechanism of controlling the estrous cycle, determining oocyte competency, and regulating ovulation. In previous studies, we performed next-gene sequencing to investigate the differentially expressed transcripts of bovine follicular granulosa cells (GCs) at the dominant follicle (DF) and subordinate follicle (SF) stages during the first follicular wave. This study aims to investigate the proteomic characterization of GCs of DF and SF in the bovine estrous cycle.

Results: In total, 3409 proteins were identified from 30,321 peptides obtained from liquid chromatograph-mass spectrometer analysis. Two hundred fifty-nine of these proteins were found to be expressed differently in DF and SF. Out of 259, a total of 26 proteins were upregulated (fold change≥2) and 233 proteins were downregulated (fold change≤0.5) in DF. Gene Ontology (GO) analysis of proteome data revealed the biological process, cellular component and molecular function of expressed proteins in DF and SF, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed important signaling pathways associated with follicular development such as the PI3K-Akt, estrogen, and insulin signaling pathways. Immunoblotting results of OGN, ROR2, and HSPB1 confirmed the accuracy of the data. Bioinformatics analysis showed that 13 proteins may be linked to follicular development.

Conclusions: Findings from this study will provide useful information for exploring follicular development and function.

Keywords: Bovine; Follicle; Label-free; Proteomic analysis.

Fig. 1

The expressed protein GO annotation. Classification of the expressed proteins in GCs of DF and SF in cattle based on the GO annotation, respectively. a: biological processes, b: molecular function, c: cellular components

Fig. 2

Biological processes of upregulated proteins and downregulated proteins

Fig. 3

Differentially expressed proteins KEGG pathways analysis. The x-coordinate is the signaling pathway names, the y-coordinate is the number of proteins

Fig. 4

Western blots for validation of OGN, ROR2 and HSPB1 abundance in DF and SF. β-actin was used as a loading control, and the abundance of proteins was corrected relatively to β-actin. On the left side of a-c: immunoblot results of OGN, ROR2, HSPB1 and β-actin in DF and SF GCs; on the right side of a-c: protein relative expression level of OGN, ROR2 and HSPB1 in DF and SF GCs. Superscript single and double asterisk indicate significantly different at the 0.05 and 0.01 levels, respectively. (n = 3 each; least square mean ± SE)