Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination

• Published in: Journal of proteomics 2025-01, Vol.310, p.105318, Article 105318
• Main Authors: Kamińska, Kinga, Świderska, Bianka, Malinowska, Agata, Grzesiak, Małgorzata
• Format: Article
• Language: English
• Subjects: Cyclin dependent kinase 1; Granulosa cells; Insulin; Liquid chromatography with tandem mass spectrometry; Scavenger receptor class B member 1; Theca interna cells; Vitamin D3
• ISSN: 1874-3919
• DOI: 10.1016/j.jprot.2024.105318

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome. [Display omitted] •VD affects granulosa cells proteome leading to promotion of cell proliferation.•I influences granulosa cells proteome and thereby affects cellular adhesion.•VD + I induce granulosa cells proliferation via DAPs increasing DNA synthesis.•VD affects theca interna cells proteome leading to decreased cholesterol transport.•Interactions of VD and I in both follicle layers are crucial for their functions.