Cell adhesion and immune response, two main functions altered in the transcriptome of seasonally regressed testes of two mammalian species

In species with seasonal breeding, male specimens undergo substantial testicular regression during the non-breeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, non-apoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK and TGF-beta signalling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the immune privilege that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.

Mediterranean Pine Vole, Microtus duodecimcostatus: A Paradigm of an Opportunistic Breeder

Most mammalian species of the temperate zones of the Earth reproduce seasonally, existing a non-breeding period in which the gonads of both sexes undergo functional regression. It is widely accepted that photoperiod is the principal environmental cue controlling these seasonal changes, although several exceptions have been described in other mammalian species in which breeding depends on cues such as food or water availability. We studied the circannual reproductive cycle in males of the Mediterranean pine vole, Microtus duodecimcostatus, in the Southeastern Iberian Peninsula. Morphological, hormonal, functional, molecular and transcriptomic analyses were performed. As reported for populations of other species from the same geographic area, male voles captured in wastelands underwent seasonal testis regression in summer whereas, surprisingly, those living either in close poplar plantations or in our animal house reproduced throughout the year, showing that it is the microenvironment of a particular vole subpopulation what determines its reproductive status and that these animals are pure opportunistic, photoperiod-independent breeders. In addition, we show that several molecular pathways, including MAPK, are deregulated and that the testicular “immune privilege” is lost in the inactive testes, providing novel mechanisms linking seasonal testosterone reduction and testis regression.

Divergent Seasonal Reproductive Patterns in Syntopic Populations of Two Murine Species in Southern Spain, Mus spretus and Apodemus sylvaticus

In most mammals with seasonal reproduction, males undergo testis regression during the non-breeding period. We performed a morphological, hormonal, functional, and molecular study of the testes of sexually inactive males of two species of murine rodents, the wood mouse, Apodemus sylvaticus, and the Algerian mouse, Mus spretus, in syntopic populations of southern Iberian peninsula. Both species reproduce during most of the year, but wood mice stop breeding in the summer whereas Algerian mice do it in winter. Sexually inactive males of A. sylvaticus show complete testis regression with reduced levels of serum testosterone and abnormal distribution of cell-adhesion molecules. Contrarily, inactive males of M. spretus maintain almost normal spermotogenesis despite a significant reduction of androgenic function. The lack of an evident explanation for the divergent seasonal breeding patterns found in southern populations of A. sylvaticus and M. spretus, compared with northern ones, implies that very subtle species/population-specific features and/or non-conspicuous environmental cues probably operate to determine their seasonal breeding pattern. These results also support the notion that multiple models of circannual testis variation are possible for different populations of the same species, showing that the mechanisms controlling seasonal reproduction are in fact very plastic and fast evolving.

Sertoli-immature spermatids disengagement during testis regression in the armadillo

In Nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and western blotting we studied key proteins of the N-cadherin/N-cadherin and α6β1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, β1-integrin, Laminin G3, N-cadherin, β-catenin, P-β-catenin-Tyr654, FAK, P-FAK-Tyr397, Src, P-Src-Tyr416 proteins present a spermatogenetic cycle-dependent localization pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, Src, and β-catenin contribute to the disassembly of the N-cadherin/N-cadherin and α6β1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

Cellular and molecular responses of adult testis to changes in nutrition: novel insights from the sheep model

This review explores the cellular and molecular mechanisms that regulate spermatogenesis in the post-pubertal testis that is regressing in response to mild undernutrition, using the sexually mature male sheep as a model. Testis regression leads to reductions in daily sperm production and in the quality of ejaculated spermatozoa (poorer movement, DNA damage). There is also a reduction in spermatogenic efficiency that appears to be caused, at least partially, by increases in germ cell apoptosis. Sertoli cell number does not change with testis regression, although about 1% of Sertoli cells do appear to retain proliferative ability after puberty. On the other hand, Sertoli cell function is disrupted during testis regression, as evidenced by a disorganization of tight junctions and indications that cell differentiation and maturation are reversed. Disrupted Sertoli cell function can explain, at least partially, the increase in germ cell apoptosis and any decrease in the rate of spermatogenesis, the two major contributors to spermatogenic efficiency. These outcomes seem to be mediated by changes in two RNA-based processes: (i) the expression of small non-coding RNAs that are involved in the regulation of Sertoli cell function, spermatogenesis and germ cell apoptosis and (ii) alternative pre-mRNA splicing that affects the regulation of spermatogenesis but does not appear to affect germ cell apoptosis, at least during testis progression induced by undernutrition in the male sheep. These research outcomes can be extended to other animal models and are relevant to issues in human male fertility.

Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8-/- mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.