{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T23:20:39Z","timestamp":1791415239487,"version":"4.3.3"},"reference-count":50,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T00:00:00Z","timestamp":1764115200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2022YFC2704600"],"award-info":[{"award-number":["2022YFC2704600"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81741038"],"award-info":[{"award-number":["81741038"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Central Government Guiding Local Science and Technology Development Foundation of Shandong Province","award":["YDZX2022096"],"award-info":[{"award-number":["YDZX2022096"]}]},{"DOI":"10.13039\/501100002858","name":"China Postdoctoral Science Foundation","doi-asserted-by":"publisher","award":["2023M742109"],"award-info":[{"award-number":["2023M742109"]}],"id":[{"id":"10.13039\/501100002858","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Infertility affects about 17.5% of couples, with male factors accounting for approximately 50% of cases. Cytoskeletal remodeling is increasingly recognized as a critical component of male reproductive function, particularly in the regulation of testosterone synthesis by Leydig cells. However, the underlying molecular mechanisms remain poorly defined. Rho-associated coiled-coil-containing kinase (ROCK), a key cytoskeletal regulator, influences actin dynamics, impacting intracellular trafficking. In this study, we investigated the roles of ROCK1 and ROCK2 in Leydig cells using the TM3 cell model. Pharmacological inhibition of ROCK activity with Y-27632 impaired actin cytoskeleton organization, reduced the phosphorylation of LIMK, COFILIN, and MLC2, and disrupted the colocalization of F-actin with StAR and cholesterol, thereby decreasing testosterone production. Furthermore, RNA-seq revealed that hCG promotes transcription of steroidogenesis-related genes, while ROCK inhibition reverses this effect. Silencing of ROCK1 via siRNA mimicked the effects of ROCK-i, suppressing steroidogenic gene expression and testosterone synthesis. In contrast, ROCK2 knockdown enhanced testosterone secretion, promoted F-actin remodeling, and increased traffic of cholesterol targeting mitochondria. These opposing effects triggered distinct responses in the SCAP\u2013SREBP2 axis, indicating a feedback mechanism regulating cholesterol homeostasis. Collectively, our findings uncover the isoform-specific roles of ROCK1 and ROCK2 in coordinating cytoskeletal dynamics and steroidogenic activity, providing new insights into the regulation of male reproductive endocrinology and identifying potential therapeutic targets for androgen deficiency and male infertility.<\/jats:p>","DOI":"10.3390\/cells14231868","type":"journal-article","created":{"date-parts":[[2025,11,26]],"date-time":"2025-11-26T13:56:34Z","timestamp":1764165394000},"page":"1868","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Deciphering the Contribution of ROCK-Dependent Actin Cytoskeleton Remodeling to Testosterone Production in Mouse Leydig Cells"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-4348-6457","authenticated-orcid":false,"given":"Ke","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China"},{"name":"Shandong Key Laboratory of Reproductive Research and Birth Defect Prevention, Shandong First Medical University, Jinan 250021, China"},{"name":"Jinan Engineering Laboratory of Reproductive Diagnosis and Treatment Technology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengze","family":"Sun","sequence":"additional","affiliation":[{"name":"School of Clinical and Basic Medicine, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan 250117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuwei","family":"Hu","sequence":"additional","affiliation":[{"name":"School of Basic Medical Sciences, Shandong University, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5494-8460","authenticated-orcid":false,"given":"Ning","family":"Hou","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shan","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China"},{"name":"Shandong Key Laboratory of Reproductive Research and Birth Defect Prevention, Shandong First Medical University, Jinan 250021, China"},{"name":"Jinan Engineering Laboratory of Reproductive Diagnosis and Treatment Technology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengzi","family":"Huang","sequence":"additional","affiliation":[{"name":"Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,26]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2024, March 31). 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