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Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1\u2010month\u2010old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3\u03b2\u2010hydroxysteroid dehydrogenase antibody. Enzyme\u2010linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in\u00a0vitro (generations 5\u20139) ranged between 1.29\u20131.67\u2009ng\/mL. Additionally, the content of the cellular autophagy signature protein microtubule\u2010associated protein 1 light chain 3 was measured at 230\u2013280\u2009pg\/mL. Through this study, we established an in\u00a0vitro system for the isolation, purification and characterization of testicular Leydig cells from 1\u2010month\u2010old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.<\/jats:p>","DOI":"10.1111\/rda.14583","type":"journal-article","created":{"date-parts":[[2024,5,15]],"date-time":"2024-05-15T11:49:51Z","timestamp":1715773791000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig"],"prefix":"10.1111","volume":"59","author":[{"given":"Hong","family":"Du","sequence":"first","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"},{"name":"Department of Reproductive Medicine Lanzhou University Second Hospital  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zunqiang","family":"Yan","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haixia","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiaoli","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoyu","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pengfei","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoli","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiaojiao","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-4615-1562","authenticated-orcid":false,"given":"Shuangbao","family":"Gun","sequence":"additional","affiliation":[{"name":"Department of College of Animal Science and Technology Gansu Agricultural University  Lanzhou Gansu China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,5,15]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/biolre\/ioz020"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mce.2009.01.023"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1210\/endo\u2010101\u20102\u2010639"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ceca.2011.02.003"},{"issue":"10","key":"e_1_2_10_6_1","first-page":"740","article-title":"Experimental study of changes in microscopic characteristics and ability of secreting testosterone of aged SD rat leydig cells","volume":"11","author":"Deng C. 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