To our knowledge, this is the first study investigating testicular histopathology in HU mice. We investigated the pathological effects of HU on mouse testes in a time-dependent manner. Our findings indicate that the HU results in a significant disruption of testicular architecture, including the expansion of luminal diameter, epithelial thinning, and reduced spermatozoa density. A depression of global molecular phenotype accompanies these changes. We also report a time-dependent effect as the four weeks of HU induced a more severe testicular disruption than two weeks of HU.
The data about testicular histopathology in HU rodents are scarce. A previous study elaborated on loosening on seminiferous tubules along with presence of necrosis (Ding et al., 2011). Additionally, they reported interstitial edema and the disappearance of germ cells in the testes. However, this study was conducted on rats. Due to limitations in creating transgenic rat models, the relative scarcity of mechanistic data from rats may reduce the translational potential of such findings in humans. Consequently, our data from mice may be more relevant to clinical studies than data from rats. Our findings of epithelial thinning, reduced spermatozoa density, and areas of necrosis are consistent with previous findings. While we did not find relevant data from clinical studies due to constraints of obtaining testes biopsy, several lines of evidence are consistent with our findings. For example, spaceflight and prolonged bed rest in humans result in reduced circulating testosterone, indicating potential damage to testicular tissues (Smith et al., 2012). Similarly, sedentary lifestyles and physical inactivity are associated with infertility in men (Foucaut et al., 2019). This indirect data indicates the potential involvement of testicular disruption in humans during unloading conditions. Thus, the HU mice at least partly mimic the features of reproductive suppression reported in physically inactive humans.
Our findings recapitulate the age-related degeneration of mouse testes. Specifically, the aged mice show a widening of the seminiferous tubular diameter, epithelial thinning, and reduced spermatozoa density (Mehraein and Negahdar, 2011). Thus, the HU may induce an accelerated aging phenotype in the mouse testes. This consequence of physical inactivity is evident in multiple body organs, which mimic age-related degeneration during HU (Globus and Morey-Holton, 2016; Qaisar et al., 2020).
We also observed a time-dependent disruption of mouse testes during HU. This sequential detriment of testicular histology indicates that the suppression of the male reproductive system is a continuous process during inactivity. These findings may help optimize timely interventions of infertility during bed rest in humans.
Raman spectra indicated a robust decline in the concentration of tryptophan and hydroxyproline in a time-dependent manner. Tryptophan is implicated in testosterone production and spermatogenesis in testes (Jimenez-Trejo et al., 2020). Thus, a reduced expression of tryptophan is consistent with histopathological disruption, including the reduced density of spermatozoa in the HU testes. Hydroxyproline is an essential component of collagen proteins in biological tissues. Among its multiple functions, it is involved in stabilizing collagens (Xu et al., 2019). Thus, the reduced hydroxyproline in HU testes may indicate instability of collagens, which may contribute to testicular pathology. The analysis of HU testes also revealed a generalized depression in the spectral peaks of all intensities. This finding indicates a reduced concentration and/or altered orientation of global molecular phenotype, which agrees with the histopathological findings. Additionally, the spectral peaks above 1000 cm-1, which indicate the C-H vibration, were suppressed in the HU testes, showing the breakdown of several amino acid molecules.
We did not investigate the molecular mechanisms driving testicular pathology in HU conditions. However, our preliminary data indicate a role for elevated necroptosis in the HU testes. Necroptosis has emerged as an underlying mechanism of multiple organs pathology. Additionally, necroptosis appears to promote age-related degeneration of mouse testes (Li et al., 2020). Thus, the degeneration of HU testes may have a potential contribution from elevated necroptosis. However, the preliminary nature of our data prevents us from establishing robust conclusions.
Taken together, our findings indicate that two and four weeks of HU result in the degeneration of mouse testes. Several characteristics alterations were observed in the histological architecture of testicular tissues, involving seminiferous tubules, epithelial thickness, and spermatozoa density. These changes were accompanied by global suppression of Raman spectra, indicating an altered concentration and/or orientation of several molecules. Lastly, our preliminary data indicate a role for necroptosis as a potential contributor to testicular disruption during HU. Further studies are required to investigate the mechanistic link between HU and testicular pathology.