3D culture system to evaluate the impact of microgravity on the male gonad

RATIONALE: One of the most important hazards of space environment is Microgravity, which causes alterations in the physiology of various systems, including the reproductive one. It has been observed that testicular cells appear to be sensitive to microgravity: 1) testicular endocrine functions are impaired during space missions since a decrease of testosterone production has been observed in astronauts; 2) even male germ cell models have been shown to be susceptible to changes in gravitational force. GENERAL OBJECTIVES: The general objective of the project is to set-up credible models to evaluate how microgravity induces alterations in testicular physiology and function. Indeed, to identify the molecular and cellular mechanisms underlying these alterations, different three-dimensional (3D) culture systems have been used. We focused our investigation on the following aspects: cell structure, cell to cell interactions, cell metabolism, cell oxidative stress, circadian rhythm genes expression level modifications, and cell endocrine/paracrine secretion. RESULTS: After 24h of Simulated Microgravity (SM) exposure, we observed in TCam-2 cell spheroids: 1) an increase of oxidative stress condition featured by high ROS levels, antioxidant barrier deregulation, and mitochondria enlargement; 2) an alteration of clock genes expression level possibly correlated with the aforementioned oxidative stress condition; 3) cell cytoskeleton and cell occluding junction alterations. After 24h of SM exposure, we observed in the testicular organ cultures: 1) an increase of testosterone and androstenedione levels in medium of culture without a significant change in Leydig cell mass; 2) an increase in protein expression level of enzymes involved in steroidogenesis: CYP17A1 and 17βHSD. CONCLUSIONS: These data indicate that TCam-2 spheroids are sensitive to SM exposure. Notably, with respect to the 2D model, investigated in previous studies, this system shows a better capacity to activate compensatory mechanisms in response to the alteration of gravitational force. Moreover, fetal testes organ cultures indicate that Fetal Leydig cells are sensible to SM being able to increase their androgen secretion probably because of the trigger of testicular mechano-sensing machinery