Immune-labeling of cytochrome P450scc and its associate electron-transferring enzymes in mouse ovarian tissue sections

Abstract

Steroid hormone biosynthesis is mediated by cytochrome P450 side-chain cleavage (cytP450scc) enzyme, a rate-limiting step involved in the conversion of cholesterol to pregnenolone in gonads. This enzyme is accompanied by electron-transferring enzymes, such as adrenodoxin and adrenodoxin reductase, all localized to the inner mitochondrial membrane. Immunostaining was performed on mouse ovarian tissue sections at various stages of folliculogenesis using antisera generated against cytP450scc, adrenodoxin, and adrenodoxin reductase. Additional staining was conducted using the CF488A-conjugated phalloidin and CF555-conjugated wheat germ agglutinin lectin to identify different stages of folliculogenesis and ovulation cycle. Staining for lectin-binding moieties and phalloidin was observed in the zona pellucida region of early preantral and antral follicles. Expression of P450scc, adrenodoxin, and adrenodoxin reductase enzymes was detected in thecal cells of primordial, primary, and secondary follicles, as well as interstitial stromal cells. The findings presented here may contribute to identifying the specific roles of cytP450scc-expressing cells in the pathogenesis of polycystic ovarian syndrome and autoimmune primary ovarian insufficiency, including cases caused by autoimmune oophoritis.

Keywords

CytP450scc / Ferredoxin / Ferredoxin reductase / Follicular granulosa cells / Thecal cells / Leydig cells

Acknowledgments

We would like to thank Prof. Israel Hanukoglu for his support during the study. We also would like to thank Ariel University for allowing the use of Central Facilities.

Funding

Sachin Sharma has been supported by a fellowship from the Graduate School of Ariel University, Israel.

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

Conceptualization: Sachin Sharma
Investigation: Sachin Sharma
Methodology: Sachin Sharma
Visualization: Sachin Sharma
Writing – original draft: All authors
Writing – review & editing: Kalimuthu Kalishwaralal

Ethics approval and consent to participate

For animal use in research conducted for this study, ethical approval was sought from the Institutional Animal Ethics Committee of Ariel University (Ariel, Israel) (permit 32_12733_b019) in accordance with Ministry of Health guidelines. This study did not involve human participants as subjects and employed the study design of humanrelated clinical trials in this research.

Consent for publication

Not applicable.

Availability of data

Microscopy data related to this study are available from the corresponding author upon request.

References

[1] Byers SL , Wiles MV , Dunn SL , Taft RA . Mouse estrous cycle identification tool and images. PLoS One. 2012; 7(4): e35538. doi: 10.1371/journal.pone.0035538

[2] McGee EA , Strauss JF . Ovarian hormone synthesis. In: Endocrinology: Adult and Pediatric. 5th ed., Vol. 2. Netherlands: Elsevier Saunders; 2010. p. 2302-2317.

[3] Strauss JF . The synthesis and metabolism of steroid hormones. In: Yen&Jaffe’s Reproductive Endocrinology. Vol. 27. Netherlands: Elsevier Health Sciences; 2009. p. 79-104.

[4] Litwack G . Steroid hormones: Chemistry, biosynthesis, and metabolism. Hormones (Fourth Edition). 2022: 29-55.

[5] Cholesterol Side-Chain Cleavage Enzyme – an Overview Science Direct Topics. Available from: https://www.sciencedirect.com/topics/medicine-and-dentistry/cholesterol-side-chain-cleavage-enzyme [Last accessed on 2025 Jan 17].

[6] Pelletier G , Li S , Luu-The V , Tremblay Y , Bélanger A , Labrie F . Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads. J Endocrinol. 2001; 171(2): 373-383. doi: 10.1677/joe.0.1710373

[7] Hanukoglu I , Suh BS , Himmelhoch S , Amsterdam A . Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. J Cell Biol. 1990; 111(4): 1373-1381. doi: 10.1083/jcb.111.4.1373

[8] Hanukoglu I , Hanukoglu Z . Stoichiometry of mitochondrial cytochromes P‐450, adrenodoxin and adrenodoxin reductase in adrenal cortex and corpus luteum. Implications for membrane organization and gene regulation. Eur J Biochem. 1986; 157(1): 27-31. doi: 10.1111/j.1432-1033.1986.tb09633.x

[9] Svitkina TM , Borisy GG . Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol. 1999; 145(5): 1009-1026. doi: 10.1083/jcb.145.5.1009

[10] Pollard TD , Borisy GG . Cellular motility driven by assembly and disassembly of actin filaments. Cell. 2003; 112(4): 453-465. doi: 10.1016/s0092-8674(03)00120-x

[11] Dunkley S , Scheffler K , Mogessie B . Cytoskeletal form and function in mammalian oocytes and zygotes. Curr Opin Cell Biol. 2022; 75: 102073. doi: 10.1016/j.ceb.2022.0200

[12] Uraji J , Scheffler K , Schuh M . Functions of actin in mouse oocytes at a glance. J Cell Sci. 2018; 131(22): jcs218099. doi: 10.1242/jcs.218099

[13] Choudhary D , Jansson I , Schenkman JB , Sarfarazi M , Stoilov I . Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues. Arch Biochem Biophys. 2003; 414(1): 91-100. doi: 10.1016/s0003-9861(03)00174-7

[14] Shimada T , Sugie A , Shindo M , et al. Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene. Toxicol Appl Pharmacol. 2003; 187(1): 1-10. doi: 10.1016/s0041-008x(02)00035-2

[15] Rajapaksa KS , Sipes IG , Hoyer PB . Involvement of microsomal epoxide hydrolase enzyme in ovotoxicity caused by 7,12-dimethylbenz[a]anthracene. Toxicol Sci. 2007; 96(2): 327-334. doi: 10.1093/toxsci/kfl202

[16] Renaud HJ , Cui JY , Khan M , Klaassen CD . Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice. Toxicol Sci. 2011; 124(2): 261-277. doi: 10.1093/toxsci/kfr240

[17] Xu Q , Song Y , Chen Y , et al. Molecular cloning and expression patterns of the cholesterol side chain cleavage enzyme (CYP11A1) gene during the reproductive cycle in goose (Anas cygnoides). J Anim Sci Biotechnol. 2015; 6(1): 54. doi: 10.1186/s40104-015-0053-9

[18] Tilly JL , Kowalski KI , Johnson AL . Cytochrome P450 side-chain cleavage (P450scc) in the hen ovary. II. P450scc messenger RNA, Immunoreactive protein, and enzyme activity in developing granulosa cells. Biol Reprod. 1991; 45(6): 967-974. doi: 10.1095/biolreprod45.6.967

[19] El Fouikar S , Van Acker N , Héliès V , et al. Folliculogenesis and steroidogenesis alterations after chronic exposure to a human-relevant mixture of environmental toxicants spare the ovarian reserve in the rabbit model. J Ovarian Res. 2024; 17(1): 134. doi: 10.1186/s13048-024-01457-6

[20] Ziegler GA , Vonrhein C , Hanukoglu I , Schulz GE . The structure of adrenodoxin reductase of mitochondrial P450 systems: Electron transfer for steroid biosynthesis. J Mol Biol. 1999; 289(4): 981-990. doi: 10.1006/jmbi.1999.2807

[21] Heinrich A , DeFalco T . Essential roles of interstitial cells in testicular development and function. Andrology. 2019; 8(4): 903-914. doi: 10.1111/andr.12703

[22] McCarty KD , Liu L , Tateishi Y , Wapshott-Stehli HL , Guengerich FP . The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive. J Biol Chem. 2024; 300(1): 105495. doi: 10.1016/j.jbc.2023.105495

[23] Miller WL . Steroid hormone synthesis in mitochondria. Mol Cell Endocrinol. 2013; 379(1-2): 62-73. doi: 10.1016/j.mce.2013.0401

[24] Hanukoglu I , Jefcoate CR . Mitochondrial cytochrome P-450scc. Mechanism of electron transport by adrenodoxin. J Biol Chem. 1980; 255(7): 3057-3061. doi: 10.1016/S0021-9258(19)85851-9

[25] Blanco LZ Jr. , Kuhn E , Morrison JC , Bahadirli-Talbott A , Smith-Sehdev A , Kurman RJ . Steroid hormone synthesis by the ovarian stroma surrounding epithelial ovarian tumors: A potential mechanism in ovarian tumorigenesis. Mod Pathol. 2017; 30(4): 563-576. doi: 10.1038/modpathol.2016.219

[26] Tong ZB , Otsuka N , Tu W , Wei Q , DeCherney AH . P450 side-chain cleavage enzyme (P450-SCC) is an ovarian autoantigen in a mouse model for autoimmune oophoritis. Reprod Sci. 2022; 29(8): 2391-2400. doi: 10.1007/s43032-022-00970-7

[27] Strauss JF , Modi B , McAllister JM . Defects in ovarian steroid hormone biosynthesis. In: Cellular Endocrinology in Health and Disease. Netherlands: Elsevier; 2014. p. 285-309.