Into the seminiferous tubules, spermatogenic cells are designed to evolve in mature spermatozoa. Many cycles for spermatozoa differentiation occur sequentially along the convoluted seminiferous tubules of the testis, resulting in pulses of sperm release that ensure the continuous production. Sertoli cells (SC) are constitutive non spermatogonia cells that have supportive and trophic functions for the spermatogonia cells of the seminiferous epithelium. SC facilitate transport of mature spermatids towards the lumen of the tubules, provide secretion of androgen binding protein and molecules with endocrine or paracrine action for spermatogenesis. They are responsible to assure the function of the blood-testis barrier. Further, they interact with intertubular endocrine Leydig cells secreting testosterone. Overall, these tasks make SC elements of primary importance for the testicular function.
In our study first evidence of hypergravity disturbance into testis environment has been obtained observing the morphology of germinative epithelium. We have observed that hypergravity affects the thickness and the general organization of germinative epithelium of seminiferous tubules, consisting in SC with small nuclei, presence of immature spermatogonia cells with large nuclear profile in the upper layer of germinative epithelium, where in control mice only cells with small hyperchromatic nuclei and differentiative cellular profile can be observed. Consequently, the maturation and production of spermatozoa can be supposed defective, in fact rarely spermatozoa were observable in the lumen of seminiferous tubules in rats exposed to hypergravity.
The good physiological performance of SC embedded in the spermatogenic layer is widely dependent on cell-cell tight junctions which are made of proteins called occludins and claudin, which interact forming a branching network between the two opposite cells resulting in a tighter seal and mechanical stability. These proteins are linked to elements of cellular cytoskeleton, being able to transduce mechanical cues in intracellular signals. Proteins of tight junctions are involved in keeping polarity, in establishing organ-specific apical domains and participate into SC assisting function during proliferation, differentiation, and migration of spermatogenic cells and realize the blood-testis barrier to control metabolic access to seminiferous tubules environment (Heiskala et al., 2001; Mruk and Cheng, 2010). Hypergravity was found to alter the expression pattern of claudins and occludin.
We hypothesize that a rearrangement of tight junction among SC has occurred, resulting in an efficiency loss of SC cells in supporting the steps of germ cells differentiation. Further, this alteration in the mechanical arrangement can be cause of the visible disassembly of the spermatogenic cells disposition with larger spaces between cells and smaller cells closer to larger ones.
Besides the tight junction complex, which allow stable linkage between adjacent cells, SC layer exhibit several gap junctions, specialized membrane areas which allow cells to communicate each other exchanging small solutes and ions. The gap junction is made up of two hemichannels, one for each cell, formed by assemblies of six channel proteins called Connexins.
This type of junction offers a pivotal advantage in cell-cell communication because it makes the SC layer working as a functional syncytium, able to make all the together SC cells responsive, optimizing in such a way the physiological behaviour of each tubule.
Following hypergravity treatment the connexin expression was markedly reduced suggesting a probable decrease in cell-cell communication, reflecting a severe trouble in coordinating cellular response aimed to spermatozoa production. Thus, the occurrence of this kind of alterations has an important negative effect on germ cells differentiation culminating in a limited reproduction rate of spermatozoa.
Although we do not have any direct evidence, we can suppose that that other epithelial layers or tissues may be subjected to intimate alterations of cell-cell junctions shattering the physiological performances under hypergravity conditions.
Blood-brain barrier (BBE) destabilization has been proved in mice following centrifugation treatment at 2g showing altered permeability characteristics which represent the main topic of the endothelial lining of the BBE to control exchanges between blood and brain parenchyma (Dubayle et al., 2020). Similarly, we expect that in the seminiferous tubules the alteration of cell-cell contact among Sertoli cells could induce an alteration in the blood-testis barrier, compromising the regular surveillance of metabolic traffic between blood and germinative epithelium.
Another important compartment in the testis anatomo-physiology is represented by Leydig cells inside the interstitial stroma representing the glandular compartment producing the bulk quantity of testosterone.
Testosterone biosynthesis is powered by different enzymes being the 3β-hydroxysteroid dehydrogenase (3β-HSD) and the 17β-hydroxysteroid dehydrogenase (17β-HSD) the main ones involved (O’Shaughnessy et al. 2000). Our data have shown that hypergravity exposition reduce widely the expression of these two enzymes, suggesting a loss of function of the glandular compartment of the testis.
Testosterone is a powerful hormone involved not only in spermatogenesis physiology, but in upper brain cognitive functions (Celec et al., 2015), bone metabolism (Sinnesael et al., 2011), cardiovascular parameters (Oskui et al., 2013), glucose homeostasis (Grossmann and Wu, 2014). Therefore, a lack in this hormone synthesis can affects multiple physiological pathways. In similar case the adrenal gland can compensate for testis function by producing another font of testosterone. At the present we don’t have any evidence concerning a direct effect of hypergravity on other endocrine glands, however, the scientific literature reports following hypergravity exposure, the alteration of the adrenal gland zona reticularis representing the secondary font of production of testosterone (Moroz et al., 2018). Following these evidence hypergravity seems to decrease widely the testosterone production, and the hypothesis of a wider involvement of hormonal balance may be advanced. Indeed, recent data show that hypergravity conditions might remodel thyrocytes cell membrane by increasing thyrotropin-receptor (TSHR) surface protein (Albi et al., 2014), but further data concerning the hypergravity effects on hormone receptor expression is still lacking.
In this view, we have analysed the receptor pattern expression for key hormones regulating the physiological function of the testis. We focused on androgen receptor (AR), interstitial cell stimulating hormone receptor (ISCHR), follicle-stimulating hormone receptor (FSHR) and we also detected the expression level of interleukin-1β. AR is a nuclear receptor which reflects the cellular response to testosterone and, in general to androgen hormones, in supporting the normal testis physiology and maintaining the male sexual phenotype. ICSHR is mainly expressed in Leydig cell where it drives testosterone production under hypothalamic-hypophysis axis control. FSHR regulates spermatogenesis and its main function is, upon binding of FSH, to increase the number of Sertoli cells by stimulation of their mitotic activity. It is also essential for tight junctions’ formation (Sluka et al., 2006). IL1β is a pleiotropic cytokine that contributes to the specific immune environment of mammalian testis and in regulating cell differentiation. Our data show only a significant reduction in AR and ICSHR expression, without changes in FSHR and IL-1β levels. These findings further could confirm the occurrence of a diminished release of testosterone from Leydig cells with the consequent reduction of AR and ICSHR expression.
SC provide key signals to support germ cell survival proceeding through spermatogenesis, and the withdrawal of ICSH and/or testosterone results in the induction of apoptosis at particular stages of germ cell development reducing spermatogenic efficiency (Walker, 2021). Lacking the effects on FSHR and IL-1β, together the specific increasing of SHBG expression on SC, could suggest the falling down of physiological function of SC toward a quiescent phase, that evolve in a loss of some SC as evidenced by apoptotic signals, leading to an altered homeostasis of the organ and reduced formation of spermatozoa.