One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell–cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of Reactive Oxygen Species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes of gravitational force even if published data on germ cell models are scarce. To gain more insights into the mechanisms of male germ cell response to altered gravity, a 3D cell culture model has been established from TCam-2 cells, a seminoma-derived cell line considered the only human cell line available to study in vitro mitotically active human male germ cells. TCam-2 cell spheroids were cultured for 24 hours under unitary gravity (Ctr) or simulated microgravity (s-microgravity) conditions, these last ones were obtained using the Random Positioning Machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels has been observed after RPM exposure. In line with these results a trend of protein and lipid oxidation increase, and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis by Transmission Electron Microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, being GPX1, NCF1, and CYBB downregulated, whereas NOX4 and HMOX1 upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated to the observed mitochondria alteration. All together, these data demonstrate TCam-2 spheroid sensitivity to acute SM exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow to overcome the exposure to altered gravitational force.

C-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium–mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

One of the hallmarks of microgravity-induced alterations in several cell models is an alteration in oxidative balance. Notably, male germ cells, sensitive to oxidative stress, have also been shown susceptibility to changes in gravitational force. To gain more insights into the mechanisms of male germ cells’ response to altered gravity, a 3D cell culture model was established from TCam-2 cells, a seminoma cell line and the only available in vitro model to study mitotically active human male germ cells. TCam-2 spheroids were cultured for 24 hours under unitary gravity (UG) or simulated microgravity conditions (SM), which was achieved using a random positioning machine (RPM). Apoptosis and necrosis analyses performed on the UG- and SM exposed samples revealed no significant differences in all of the cell death markers. Notably, the Mitosox assay revealed significant oxidation of mitochondria, after microgravity exposure, at least at this culture time. In the SM-treated samples, gene expression levels (evaluated by real-time PCR) of the main enzymes of the antioxidant barrier, GPX1 and NCF1, were reduced, indicating an influence of SM on mitochondrial function. Notably, the expression of HMOX, involved in the heme catabolism of mitochondrial cytochromes, was increased. The SOD, XDH, CYBA, NCF-2, TXN, and TXNRD genes were not affected. The ultrastructural analysis by transmission electron microscopy revealed that SM significantly altered TCam-2 spheroid mitochondria, which appeared swollen and, in some cases, disrupted. Indeed, mitophagy, or mitochondrial autophagy, appears to be more represented in the samples exposed to simulated microgravity. This result seems to be in line with the increase, mediated by the simulated microgravity, in the enzyme HMOX. All together, these preliminary data demonstrate TCam-2 spheroids’ sensitivity to acute SM exposure, strongly indicating a microgravity-dependent modulation of mitochondrial morphology and activity and encouraging us to perform further investigations on the chronical exposure to SM of TCam-2 spheroids.

The past few decades have seen an increasing number of both space travels and studies aimed at investigating the effects induced by space flights and the environment on humans. One of the main features of these conditions is the presence of altered gravity, mostly represented by microgravity experienced by astronauts. Microgravity is well known to induce deleterious effects at cellular, organ and systemic levels, including alterations in the male and female reproductive systems. In the present study, we investigated the effect of simulated microgravity on the metabolic activity of male germ cells using TCam-2 line as a cell model. These cells were cultured in the Random Positioning Machine that simulated microgravity conditions, and were grown as 2D monolayers or 3D spheroids to assay the effects on single cells or on organ-like structures. After a 24 hour-exposure to simulated microgravity, TCam-2 monolayers showed: (1) a decreased proliferation rate and a delay in cell cycle progression; (2) increased anaerobic metabolism; (3) increased levels of reactive oxygen species and superoxide anion; (4) modifications in mitochondrial morphology. After the same 24 hour-exposure, TCam-2 spheroids showed: (1) an increased anaerobic and aerobic activity in 40% and 26% of samples, respectively; (2) alterations in the redox balance with a decrease in catalase activity in about 65% of cell samples, and therefore, a deficit in the cellular antioxidant capacity; (3) increases in oxidative damage to proteins and lipids in more than 50% of cell samples. In conclusion, these data demonstrated a clear inference of simulated microgravity on the metabolic activity of TCam-2 cells, which is expressed through the activation of an oxidative stress state, that, if not compensated for, could be deleted over time.

One of the most important hazards of the space environment is microgravity, which causes an alteration in the physiology of different systems, including the reproductive one. It is widely accepted that cytoskeleton is the microgravity-sensitive apparatus of the cells, and that cytoskeletal modifications are responsible for microgravity-triggered cell alterations. We established a 3D free-floating culture system from TCam-2 cell, a human seminoma cell line, and then exposed the obtained TCam-2 spheroids for 24 h at unitary gravity (UG), or under a simulated microgravity condition (SM), using the random position machine (RPM). We tested the cytoskeletal and junctional features of these samples using Western blot and confocal microscopy analysis to elucidate the impact of microgravity on the adherent and occluding junctions of TCam-2 spheroids. The junctional ultrastructure was studied using transmission electron microscopy (TEM). TEM analysis revealed the presence of occluding junctions both in UG or SM samples. Even if Western blot revealed no quantitative difference in actin and occludin proteins both in UG and SM exposed samples, fluorescence colocalization analysis showed a significative increase in the colocalization area of occludin and actin proteins in the superficial layer of TCam-2 spheroids grown in RPM conditions. This result let us speculate that tight junction functionality is different in UG and SM exposed spheroids. As far as adherent junctions are concerned, TEM analysis revealed adherent junctions both in UG or SM samples. Moreover, we observed by Western blot a trend in terms of the increase in the vimentin expression in SM exposed spheroids. Confocal microscopy analyses confirmed this significant increase. All together, these data suggest that simulated microgravity conditions in TCam-2 spheroids alter the tight junction assembly, while the increase in the intermediate filament’s structures can in part be associated with an enrichment in the adherent junctions. A functional investigation is needed to more deeply clarify this hypothesis.