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

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.

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.

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.

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.