Mecanismos de regresión y activación testicular en mamíferos con reproducción estacionalcontrol genético y susceptibilidad a cambios ambientales

Résumé

ABSTRACT Seasonal reproduction is the reproduction that occurs discontinuously throughout the year as a result of adaptation to environmental fluctuations resulting from climatic seasons. This behaviour is exhibited by numerous vertebrate species inhabiting temperate regions of the Earth, thus concentrating their reproductive effort in those seasons of the year that offer the best environmental conditions and ensuring that the offspring born and grow in the most favourable periods. Species with seasonally reproduction are able to stop and resume gametogenesis cyclically. Both males and females undergo substantial changes in their gonads during the transition between reproductive and non-reproductive periods. In males, a process of testicular regression takes place which involves major changes in testicular function, altering spermatogenesis, androgenesis and the local immune system. Most of the studies to date are incomplete due to they only focus on very particular aspects of the process in many cases (apoptosis, hormonal variations, ultrastructure, morphological changes, dynamics of cell adhesion molecules). Complete studies including all these features have only been carried out on a small number of species, such as the Iberian mole, the large hairy armadillo and the Mediterranean vole. In the present PhD thesis, we have carried out a comprehensive study of the process of testicular regression in a new species, the longeared hedgehog, Hemiechinus auritus captured from northern Egypt. Our results show that germ cell desquamation is the main effector of testicular regression in this species, as previously described in two studies in the Iberian mole Talpa occidentalis and the large hairy armadillo C. villosus. The presence of abundant primary spermatocytes in the lumen of seminiferous tubules and epididymis indicates that immature meiotic cells detach early from the seminiferous epithelium. Subsequently, these detached germ cells are eliminated via the distal section of the genito- urinary tract. In addition we found that apoptosis is not the main cause of germ cell loss in the regressing testis of H. auritus, as very small numbers of apoptotic cells are present in these testes. However, apoptosis has an important role in the quiescent testis, because it is the main mechanism that eliminates primary spermatocytes that reach the pachytene stage. On the other hand, we have found that the expression of cell adhesión molecules is altered in both the germinal epithelium of regressing and inactive testes, suggesting that Sertoli cell-cell adhesion and Sertoli cellgerm cell adhesion is compromised in regressing testes, thus explaining the germ cell detachment that occurs during this period. Finally, as happens in all species studied to date, circulating testosterone levels are reduced in reproductively inactive H. auritus individuals, indicating that reduced androgen concentration is probably the hormonal signal that induces seasonal germ cell depletion and loss of cell adhesion in the mammalian germ cell epithelium. From the different studies carried out in mammals, including the present one, it is clear that there is not a unique mechanism of testis regression. Two main cellular processes by which germ cells are massively depleted during the process of testicular regression have now been identified: apoptosis and germ cell desquamation. Despite this, the genetic control of these testicular changes is poorly understood, mainly because of the complexity of the genetic pathways operating in a specific testicular cell type and the cross-signalling between different cell types. In this sense, expression profiling studies can provide an integrated view of the interacting molecular pathways that act on the testis, and can also give us information on which of them are affected in the regressing testis. In this PhD thesis we have analysed the testicular transcriptome of two species in which we had previously carried out histological, immunohistological, functional and hormonal studies throughout their reproductive cycle: the Mediterranean vole, Microtus duodecimcostatus, and the Iberian vole, T. occidentalis. In M. duodecimcostatus, we found that MAPK/ERK1/2, WNT, TGF-Beta , cytosolic Ca2+ and PI3K signalling pathways are downregulated in the inactive testis. All these pathways operate in Sertoli cells and are involved in the regulation of spermatogenesis and in the dynamics of tight and adherens junctions present in the BTB. Our analysis demonstrated that MAPK/ERK1/2, a signalling pathway required for mitotic and meiotic cell proliferation, plays a central role in this process. Previous studies have shown that testosterone modulates the activity of the MAPK/ERK signalling pathway in Sertoli cells, indicating that decreased serum testosterone levels in inactive M. duodecimcostatus males affect Sertoli cell regulation of several interconnected molecular pathways leading to: a) alterations in the spermatogenic cycle, b) deregulation of cell adhesion molecules and c) alteration of BTB dynamics. The testis is subjected to a special immunological environment, known as the immuno-privilege, that protects the germ cells from autoinmune response. This immuno-privilege is based on: a) the formation of BTB in the germinal epithelium, b) the decreased ability of the testicular macrophage population to induce an inflammatory response and c) the constitutive expression of anti-inflammatory cytokines by immune and somatic cells. Our transcriptomic analysis indicated that the macrophage population was activated in the inactive testis of M. duodecimcostatus. Previous studies have shown that testosterone is essential to maintaining testicular immuno-privilege under physiological conditions, so we can conclude that reduced testosterone levels in the inactive testis of M. duodecimcostatus may induce the secretion of pro-inflammatory cytokines by macrophages and, probably, by other somatic cell types (e.g. Sertoli and peritubular myoid cells) that activate macrophages. Interestingly, transcriptomic study of T. occidentalis testis also revealed that biological processes such as cell adhesion and cell junction assembly, as well as several molecular pathways, including MAPK, ERK1/2, TGF-Beta , cytosolic Ca2+, PI3K, GTPase and TNF were altered in the quiescent testis. On the other hand, transcriptomic analysis also revealed that the immune system was activated in the inactive testis of T. occidentalis. According to all these observations, we suggest that, as in M. duodecimcostatus, low testosterone levels in the inactive testis of T. occidentalis lead to the loss of the immunoprivilege operating in the active testis, as manifested by permeability of the BTB and increased cytokine production by the macrophage population (and perhaps other somatic cell). Definitely, all of these processes could contribute to the maintenance the quiescent state of the mole gonads during the non-reproductive period. In T. occidentalis we also analysed the expression levels of genes belonging to the gene expression profile of early spermatogenic clusters and found that several biological processes are altered in inactive testes, in particular the protein ubiquitination at the spermatogonia stage. However, it is difficult to know if this alteration in expression profiles is due to the testicular environment of the quiescent testis, in which both BTB and cell adhesion function are compromised, or to currently unknown mechanisms directly affecting germ cell expression, or both. Finally, we compared the tanscriptomic data of both the Iberian mole and the Mediterranean pine vole. We found a large number of genes that are deregulated in the inactive testes of both species, and found two notable coincidences: 1) many of these genes are involved in the control of cell adhesion, including those involved in molecular pathways such as MAPK, ERK1/2, TGF-Beta , GTPase and TNF, which control cell junctions in the germinative epithelium, and 2) we also found a shared set of genes involved in the regulation of the immune response. These overlaps are relevant because the inactive testes of these two species do not have identical features. For example, the initiation of meiosis by spermatogonia is completely abolished in the inactive testes of M. duodecimcostatus, but not in those of T. occidentalis, where spermatogonia continue enterig meiosis and spermatogenesis progresses to zygotene-pachytene. In addition, the quiescent seminiferous tubules of M. duodecimcostatus remain adjacent to each other, whereas those of T. occidentalis are widely separated by Leydig cell intervention. Despite these differences, here we have found that two important testicular functions, cell adhesion and immune response, are dysregulated in the inactive testes of these two species, suggesting that there are conserved molecular mechanisms associated with seasonal testicular involution in mammals. Therefore, similar studies should be conducted in other species to test this hypothesis.