title: Enrichment and in vitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls.
abstract: Enrichment and propagation of gonocytes or spermatogonial stem cells (SSCs) from cryopreserved testicular tissue is essential to apply SSCs-related techniques in large domestic animals. We previously reported the cryopreservation of adult bovine testicular tissue. Here, we conducted the enrichment and culture of putative gonocytes from cryopreserved testicular tissues of post-natal 1-day-old bulls. The testicular structure was well maintained after freezing and thawing. Higher mRNA levels of gonocyte/SSCs markers (PLZF, GFRα1, and UCHL-1) than those of pluripotency genes (Oct4, Sox2, and Nanog) were detected in the frozen-thawed sex cords. GFRα1 was specifically detected in the membrane and cytoplasm of gonocytes by immunostaining. Differential plating provided 40-50% enrichment of putative gonocytes. They were single, paired-, aligned-cells, or grape cluster-like colonies in minimum essential medium (MEM) containing 2.5% FBS + 2 mM glutamine + 100 IU/mL penicillin-streptomycin + 40 μg/mL gentamycin + 15 mM HEPES + 10 mM β-mercaptoethanol + 0.1 mM non-essential amino acids + 1 mM sodium pyruvate. On day 3, gonocyte progeny increased and the contaminated somatic cells spread and concurrently divided slowly. On day 5, gonocyte progeny proliferated continuously and typical intercellular bridges formed by incomplete cytokinesis in paired-cells or aligned-cysts were observed. Immunochemically, they were still GFRα1 and PLZF positive. These cells expressed significantly higher gonocyte/SSCs marker mRNAs than pluripotency gene mRNAs, concomitant with a higher level of differentiated spermatogonia marker c-kit. With time, gonocyte progeny colonies appeared in varied sizes and expanded dramatically on day 7. After cultured for 9-10 days, however, large colonies collapsed and dispersed as some single cells and small syncytial cysts. Together, MEM containing 10% dimethyl sulfoxide + 2.5% newborn calf serum provides efficient cryoprotection for the testicular tissue from 1-day-old neonatal bulls. Putative gonocytes enriched from these nascent tissues present robust proliferation capacity, conserved gonocyte/SSCs markers, and SSCs-like in vitro features.
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: H
authors.initials: H
authors.lastname: Cai
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: B
authors.initials: B
authors.lastname: Tang
authors.affiliation: Department of Laboratory Medicines, The 90th Hospital of Jinan, Jinan, Shandong, China.
authors.forename: J Y
authors.initials: JY
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: X X
authors.initials: XX
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: Z Z
authors.initials: ZZ
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: X L
authors.initials: XL
authors.lastname: An
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.|Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
authors.forename: L X
authors.initials: LX
authors.lastname: Lai
authors.affiliation: State & Local Joint Engineering Laboratory for Animal Models of Human Diseases, The First Hospital, Jilin University, Changchun, Jilin, China.
authors.forename: Z Y
authors.initials: ZY
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Jilin University, Changchun, Jilin, China.
authors.forename: X M
authors.initials: XM
authors.lastname: Zhang
mesh_terms: D000072956:Adult Germline Stem Cells / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D015925:Cryopreservation; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000235:genetics / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008297:Male; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013093:Spermatogonia / Q000166:cytology / Q000378:metabolism*; D013737:Testis / Q000166:cytology / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000235:genetics / Q000378:metabolism
chemical_list: D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D051741:Kruppel-Like Transcription Factors; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D043222:Ubiquitin Thiolesterase
keywords: cell culture; cryopreservation; enrichment; gonocyte; neonatal bull; testicular tissue