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

Immune-labeling of cytochrome P450scc and its associate electron transferring enzymes in mouse ovary tissue-sections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immune-labeling of cytochrome P450scc and its associate electron transferring enzymes in mouse ovary tissue-sections Sachin Sharma, Kalimuthu Kalishwarlal Kali This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6122209/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Steroid biosynthesis is mediated by cytochrome P450scc enzyme, a rate-limiting step involved in the conversion of cholesterol to pregnenolone in gonads. The cytochrome P450scc enzyme is accompanied by electron transferring enzyme adrenodoxin and adrenodoxin reductase, all present on mitochondrial integral membrane. This research article basically reveals the sites of expression of cytochrome P450scc, adrenodoxin, and adrenodoxin reductase in mouse ovarian system using high-resolution microscopy. The study has covered thorough expression of these mitochondrial systems in different phases of follicle formation and surrounding interstitial stromal cells. Methods We stained mouse ovary sections at various stages of their folliculogenesis using antisera generated against cytochrome P450scc, Adrenodoxin, and Adrenodoxin reductase enzymes. We also stained ovary sections with phalloidin conjugates 488 and WGA-Lectin conjugates 555 to identify different stages of folliculogenesis/ovulation cycle. Results The current study showed staining for lectin binding moieties and phalloidin on the zona pellucida region of early-preantral and antral follicles. We identified the expression of P450scc, adrenodoxin, and adrenodoxin reductase enzymes in thecal cells of primordial follicles, primary follicles, secondary follicles, and interstitial stromal cells. Conclusion The information generated here might be useful in identifying specific role of cytochrome P450scc expressing cells in Addison's disease and autoimmune primary ovarian insufficiency. Cytochrome P450scc Ferredoxin Ferredoxin reductase follicular granulosa cells thecal cells Leydig cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background In mouse, estrous cycle is divided into four stages (proestrus, estrus, metestrus, and diestrus) which repeat for every 4–5 days unless there is interruption by pregnancy, pseudo-pregnancy, or anestrus ( 1 ). Mouse ovary consists of ovarian follicle cells that convert into oocytes and somatic cells differentiate into granulosa and theca cells. Several evidence indicates that steroidal and non-steroidal factors produced by granulose and theca cells play an important part in development or differentiation of both cell types during folliculogenesis ( 2 ). Cholesterol side chain cleavage enzymes are encoded by gene CYP11A1. The cytochrome P450scc enzyme localized on thecal, stromal, and interstitial cells in ovarian tissue, catalyzes the cleavage of side chain of cholesterol along with the help of adrenodoxin reductase and adrenodoxin which shuttles electrons to cytochrome P450scc. Adrenodoxin reductase is a member of flavoproteins reductase and adrenodoxin is a member of iron-sulphoprotein ( 3 – 5 ). The subcellular localization of cytochome p450scc was found within the mitochondrial space of theca interna cells and corpus luteum cells as labeled using the immunogold method ( 6 ). In subclutured granulose cells transformed with Ha-ras oncogene (PO-GRS cell line) also showed mitochondrial localization of cytochrome p450scc, adrenodoxin, and adrenodoxin reductase enzymes using electron microscopic technique ( 7 ). Although, none of these techniques have severe flaws and lack clear in situ visualization of these enzymes using specific probes. In the current research article, first, we mapped cellular architecture of ovary tissue and then, examined in-situ localization of mitochondrial cytochrome p450scc, adrenodoxin, and adrenodoxin reductase enzymes using high resolution microscopy imaging. Materials and methods a.) Antibodies and fluorescent tags The antisera against Adrenodoxin, Adrenodoxin reductase, and cytochrome P450scc were generated in the laboratory of Prof. Israel Hanukoglu ( 8 ). The secondary antisera Alexa Fluor 555 goat anti-rabbit IgG (H + L) (A21428) was purchased from the Life Technologies. The CF488A conjugates phalloidin (BTM-00042) and CF-555 conjugate wheat-germ-agglutinin (WGA) (BTM-29076-1) were purchased from Biotium Inc. The FITC conjugate WGA (L4895-2MG) was purchased from Sigma-Aldrich. b.) Animals Three-months-old Sabra female mice were housed according to the standard laboratory conditions with ab-libitum access to food and water. The study protocol was approved by the institutional animal ethics committee of Ariel University (Ariel, Israel) (permit 32_12733_b019), according to the Ministry of Health guidelines. The ovaries were surgically removed immediately after CO2 asphyxiation of each animal. The samples were kept in 4% paraformaldehyde in phosphate buffered saline (PBS) (10 mM potassium phosphate, pH 7.4, and 150mM NaCl) overnight for immunofluorescence studies. Samples were then transferred to 30% sucrose and kept at 4°C for at least 24 h. Tissues were later embedded in OCT compound (Tissue-Tek, Sakura, Netherlands) and stored at − 80°C. c.) Cryotomy and immunofluorescence staining The tissue blocks were sliced to 30 µm thick-sections on a cryostat (Leica Jung Frigocut 2000, Wetzlar, Germany) at − 25°C and collected in PBS containing 0.1% sodium azide. For immunofluorescence labeling, tissues sections were permeabilized with 0.1% Tween-20 (Sigma-Aldrich) in PBS for 10 min and washed three times in PBS for 5 min each. The sections were blocked in 300 µl 4% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 20 min and incubated at room temperature for 1 h separately with different antisera of anti-adrenodoxin, anti-adrenodoxin reductase, and anti-cytochrome P450scc (dilution 1:50) in PBS containing 2% BSA. Sections were then washed six times in PBS (5 min each) and incubated with the secondary antiserum goat-anti-rabbit IgG (dilution 1:200) in PBS containing 2% BSA for overnight at 4°C. After six washes for 5 minutes each with PBS, nuclei were stained using the DAPI (4’6-diamidino-2-phenylindole) for 2 min. Unless otherwise indicated, all the steps were carried out at room temperature. In control experiments, the same protocol was followed except primary antiserum that was either replaced by normal rabbit serum or omitted from the reactions. In such control slides, only the blue DAPI staining was visible. To stain the actin filaments, tissue slices reacted with CF488A phalloidin conjugate (dilution 1:20) in PBS for 45 min at room temperature. The lectin binding sites in the sections were stained using the FITC conjugate WGA and CF-555 conjugate WGA (dilution 5:1000) in PBS for 30–45 min. The sections were then transferred onto x-tra Adhesive slides (Leica Biosystems, Peterborough, UK), mounted with anti-fade reagent glycerol containing n-propyl gallate (Sigma-Aldrich) in 100mM phosphate buffer (pH 7.2). Experiments were performed at least thrice with independent results. d.) Confocal microscopy High-resolution fluorescent images were acquired using an LSM 700 confocal microscope (Carl-Zeiss, Germany). The laser diodes used in the image acquisition were 405 nm for DAPI, 488 nm for CF488A, 555 nm for Alexa Fluor 555 and DyLight ™ 554. The fluorescence and bright-field illumination modes were used during the image acquisition process. Samples were visualized through LCI Plan-Apochromat 25x/0.8, EC Plan-Neofluar 40x/1.30, and Plan-Apochrom at 63x/1.40 oil objective lenses. The composite images were generated using the tile-scan image overview mode. Results The current study has identified subcellular localization of cytochrome P450scc, adrenodoxin, and adrenodoxin reductase enzymes in mouse ovary sections. We described all results in only one section presented below. a. Actin and lectin localization Actin filaments refer to the fundamental cytoskeleton organizing structural part of cells and tissues, formed by polymerization of actin moieties that regulate cellular functions such as cell migration, cell adhesion, and cell protrusion formations. Actin polymerization adapts different conformational changes, from densely packed lamellipodia to large and contracting bundles in mesenchymal cells ( 9 , 10 ). Actin filaments have also known to play vital roles in oocyte maturation and role of F-actin has been confirmed in chromosome segregation during oocyte meiosis ( 11 ). Moreover, actin network has been linked to long-range vesicle transport, nuclear repositioning, spindle migration, and anchorage in mammalian oocytes ( 12 ). We stained 30µm thick tissue-sections with CF-555 conjugate wheat-germ-agglutinin and Alexa-488 phalloidin (Phalloidin, a class of toxins belongs to phallotoxins, found on death-cap mushrooms) to stain actin filaments across the ovary-sections (Fig. 1 ). We observed staining for lectin binding moieties and phalloidin on the zona pellucida region of early-preantral and antral follicles (Fig. 1 A, B). However, we could not find lectin binding staining in late atretic follicles (Fig. 1 A). In phalloidin staining along with DAPI has clearly explained different phases of ovarian follicle development (Fig. 1 B, C, and D). The current image shows primordial and primary follicles, early preantral and antral phase, and late atretic follicles at their development stages. b. Cytochrome P450scc Several enzymes from the P450 family are expressed in mice gonads. Some of them are involved in steroidogenic hormone biosynthesis (Cyp11a and Cyp17a1) and few of them are involved in metabolizing the xenobiotic compounds ( 13 , 14 ). Three Cyp proteins (Cyp2b19, 3a57, and 4f39) were found to be highly expressed in mouse testis and Cyp1b1 in ovaries ( 15 , 16 ). Cyp11a1 gene belonging to the family of 11 of cytochrome P450 catalyze crucial step in the conversion of cholesterol to pregnenolone in developing avian granulosa cells ( 17 , 18 ). In this research article, we stained mouse ovary sections with Cytochrome P450scc antibodies along with phalloidin. We found strong staining of phalloidin in the zona pellucida region of primary follicles (Fig. 2 B). We identified the expression of P450scc enzymes in thecal cells of primordial follicles, primary follicles, secondary follicles, and interstitial stromal cells, but negative for granulosa cells of mouse ovary cross-sections (Fig. 2 C, D). However, we found expressions of cytP450scc enzyme in follicular thecal and follicular granulosa cells of corpora lutea (Fig. 3 A, B). Corpora lutea is a source of the hormone progesterone and it is formed once the ovum is released from follicles in ovary ( 19 ). c. Adrenodoxin reductase and Adrenodoxin Adrenodoxin reductase is a monomeric 51kDa flavoenzyme that is used in all steroidogenic tissues and serves as the first electron transfer protein in all the mitochondrial P450 system including side chain cleavage. Adrenodoxin reductase receives a two-electron package from NADPH and converts it into two single electrons via adrenodoxin to mitochondrial cytochromes P450 ( 20 ). In our current study, we stained mouse 25µm thick ovary sections with adrenodoxin reductase and adrenodoxin antibodies. We observed staining of adrenodoxin reductase and adrenodoxin antibodies in thecal cells and interstitial stromal cells lining the follicles around (Fig. 4 B, C and Fig. 5 B, C). These staining patterns were similar to cytochrome P450scc staining in mouse ovary sections. However, we could not observe staining in granulosa cells of either of these follicles that were present at different stages. To cross-check specificity of these steroidogenic mitochondrial enzymes, we stained mouse testicular parenchyma with adrenodoxin antibodies at different stages of sperm maturation. We found that adrenodoxin staining was present only in interstitial cells (Fig. 6 C, D) of mouse testicular sections. However interstitial cells present in testicular parenchyma comprised of Leydig cells, immune cells, and fibroblasts ( 21 ). In our high-resolution images of testicular parenchyma stained for adrenodoxin resulted specifically staining of Leydig cells (Fig. 7 C, D). These results were confirmatory of expression of Cytochrome P450scc, Adrenodoxin reductase, and Adrenodoxin, all parts of steroidogenic mitochondrial system, in the gonadal cells that are involved in steroid biosynthesis. Discussion Cytochrome P450scc is a classical cholesterol side chain cleavage protein that removes six carbons of the side chain of cholesterol and converts into pregnenolone, the first rate-limiting step in mammalian steroid biosynthesis ( 22 , 23 ) along with adrenodoxin and adrenodoxin reductase. These mitochondrial enzymes are present on an integral mitochondrial membrane while adrenodoxin is predominantly located on inner mitochondrial membrane as evident by electron microscope ( 7 ). Adrenodoxin that transfers electrons to cytochrome P450scc has been widely studied and highly essential for steroidal biosynthesis ( 24 ). Ovaries secret multiple steroid hormones such as pregnenolone, progesterone, 17α-progesterone, androstenedione, testosterone, estrone, and estradiol that all depend on the estrus cycle. Two somatic cell types, thecal and follicular granulosa cells produce steroid hormones, but interstitial stromal cells also stained positive for cytP450scc enzyme (Fig. 2 D, 4 C, 5 C) and already known to produce steroids in epithelial ovarian tumor cells ( 25 ). The human genome contains a total of 57 P450 genes categorized into two classes: type I and type II. Type I enzymes localized on mitochondrial membrane receive electrons from NADPH mediated by ferredoxin and ferredoxin reductase. Type II enzymes are located on endoplasmic reticulum and receive electrons from NADPH mediated by P450 oxidoreductase. CytP450scc expression is required for steroids production but also considered as antigens associated with Addison's disease and autoimmune primary ovarian insufficiency (POI) ( 26 ). Hence, we believe that current study will be important and highly useful in determining cytP450 system’s role in disease and several other regulatory aspects of steroid biosynthesis. Conclusion The current study evaluates the localization of cytochrome p450scc, Adrenodoxin, and Adrenodoxin reductase (rate-limiting enzymes) involved in the biosynthesis of steroids production in mammalian gamut. Results clearly indicate that identifying cell-types in mouse ovary tissues that express these enzymes would be greatly beneficial in understanding the pathogenesis of autoimmune disorder of steroids producing cells such as autoimmune oophoritis. Abbreviations CYP11A1 : cytochrome P450 11A1 Ha-ras : Harvey Rat sarcoma virus WGA : Wheat-Germ-Agglutinin (WGA) FITC : Fluorescein IsothioCyanate PBS : Phosphate Buffered Saline BSA : Bovine Serum Albumin (BSA) DAPI : 4',6-diamidino-2-phenylindole NADPH : Nicotinamide Adenine Dinucleotide Phosphate Declarations a. Ethical approval and consent to participate For animal use in research conducted for this study, ethical approval was sought from institutional animal ethics committee of Ariel University (Ariel, Israel) (permit 32_12733_b019), according to the Ministry of Health guidelines. This study does not involve the usage of any human participants and human-related clinical trials. b. Consent for publication This section does not apply to the work carried out in this manuscript. c. Availability of data and materials Data related to this study are available upon request from the corresponding author. d. Competing interests The authors have declared that there are no competing interests to be found and no other disclosure to be announced. e. Funding interests The study has been partially funded by Ariel University Research Grants, Israel. f. Authors contribution SS has conceptualized study, designed experiments, and prepared figures and drafted the manuscript. KK has edited and reviewed manuscript-draft. g. Acknowledgements We would like to thank Dr. Girishkumar Kaitholil Kumaran (Scientist – Oxford University) for the fruitful discussion during the data generation and data presentation. This research article is dedicated to Prof. Israel Hanukoglu for his contributions to steroid biochemistry and his work to produce antibodies against enzymes (CytP450scc, Adrenodoxin, and Adrenodoxin reductase). References Byers SL, Wiles M V., Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One. 2012 Apr 13;7(4). McGee EA, Strauss JF. Ovarian Hormone Synthesis. Endocrinology: Adult and Pediatric, Sixth Edition. 2010 Jan 1;2:2302–17. Strauss JF. The synthesis and metabolism of steroid hormones. Yen & Jaffe’s Reproductive Endocrinology: Expert Consult - Online and Print. 2009 May 27;79–104. Litwack G. Steroid hormones: chemistry, biosynthesis, and metabolism. Hormones. 2022;29–55. 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Available from: https://www.nature.com/articles/modpathol2016219 Tong Z Bin, 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 [Internet]. 2022 Aug 1 [cited 2025 Jan 22];29(8):2391–400. Available from: https://pubmed.ncbi.nlm.nih.gov/35585293/ Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6122209","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":422254516,"identity":"8e69c09b-461c-406c-99a3-9199998aa59b","order_by":0,"name":"Sachin 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cross-sectional part of mouse ovary. \u003cstrong\u003eC\u003c/strong\u003e) DAPi staining of cell nuclei. \u003cstrong\u003eD\u003c/strong\u003e) A merge image of sections A, B, and C.\u003c/p\u003e","description":"","filename":"Figure1Actinandlectin.pdf.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/e796221ab5bc8e7238881dbc.png"},{"id":77635868,"identity":"052d44c8-b1d6-4bd6-916a-267df5daa17f","added_by":"auto","created_at":"2025-03-03 18:37:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3746724,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of cytochrome P450scc expression in mouse ovary follicles along with lectin staining. \u003cstrong\u003eA\u003c/strong\u003e) The red color staining shows specific localization of cytochrome P450scc enzymes within the thecal cells and interstitial stromal cells. \u003cstrong\u003eB\u003c/strong\u003e) Lectin localization on the zona pellucida region of secondary follicle. \u003cstrong\u003eC\u003c/strong\u003e) DAPi staining the nuclei. \u003cstrong\u003eD\u003c/strong\u003e) A merge image of sections A, B, and C.\u003c/p\u003e","description":"","filename":"Figure2cytP450.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/9739ed3d87904bcc619ff276.png"},{"id":77636343,"identity":"b9989835-8727-4cf4-b29c-626b2e1a0646","added_by":"auto","created_at":"2025-03-03 18:45:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4113243,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of cytochrome P450scc expression in corpora lutea. \u003cstrong\u003eA\u003c/strong\u003e) Cytochrome P450 localization in follicular granulosa cells. \u003cstrong\u003eB\u003c/strong\u003e) DAPi staining the cell nuclei. \u003cstrong\u003eC\u003c/strong\u003e) A merge image of A and B sections.\u003c/p\u003e","description":"","filename":"Figure3cytP450.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/a3535af05f74219ab326d3cb.png"},{"id":77636346,"identity":"38a88c4e-41a8-4eb0-a868-08a87a4c0a6b","added_by":"auto","created_at":"2025-03-03 18:45:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2468847,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of mouse ovary cross-section stained with adrenodoxin reductase antibodies. \u003cstrong\u003eA\u003c/strong\u003e) DAPi staining the mouse ovary cross-section. \u003cstrong\u003eB\u003c/strong\u003e) Adrenodoxin reductase staining was observed mostly on thecal cells and interstitial cells. \u003cstrong\u003eC\u003c/strong\u003e) A merge image of sections A and B.\u003c/p\u003e","description":"","filename":"Figure4Adxredu.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/cc6ab76cde106aa4b896db83.png"},{"id":77635870,"identity":"5e467a5e-e1c4-4c7a-b01d-1ad438fedb01","added_by":"auto","created_at":"2025-03-03 18:37:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2374203,"visible":true,"origin":"","legend":"\u003cp\u003eAn image of mouse ovary cross-section stained with adrenodoxin antibodies. \u003cstrong\u003eA\u003c/strong\u003e) DAPi staining the mouse ovary cross-section. \u003cstrong\u003eB\u003c/strong\u003e) Adrenodoxin staining was observed mostly on thecal cells of mouse ovary follicles and interstitial stromal cells. \u003cstrong\u003eC\u003c/strong\u003e) A merge image of sections A and B.\u003c/p\u003e","description":"","filename":"Figure5Adx.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/7088e6cf850aa7ee108ec846.png"},{"id":77635872,"identity":"e2ef7c5b-8b6c-4af8-8ff7-114dd03df457","added_by":"auto","created_at":"2025-03-03 18:37:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3704326,"visible":true,"origin":"","legend":"\u003cp\u003eA comprehensive image of mouse testicular parenchyma. \u003cstrong\u003eA\u003c/strong\u003e) DAPi staining. \u003cstrong\u003eB\u003c/strong\u003e) Actin localization in mouse testes cross-section showing different stages of sperm development. \u003cstrong\u003eC\u003c/strong\u003e) Adrenodoxin staining the Leydig cells present within the interstitial cells surrounding the seminiferous tubules. \u003cstrong\u003eD\u003c/strong\u003e) A merge image of sections A, B, and C.\u003c/p\u003e","description":"","filename":"Figure6AdxMustestis.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/f2fd83bb444a26b7ae9bd517.png"},{"id":77636345,"identity":"a8ef9ee1-9165-44fe-aa52-860a5ab72c27","added_by":"auto","created_at":"2025-03-03 18:45:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2884367,"visible":true,"origin":"","legend":"\u003cp\u003eA high-resolution image of adrenodoxin localization in Leydig cells in the population of interstitial cells comprising of fibroblasts, macrophage, and major steroid enzyme producing Leydig cells.\u003c/p\u003e","description":"","filename":"Figure7AdxMustestiszoom.png","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/e48f90f1307336663e62bf52.png"},{"id":79023445,"identity":"712a640f-a2b5-485c-b545-1b9759f00dc8","added_by":"auto","created_at":"2025-03-22 18:46:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36503870,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6122209/v1/7c97923f-d3ad-44dd-b180-e29b5e31cd1a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immune-labeling of cytochrome P450scc and its associate electron transferring enzymes in mouse ovary tissue-sections","fulltext":[{"header":"Background","content":"\u003cp\u003eIn mouse, estrous cycle is divided into four stages (proestrus, estrus, metestrus, and diestrus) which repeat for every 4\u0026ndash;5 days unless there is interruption by pregnancy, pseudo-pregnancy, or anestrus (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Mouse ovary consists of ovarian follicle cells that convert into oocytes and somatic cells differentiate into granulosa and theca cells. Several evidence indicates that steroidal and non-steroidal factors produced by granulose and theca cells play an important part in development or differentiation of both cell types during folliculogenesis (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCholesterol side chain cleavage enzymes are encoded by gene CYP11A1. The cytochrome P450scc enzyme localized on thecal, stromal, and interstitial cells in ovarian tissue, catalyzes the cleavage of side chain of cholesterol along with the help of adrenodoxin reductase and adrenodoxin which shuttles electrons to cytochrome P450scc. Adrenodoxin reductase is a member of flavoproteins reductase and adrenodoxin is a member of iron-sulphoprotein (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The subcellular localization of cytochome p450scc was found within the mitochondrial space of theca interna cells and corpus luteum cells as labeled using the immunogold method (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). In subclutured granulose cells transformed with Ha-ras oncogene (PO-GRS cell line) also showed mitochondrial localization of cytochrome p450scc, adrenodoxin, and adrenodoxin reductase enzymes using electron microscopic technique (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Although, none of these techniques have severe flaws and lack clear in situ visualization of these enzymes using specific probes.\u003c/p\u003e \u003cp\u003eIn the current research article, first, we mapped cellular architecture of ovary tissue and then, examined in-situ localization of mitochondrial cytochrome p450scc, adrenodoxin, and adrenodoxin reductase enzymes using high resolution microscopy imaging.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ea.) Antibodies and fluorescent tags\u003c/h2\u003e \u003cp\u003eThe antisera against Adrenodoxin, Adrenodoxin reductase, and cytochrome P450scc were generated in the laboratory of Prof. Israel Hanukoglu (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The secondary antisera Alexa Fluor 555 goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (A21428) was purchased from the Life Technologies. The CF488A conjugates phalloidin (BTM-00042) and CF-555 conjugate wheat-germ-agglutinin (WGA) (BTM-29076-1) were purchased from Biotium Inc. The FITC conjugate WGA (L4895-2MG) was purchased from Sigma-Aldrich.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eb.) Animals\u003c/h3\u003e\n\u003cp\u003eThree-months-old Sabra female mice were housed according to the standard laboratory conditions with \u003cem\u003eab-libitum\u003c/em\u003e access to food and water. The study protocol was approved by the institutional animal ethics committee of Ariel University (Ariel, Israel) (permit 32_12733_b019), according to the Ministry of Health guidelines. The ovaries were surgically removed immediately after CO2 asphyxiation of each animal. The samples were kept in 4% paraformaldehyde in phosphate buffered saline (PBS) (10 mM potassium phosphate, pH 7.4, and 150mM NaCl) overnight for immunofluorescence studies. Samples were then transferred to 30% sucrose and kept at 4\u0026deg;C for at least 24 h. Tissues were later embedded in OCT compound (Tissue-Tek, Sakura, Netherlands) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003ec.) Cryotomy and immunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eThe tissue blocks were sliced to 30 \u0026micro;m thick-sections on a cryostat (Leica Jung Frigocut 2000, Wetzlar, Germany) at \u0026minus;\u0026thinsp;25\u0026deg;C and collected in PBS containing 0.1% sodium azide. For immunofluorescence labeling, tissues sections were permeabilized with 0.1% Tween-20 (Sigma-Aldrich) in PBS for 10 min and washed three times in PBS for 5 min each. The sections were blocked in 300 \u0026micro;l 4% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS for 20 min and incubated at room temperature for 1 h separately with different antisera of anti-adrenodoxin, anti-adrenodoxin reductase, and anti-cytochrome P450scc (dilution 1:50) in PBS containing 2% BSA. Sections were then washed six times in PBS (5 min each) and incubated with the secondary antiserum goat-anti-rabbit IgG (dilution 1:200) in PBS containing 2% BSA for overnight at 4\u0026deg;C. After six washes for 5 minutes each with PBS, nuclei were stained using the DAPI (4\u0026rsquo;6-diamidino-2-phenylindole) for 2 min.\u003c/p\u003e \u003cp\u003eUnless otherwise indicated, all the steps were carried out at room temperature. In control experiments, the same protocol was followed except primary antiserum that was either replaced by normal rabbit serum or omitted from the reactions. In such control slides, only the blue DAPI staining was visible. To stain the actin filaments, tissue slices reacted with CF488A phalloidin conjugate (dilution 1:20) in PBS for 45 min at room temperature. The lectin binding sites in the sections were stained using the FITC conjugate WGA and CF-555 conjugate WGA (dilution 5:1000) in PBS for 30\u0026ndash;45 min.\u003c/p\u003e \u003cp\u003eThe sections were then transferred onto x-tra Adhesive slides (Leica Biosystems, Peterborough, UK), mounted with anti-fade reagent glycerol containing n-propyl gallate (Sigma-Aldrich) in 100mM phosphate buffer (pH 7.2). Experiments were performed at least thrice with independent results.\u003c/p\u003e\n\u003ch3\u003ed.) Confocal microscopy\u003c/h3\u003e\n\u003cp\u003eHigh-resolution fluorescent images were acquired using an LSM 700 confocal microscope (Carl-Zeiss, Germany). The laser diodes used in the image acquisition were 405 nm for DAPI, 488 nm for CF488A, 555 nm for Alexa Fluor 555 and DyLight\u003csup\u003e\u0026trade;\u003c/sup\u003e 554. The fluorescence and bright-field illumination modes were used during the image acquisition process. Samples were visualized through LCI Plan-Apochromat 25x/0.8, EC Plan-Neofluar 40x/1.30, and Plan-Apochrom at 63x/1.40 oil objective lenses. The composite images were generated using the tile-scan image overview mode.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe current study has identified subcellular localization of cytochrome P450scc, adrenodoxin, and adrenodoxin reductase enzymes in mouse ovary sections. We described all results in only one section presented below.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ea. Actin and lectin localization\u003c/h2\u003e \u003cp\u003eActin filaments refer to the fundamental cytoskeleton organizing structural part of cells and tissues, formed by polymerization of actin moieties that regulate cellular functions such as cell migration, cell adhesion, and cell protrusion formations. Actin polymerization adapts different conformational changes, from densely packed lamellipodia to large and contracting bundles in mesenchymal cells (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Actin filaments have also known to play vital roles in oocyte maturation and role of F-actin has been confirmed in chromosome segregation during oocyte meiosis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Moreover, actin network has been linked to long-range vesicle transport, nuclear repositioning, spindle migration, and anchorage in mammalian oocytes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe stained 30\u0026micro;m thick tissue-sections with CF-555 conjugate wheat-germ-agglutinin and Alexa-488 phalloidin (Phalloidin, a class of toxins belongs to phallotoxins, found on death-cap mushrooms) to stain actin filaments across the ovary-sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We observed staining for lectin binding moieties and phalloidin on the zona pellucida region of early-preantral and antral follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). However, we could not find lectin binding staining in late atretic follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In phalloidin staining along with DAPI has clearly explained different phases of ovarian follicle development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C, and D). The current image shows primordial and primary follicles, early preantral and antral phase, and late atretic follicles at their development stages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eb. Cytochrome P450scc\u003c/h3\u003e\n\u003cp\u003eSeveral enzymes from the P450 family are expressed in mice gonads. Some of them are involved in steroidogenic hormone biosynthesis (Cyp11a and Cyp17a1) and few of them are involved in metabolizing the xenobiotic compounds (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Three Cyp proteins (Cyp2b19, 3a57, and 4f39) were found to be highly expressed in mouse testis and Cyp1b1 in ovaries (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Cyp11a1 gene belonging to the family of 11 of cytochrome P450 catalyze crucial step in the conversion of cholesterol to pregnenolone in developing avian granulosa cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this research article, we stained mouse ovary sections with Cytochrome P450scc antibodies along with phalloidin. We found strong staining of phalloidin in the zona pellucida region of primary follicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We identified the expression of P450scc enzymes in thecal cells of primordial follicles, primary follicles, secondary follicles, and interstitial stromal cells, but negative for granulosa cells of mouse ovary cross-sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). However, we found expressions of cytP450scc enzyme in follicular thecal and follicular granulosa cells of corpora lutea (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Corpora lutea is a source of the hormone progesterone and it is formed once the ovum is released from follicles in ovary (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ec. Adrenodoxin reductase and Adrenodoxin\u003c/h3\u003e\n\u003cp\u003eAdrenodoxin reductase is a monomeric 51kDa flavoenzyme that is used in all steroidogenic tissues and serves as the first electron transfer protein in all the mitochondrial P450 system including side chain cleavage. Adrenodoxin reductase receives a two-electron package from NADPH and converts it into two single electrons via adrenodoxin to mitochondrial cytochromes P450 (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our current study, we stained mouse 25\u0026micro;m thick ovary sections with adrenodoxin reductase and adrenodoxin antibodies. We observed staining of adrenodoxin reductase and adrenodoxin antibodies in thecal cells and interstitial stromal cells lining the follicles around (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). These staining patterns were similar to cytochrome P450scc staining in mouse ovary sections. However, we could not observe staining in granulosa cells of either of these follicles that were present at different stages. To cross-check specificity of these steroidogenic mitochondrial enzymes, we stained mouse testicular parenchyma with adrenodoxin antibodies at different stages of sperm maturation. We found that adrenodoxin staining was present only in interstitial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D) of mouse testicular sections. However interstitial cells present in testicular parenchyma comprised of Leydig cells, immune cells, and fibroblasts (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In our high-resolution images of testicular parenchyma stained for adrenodoxin resulted specifically staining of Leydig cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). These results were confirmatory of expression of Cytochrome P450scc, Adrenodoxin reductase, and Adrenodoxin, all parts of steroidogenic mitochondrial system, in the gonadal cells that are involved in steroid biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCytochrome P450scc is a classical cholesterol side chain cleavage protein that removes six carbons of the side chain of cholesterol and converts into pregnenolone, the first rate-limiting step in mammalian steroid biosynthesis (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) along with adrenodoxin and adrenodoxin reductase. These mitochondrial enzymes are present on an integral mitochondrial membrane while adrenodoxin is predominantly located on inner mitochondrial membrane as evident by electron microscope (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Adrenodoxin that transfers electrons to cytochrome P450scc has been widely studied and highly essential for steroidal biosynthesis (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOvaries secret multiple steroid hormones such as pregnenolone, progesterone, 17α-progesterone, androstenedione, testosterone, estrone, and estradiol that all depend on the estrus cycle. Two somatic cell types, thecal and follicular granulosa cells produce steroid hormones, but interstitial stromal cells also stained positive for cytP450scc enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and already known to produce steroids in epithelial ovarian tumor cells (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The human genome contains a total of 57 P450 genes categorized into two classes: type I and type II. Type I enzymes localized on mitochondrial membrane receive electrons from NADPH mediated by ferredoxin and ferredoxin reductase. Type II enzymes are located on endoplasmic reticulum and receive electrons from NADPH mediated by P450 oxidoreductase. CytP450scc expression is required for steroids production but also considered as antigens associated with Addison's disease and autoimmune primary ovarian insufficiency (POI) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Hence, we believe that current study will be important and highly useful in determining cytP450 system\u0026rsquo;s role in disease and several other regulatory aspects of steroid biosynthesis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study evaluates the localization of cytochrome p450scc, Adrenodoxin, and Adrenodoxin reductase (rate-limiting enzymes) involved in the biosynthesis of steroids production in mammalian gamut. Results clearly indicate that identifying cell-types in mouse ovary tissues that express these enzymes would be greatly beneficial in understanding the pathogenesis of autoimmune disorder of steroids producing cells such as autoimmune oophoritis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCYP11A1 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: cytochrome P450 11A1\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHa-ras \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;:\u0026nbsp;\u003c/em\u003eHarvey Rat sarcoma virus\u003c/p\u003e\n\u003cp\u003eWGA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: Wheat-Germ-Agglutinin (WGA)\u003c/p\u003e\n\u003cp\u003eFITC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: Fluorescein IsothioCyanate\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: Phosphate Buffered Saline\u003c/p\u003e\n\u003cp\u003eBSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Bovine Serum Albumin (BSA)\u003c/p\u003e\n\u003cp\u003eDAPI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;: 4\u0026apos;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eNADPH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;: Nicotinamide Adenine Dinucleotide Phosphate\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003ea. \u0026nbsp;Ethical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor animal use in research conducted for this study, ethical approval was sought from institutional animal ethics committee of Ariel University (Ariel, Israel) (permit 32_12733_b019), according to the Ministry of Health guidelines. This study does not involve the usage of any human participants and human-related clinical trials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis section does not apply to the work carried out in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec.\u0026nbsp;\u0026nbsp;Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData related to this study are available upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed.\u0026nbsp;Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that there are no competing interests to be found and no other disclosure to be announced.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee. \u0026nbsp;Funding interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been partially funded by Ariel University Research Grants, Israel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef.\u0026nbsp; \u0026nbsp;Authors contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSS has conceptualized study, designed experiments, and prepared figures and drafted the manuscript. KK has edited and reviewed manuscript-draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg.\u0026nbsp;Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Girishkumar Kaitholil Kumaran (Scientist \u0026ndash; Oxford University) for the fruitful discussion during the data generation and data presentation. This research article is dedicated to Prof. Israel Hanukoglu for his contributions to steroid biochemistry and his work to produce antibodies against enzymes (CytP450scc, Adrenodoxin, and Adrenodoxin reductase).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eByers SL, Wiles M V., Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One. 2012 Apr 13;7(4). \u003c/li\u003e\n\u003cli\u003eMcGee EA, Strauss JF. Ovarian Hormone Synthesis. Endocrinology: Adult and Pediatric, Sixth Edition. 2010 Jan 1;2:2302\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eStrauss JF. 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Available from: https://europepmc.org/article/med/11691658\u003c/li\u003e\n\u003cli\u003eHanukoglu I, Suh BS, Himmelhoch S, Amsterdam A. Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. Journal of Cell Biology [Internet]. 1990 Oct 1 [cited 2025 Jan 20];111(4):1373\u0026ndash;81. Available from: https://dx.doi.org/10.1083/jcb.111.4.1373\u003c/li\u003e\n\u003cli\u003eHANUKOGLU 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\u0026ndash;31. \u003c/li\u003e\n\u003cli\u003eSvitkina TM, Borisy GG. Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia. Journal of Cell Biology. 1999 May 31;145(5):1009\u0026ndash;26. \u003c/li\u003e\n\u003cli\u003ePollard TD, Borisy GG. Cellular Motility Driven by Assembly and Disassembly of Actin Filaments. Cell. 2003 Feb 21;112(4):453\u0026ndash;65. \u003c/li\u003e\n\u003cli\u003eDunkley S, Scheffler K, Mogessie B. Cytoskeletal form and function in mammalian oocytes and zygotes. Curr Opin Cell Biol. 2022 Apr 1;75:102073. \u003c/li\u003e\n\u003cli\u003eUraji J, Scheffler K, Schuh M. Functions of actin in mouse oocytes at a glance. J Cell Sci [Internet]. 2018 Nov 1 [cited 2025 Jan 20];131(22). Available from: https://dx.doi.org/10.1242/jcs.218099\u003c/li\u003e\n\u003cli\u003eChoudhary 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 [Internet]. 2003 Jun 1 [cited 2025 Jan 21];414(1):91\u0026ndash;100. Available from: https://europepmc.org/article/MED/12745259\u003c/li\u003e\n\u003cli\u003eShimada T, Sugie A, Shindo M, Nakajima T, Azuma E, Hashimoto 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 [Internet]. 2003 Feb 1 [cited 2025 Jan 21];187(1):1\u0026ndash;10. Available from: https://europepmc.org/article/MED/12628579\u003c/li\u003e\n\u003cli\u003eRajapaksa KS, Sipes IG, Hoyer PB. involvement of microsomal epoxide hydrolase enzyme in ovotoxicity caused by 7,12-dimethylbenz[a]anthracene. Toxicol Sci [Internet]. 2007 Jan 4 [cited 2025 Jan 21];96(2):327\u0026ndash;34. Available from: https://europepmc.org/article/MED/17204581\u003c/li\u003e\n\u003cli\u003eRenaud HJ, Cui JY, Khan M, Klaassen CD. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice. Toxicol Sci [Internet]. 2011 Sep 13 [cited 2025 Jan 21];124(2):261\u0026ndash;77. Available from: https://europepmc.org/articles/PMC3216415\u003c/li\u003e\n\u003cli\u003eXu Q, Song Y, Chen Y, Liu R, Zhang Y, Li 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 [Internet]. 2015 Dec 22 [cited 2025 Jan 21];6(1):54. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4688999/\u003c/li\u003e\n\u003cli\u003eTilly 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 [Internet]. 1991 Dec 1 [cited 2025 Jan 21];45(6):967\u0026ndash;74. 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Essential Roles of Interstitial Cells in Testicular Development and Function. Andrology [Internet]. 2019 Jul 1 [cited 2025 Jan 22];8(4):903. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC7036326/\u003c/li\u003e\n\u003cli\u003eMcCarty 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. Journal of Biological Chemistry [Internet]. 2024 Jan 1 [cited 2025 Jan 22];300(1):105495. Available from: http://www.jbc.org/article/S0021925823025231/fulltext\u003c/li\u003e\n\u003cli\u003eMiller WL. Steroid hormone synthesis in mitochondria. Mol Cell Endocrinol [Internet]. 2013 Oct 15 [cited 2025 Jan 22];379(1\u0026ndash;2):62\u0026ndash;73. Available from: https://pubmed.ncbi.nlm.nih.gov/23628605/\u003c/li\u003e\n\u003cli\u003eHanukoglu I, Jefcoate CR. Mitochondrial cytochrome P-450scc. Mechanism of electron transport by adrenodoxin. J Biol Chem [Internet]. 1980 Apr 10 [cited 2025 Jan 22];255(7):3057\u0026ndash;61. Available from: https://pubmed.ncbi.nlm.nih.gov/6766943/\u003c/li\u003e\n\u003cli\u003eBlanco LZ, 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. Modern Pathology 2017 30:4 [Internet]. 2017 Jan 6 [cited 2025 Jan 22];30(4):563\u0026ndash;76. Available from: https://www.nature.com/articles/modpathol2016219\u003c/li\u003e\n\u003cli\u003eTong Z Bin, 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 [Internet]. 2022 Aug 1 [cited 2025 Jan 22];29(8):2391\u0026ndash;400. Available from: https://pubmed.ncbi.nlm.nih.gov/35585293/\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cytochrome P450scc, Ferredoxin, Ferredoxin reductase, follicular granulosa cells, thecal cells, Leydig cells","lastPublishedDoi":"10.21203/rs.3.rs-6122209/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6122209/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSteroid biosynthesis is mediated by cytochrome P450scc enzyme, a rate-limiting step involved in the conversion of cholesterol to pregnenolone in gonads. The cytochrome P450scc enzyme is accompanied by electron transferring enzyme adrenodoxin and adrenodoxin reductase, all present on mitochondrial integral membrane. This research article basically reveals the sites of expression of cytochrome P450scc, adrenodoxin, and adrenodoxin reductase in mouse ovarian system using high-resolution microscopy. The study has covered thorough expression of these mitochondrial systems in different phases of follicle formation and surrounding interstitial stromal cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe stained mouse ovary sections at various stages of their folliculogenesis using antisera generated against cytochrome P450scc, Adrenodoxin, and Adrenodoxin reductase enzymes. We also stained ovary sections with phalloidin conjugates 488 and WGA-Lectin conjugates 555 to identify different stages of folliculogenesis/ovulation cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study showed staining for lectin binding moieties and phalloidin on the zona pellucida region of early-preantral and antral follicles. We identified the expression of P450scc, adrenodoxin, and adrenodoxin reductase enzymes in thecal cells of primordial follicles, primary follicles, secondary follicles, and interstitial stromal cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe information generated here might be useful in identifying specific role of cytochrome P450scc expressing cells in Addison's disease and autoimmune primary ovarian insufficiency.\u003c/p\u003e","manuscriptTitle":"Immune-labeling of cytochrome P450scc and its associate electron transferring enzymes in mouse ovary tissue-sections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 18:37:38","doi":"10.21203/rs.3.rs-6122209/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2169a24-8a64-4eb1-b522-28b8c25bd9ad","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-22T18:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 18:37:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6122209","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6122209","identity":"rs-6122209","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}