Abstract
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.
Author Contributions
Conceptualization, A.C., G.R., L.G. and M.B.; methodology, M.B., L.G., M.A.M., C.M. and S.G.; software, A.C., G.R., M.B. and L.G.; validation, A.C., G.R., M.B. and L.G.; formal analysis, A.C., G.R., M.B. and L.G.; investigation, A.C., G.R., M.B. and L.G.; resources, A.C., G.R., M.B. and L.G.; data curation, A.C., G.R., M.B. and L.G.; writing—original draft preparation, A.C. and G.R.; writing—review and editing, M.B., L.G., M.A.M., C.M. and S.G.; visualization, A.C. and G.R.; supervision, F.F.; project administration, A.C. and F.F.; funding acquisition, A.C. and G.R. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by grants from ASI–Italian Space Agency, number 2020-24-HH.0.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).