Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Yashar Esmaeilian, Sevgi Yusufoglu, Gamze Bildik, Ozgur Oktem, Ece Iltumur

Journal: Methods in molecular biology (Clifton, N.J.) 2025;2879():151-171

PMID: 38411886

Abstract

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

Address: Research Center for Translational Medicine, Koç University, Istanbul, Turkey.; The Graduate School of Health Sciences, Koç University, Istanbul, Turkey.; The Graduate School of Health Sciences, Koç University, Istanbul, Turkey.; Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.; Research Center for Translational Medicine, Koç University, Istanbul, Turkey. [email protected].; The Graduate School of Health Sciences, Koç University, Istanbul, Turkey. [email protected].; Department of Obstetrics and Gynecology, Koç University School of Medicine, Istanbul, Turkey. [email protected].

DOI: 10.1007/7651_2024_520

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https://doi.org/10.1007/7651_2024_520

MeSH Terms: Humans, Female, Male, Testis, Autophagy, Fluorescent Antibody Technique, Ovary, Mitochondria, Gonadal Steroid Hormones, Cholesterol, Lysosomes, Granulosa Cells, Lipid Droplets