{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,11]],"date-time":"2025-07-11T10:26:57Z","timestamp":1752229617185,"version":"3.40.5"},"reference-count":24,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2023,1,16]],"date-time":"2023-01-16T00:00:00Z","timestamp":1673827200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Andrology"],"published-print":{"date-parts":[[2023,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>The sperm retrieval rate of microdissection testicular sperm extraction varies from 25% to 60%. Therefore, it is necessary to establish objective selection criteria for identifying seminiferous tubules with spermatozoa.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objectives<\/jats:title><jats:p>Our aim was to develop a method for identifying spermatogenesis without sectioning testicular tissues.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and methods<\/jats:title><jats:p>Testicular tissues of 10\u2010week\u2010old normal rats were fixed with 4% paraformaldehyde. Fluorescent labeling of seminiferous tubule nuclei and F\u2010actin was performed, and the specimens were observed without sectioning using a multiphoton microscope. Cryptorchid rats were used as a model lacking elongated spermatids. Multiphoton images were compared with images of normal seminiferous tubules. In addition, seminiferous tubules of 10\u2010week\u2010old normal rats were labeled by testicular interstitial injection of fluorescent probes and observed by a multiphoton microscope without fixation. Terminal deoxynucleotidyl transferase dUTP nick end labeling\u2010stained images of normal and probe\u2010injected testes were compared.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In fixed seminiferous tubules, elongated spermatids were identified. In addition, F\u2010actin of apical ectoplasmic specialization was observed around elongated spermatids. Furthermore, spermatogenic stages were identified by an array of nuclei or F\u2010actin. In cryptorchid testes, elongated spermatids and F\u2010actin of the apical ectoplasmic specialization were not observed. In testes injected with fluorescent probes, F\u2010actin of the apical ectoplasmic specialization was observed, and spermatogenic stages were identified without fixation. There was no significant difference in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling\u2010positive cells per seminiferous tubule between normal and probe\u2010injected testes.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Seminiferous epithelium could be observed without sectioning of tissues by fluorescent probes and a multiphoton microscope. Active spermatogenesis was observed by labeling F\u2010actin with and without fixation. Moreover, the toxicity of fluorescent probes was limited. Our method has a potential for live imaging of testicular tissue.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.13379","type":"journal-article","created":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T04:54:54Z","timestamp":1672808094000},"page":"1147-1156","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Identification of active spermatogenesis using a multiphoton microscope"],"prefix":"10.1111","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5827-7286","authenticated-orcid":false,"given":"Tomoki","family":"Takeda","sequence":"first","affiliation":[{"name":"Department of Nephro\u2010urology Nagoya City University Graduate School of Medical Sciences  Nagoya Aichi Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shoichiro","family":"Iwatsuki","sequence":"additional","affiliation":[{"name":"Department of Nephro\u2010urology Nagoya City University Graduate School of Medical 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