Generation and Characterization of Bovine Testicular Organoids Derived from Primary Somatic Cell Populations

SIMPLE SUMMARY: Organoids are 3D-cell culture systems composed of tissue-specific cells that create structures similar to those of their tissue of origin. Organoids have generated great interest in recent years, since they are considered a useful tool to perform laboratory studies in different scientific areas including reproduction. Testicular organoids (TOs) may provide an innovative model for the study of testicular physiology in various species; however, no previous studies have reported on the production of TOs in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs, derived from testicular cell populations that include Leydig, Sertoli and peritubular myoid cells. After isolation from testes and characterization, testicular cells were cultured in ultra-low attachment dishes. Testicular cells formed TOs after 3 days of culture. Leydig, Sertoli and peritubular myoid cells displayed specific locations and changed in number in TOs. Moreover, bovine TOs were able to produce and increase the concentration of testosterone after 27 days of culture. The present study represents the first report on the generation and characterization of bovine TOs. These TOs could be useful tools to evaluate the impact of exogenous factors on the physiology of sperm production and testis development in domestic and wild cattle. ABSTRACT: Organoids are 3D-culture systems composed of tissue-specific primary cells that self-organize and self-renew, creating structures similar to those of their tissue of origin. Testicular organoids (TOs) may recreate conditions of the testicular niche in domestic and wild cattle; however, no previous TO studies have been reported in the bovine species. Thus, in the present study, we sought to generate and characterize bovine TOs derived from primary testicular cell populations including Leydig, Sertoli and peritubular myoid cells. Testicular cells were isolated from bovine testes and cultured in ultra-low attachment (ULA) plates and Matrigel. TOs were cultured in media supplemented from day 3 with 100 ng/mL of BMP4 and 10 ng/mL of FGF2 and from day 7 with 15 ng/mL of GDNF. Testicular cells were able to generate TOs after 3 days of culture. The cells positive for STAR (Leydig) and COL1A (peritubular myoid) decreased (p < 0.05), whereas cells positive for WT1 (Sertoli) increased (p < 0.05) in TOs during a 28-day culture period. The levels of testosterone in media increased (p < 0.05) at day 28 of culture. Thus, testicular cells isolated from bovine testes were able to generate TOs under in vitro conditions. These bovine TOs have steroidogenic activity characterized by the production of testosterone.