Evaluation of Apoptosis-Related Genes and Hormone Secretion Profiles Using Three Dimensional Culture System of Human Testicular Organoids

Trends and Emerging Therapies in Chemotherapy of HER2-positive BC

Authors

  • Mehdi Abbasi Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Aghbibi Nikmahzar Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Farnaz Khadivi Department of Anatomy, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
  • Morteza Koruji tem Cell and Regenerative Center, Iran University of Medical Sciences, Tehran, Iran/ Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
  • Masoomeh Dehghan Tarazjani Vali-E-Asr Reproductive Research Center, Family Research Institute, Tehran University of Medical Sciences, Tehran, Iran
  • Mehrdad Jahanshahi Neuroscience Research Center, Department of Anatomy, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran
  • Maryam Shabani Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
  • Yasaman Abbasi Program in Neuroscience, Center to Advance Chronic Pain Research, Department of Neural and Pain Sciences, School of Dentistry, University of Maryland, Baltimore, MD, United States

DOI:

https://doi.org/10.31661/gmj.v12i.2805

Keywords:

permatogonial Stem Cells, Organoid Culture, Apoptosis, 3D Culture

Abstract

Background: In reproductive biology, testicular organoids can be used to treat infertility and to study testicular development and spermatogonial stem cells (SSCs) differentiation. Generating organoid from primary cells is challenging. In this study, testicular organoids were created using human primary testicular cells and evaluated the apoptotic gene expression and hormone secretion profiles of the organoids. Materials and Methods: Primary human testicular cells were isolated using 2-step enzymatic digestion from three brain-dead donors. Immunocytochemistry and flow cytometry analyses were performed to confirm human SSCs. Isolated cells were cultured in three experimental groups: control group (2 dimensional (2D)), group 1 (organoid culture after 2D culture), and group 2 (organoid culture immediately after enzymatic digestion). Testicular organoids were cultured in DMEM/F-12 media supplemented with follicle-stimulating hormone (FSH) and fetal bovine serum (FBS) for four weeks. After 24 hours and four weeks of culture, reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the relative expression of apoptotic genes (caspase 3, 9, Bax, and Bcl-2). At 24 hours, two weeks, and four weeks after culture, enzyme-linked immunoassay (ELISA) was used to determine the testosterone and inhibin B concentrations. Light microscopy and toluidine blue staining were also used for morphological analysis. Results: RT-qPCR results revealed that pro-apoptotic (caspase 3, 9, Bax) gene expression levels were highest in group 2 after 24 h and four weeks of culture. In contrast, the expression level of Bcl-2 (anti-apoptotic) was lower in group 2 compared to other groups. The hormone secretion levels decreased in a time-dependent manner during the cultivation. According to morphological evaluations, testicular organoids are compact, spherical structures with two to three elongated cells organized along their border. Conclusion: Our findings revealed that the testicular organoid culture system maintained hormonal secretory abilities, demonstrating the function of Sertoli and Leydig cells in the absence of testis-specific environments.

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2023-08-26

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