Titolo della tesi: Unveiling the spermatogonial stem cell niche in primates: a histological approach
The maintenance of seminiferous epithelium function depends on the intricate molecular and cellular interactions between spermatogonial stem cells (SSCs) and their cognate niche. To sustain the continuous production of sperm, a delicate balance must prevail among the various niche factors that control the fate decisions of SSCs by promoting either self-renewal or spermatogenic commitment of undifferentiated spermatogonia (undiff-SPG). Despite its critical importance, our understanding of SSC homeostasis remains limited, particularly regarding the spatial distribution of SSCs within the testicular microenvironment. Here, we aim to characterize the SSC niche and its regulation in primates by identifying novel putative cell-cell interactions between somatic cells and SSCs, and by evaluating the histological distribution of different spermatogonia subpopulations during the seminiferous epithelium cycle. To this end, we analyzed the expression of established markers as well as novel markers identified by sc-RNAseq in the spermatogonial compartment of humans and Macaca fascicularis. Specifically, we sought to analyze the topological distribution of positive cells within the tubules (i.e., tubule-tubule or tubule-interstitium contact regions) during different stages of the seminiferous epithelium cycle. To validate protein expression, intact seminiferous tubules and sections from Macaca fascicularis and human testis biopsies were stained by immunofluorescence and analyzed by confocal microscopy. To analyze the topological position of spermatogonia in the seminiferous tubules, immunohistochemistry was performed on Macaca fascicularis Bouin-fixed samples, which were analyzed by light microscopy. Strauss’s linear selectivity index (Linear Index, Li) was employed to analyze region selection by the various spermatogonia subpopulations in the basal compartment of seminiferous tubules in monkeys.
The analysis of protein expression of the published L-R pairs we selected (BMP4-BMPR1B and WNT5A-RYK) revealed that only the BMP4-BMPR1B pair showed a positive correlation between RNA and protein expression in both human and monkey testis. Nevertheless, protein analysis showed that BMPR1B has a wider expression domain compared to scRNAseq data, as it is expressed by all undiff-SPG and in a small percentage of differentiating spermatogonia (diff-SPG). Interestingly, BMP4 was found to be expressed in the interstitial Leydig cells in both human and monkey testis, suggesting that the BMP4-BMPR1B pair represents a functional putative somatic-to-germ cell interaction. Remarkably, our examination of SPG subsets' relative positioning along the basal membrane in relation to interstitial tissue revealed significant distinctions. While PIWIL4+ undiff-SPG exhibited random distribution along the basal compartment across all the stages of the seminiferous epithelium cycle, GFRA1+ and NANOS3+ undiff-SPG, as well as c-KIT+ diff-SPG, displayed stage-dependent localization patterns. During the first half of the seminiferous epithelium cycle, GFRA1+ undiff-SPG occupied tubular regions adjacent to the interstitium, whereas in the second half they preferentially resided in tubule-tubule contact regions. NANOS3+ undiff-SPG, on the other hand, preferentially resided in regions facing the interstitium only at stages VI-VII, while from stage X to stage I, they predominantly inhabited tubule-tubule contact regions. Finally, c-KIT+ diff-SPG also preferentially resided in regions facing the interstitium, but only at stage VI-VII. The spatial organization of different SPG subsets appears to be coordinated with the stages of the seminiferous epithelium cycle, suggesting dynamic regulation of SSC behavior throughout sperm production. Our study contributes to the growing body of literature aimed at deciphering the complexities of SSC biology and the regulation of spermatogenesis in mammalian species, with implications for understanding male fertility.