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You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (PD53)1 May 2024PD53-03 3D HUMAN TESTIS ORGANOID SYSTEM TO INVESTIGATE DIRECT EFFECTS OF CANNABINOIDS ON DIFFERENT TESTICULAR CELL TYPES Megan Escott, Evan Zelt, Janmejay Hingu, Salome Amdokze, Chenchu Nagarakanti, Jerri McLemore, Bita Nickkholgh, Allyn C. Howlett, and Hooman Sadri-Ardekani Megan EscottMegan Escott , Evan ZeltEvan Zelt , Janmejay HinguJanmejay Hingu , Salome AmdokzeSalome Amdokze , Chenchu NagarakantiChenchu Nagarakanti , Jerri McLemoreJerri McLemore , Bita NickkholghBita Nickkholgh , Allyn C. HowlettAllyn C. Howlett , and Hooman Sadri-ArdekaniHooman Sadri-Ardekani View All Author Informationhttps://doi.org/10.1097/01.JU.0001009456.01770.91.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Previous work demonstrated components of the endocannabinoid system in the human testis and investigated its role in male reproduction. We previously reported in vitro effects of selective cannabinoids on human testicular 2D cell cultures of three adult patients. However, given organoids better recapitulate native tissue environments, in this work we use 3D human testis organoid (HTO) systems from the same patient cells to better characterize the effects of cannabinoids on male reproductive function. METHODS: Testicular biopsy specimens from 3 adults with complete spermatogenesis were formed into HTOs using previously published methods. HTOs were exposed to ligand concentrations of 100 nM. Experimental conditions were: (1) no treatment (2) CP55,940 (cannabinoid agonist), (3) SR141716 (CB1 inverse agonist), (4) SR144528 (CB2 inverse agonist) (conditions 1 to 4 exposure time was 2 hours) (5) SR141716 for 5 minutes then CP55,940 for 2 hours and (6) SR144528 for 5 minutes then CP55,940 for 2 hours. RNA was isolated using Qiagen's RNeasy Kit. cDNA was synthesized using Applied Biosystems' high-capacity cDNA reverse transcription kit. RT-qPCR determined relative gene expression for markers of undifferentiated spermatogonial cells (ZBTB16), Sertoli cells (SOX9), Leydig cells (STAR), and peritubular cells (ACTA2) using TaqMan gene expression assay with technical duplicates. Statistics consisted of ANOVA with multiple comparisons. RESULTS: In 2D cell culture, no significant changes in gene expression were observed in any cell marker after cannabinoid exposure. In HTOs, however, significant changes were observed in the expression of ZBTB16 and ACTA2 across most conditions (Table 1). Two of three biological replicants had similar upregulation or downregulation responses across conditions. CONCLUSIONS: We found that cannabinoid exposure resulted in significantly different gene expression for some cell markers in HTOs in contrast to previous results in 2D cultures of the same patients' cells, highlighting the need for further investigation. We expect variations in individual patient responses, as seen in any analysis of pharmacologic efficacy. Future work will include analysis of ex vivo models, various cannabinoid concentrations, and a validated positive control, like brain tissue. Source of Funding: Institutional (Internal) Funding © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e1139 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information Megan Escott More articles by this author Evan Zelt More articles by this author Janmejay Hingu More articles by this author Salome Amdokze More articles by this author Chenchu Nagarakanti More articles by this author Jerri McLemore More articles by this author Bita Nickkholgh More articles by this author Allyn C. Howlett More articles by this author Hooman Sadri-Ardekani More articles by this author Expand All Advertisement PDF downloadLoading ...
Escott et al. (Mon,) studied this question.