(Phospho-)proteomic Signaling Responses of Human Male Germ Cell Lines to Simulated Microgravity and Hypogravity.

Introduction: Access to space has significantly increased over the last decade, and with this comes the need for comprehensive knowledge about the effects exerted by altered gravitational conditions on human physiology and reproductive health. However, it should be highlighted that our knowledge about the effects of altered gravitational force on germ cells is still very poor. In this study, we exploited Reverse-Phase Protein microArrays (RPPAs), a biased (phospho-)proteomic approach, to investigate the impact of simulated microgravity (SμG) and hypogravity (ShG) on two human male germ cell lines, TCam-2 and NT2D1. Methods: TCam-2 and NT2D1 cell lines were exposed to SμG and ShG conditions using a Random Positioning Machine for 3, 24, and 72 hours. RPPA analysis was conducted using a panel of 130 antibodies selected to investigate a broad number of pathways potentially affected by altered gravity conditions. Results: The data analysis revealed that the exposure of both TCam-2 and NT2D1 cells to altered gravity induced significant (phospho-)proteomic changes. In particular, SμG induced early-phase alterations (3–24 hours), mostly characterized by the upregulation of some key regulators of signaling pathways, whereas longer SμG exposure (72 hours) resulted in the downregulation of other signaling proteins. ShG elicited minor changes, mostly characterized by reduced protein expression. The key pathways affected included cytoskeletal dynamics, proliferation, apoptosis, and autophagy. Notably, cell viability was not significantly impacted, suggesting compensating adaptation mechanisms to altered gravitational conditions. Conclusions: These findings indicate that (phospho-)proteomic responses to simulated gravity conditions were transient and non-persistent, demonstrating that human male germ cells exhibit resilience and adaptative capacity to cope with altered gravitational environments. This study provides valuable preliminary insights into the cellular and molecular mechanisms involved in gravity sensing and adaptation, which is crucial for developing countermeasures to ensure reproductive health and functionality during long-duration space missions for astronauts but also for the health of their future offspring.