Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Characterization of non-human primate spermatogonial compartment

Background: Human and non-human primate spermatogonia (SPG) share important similarities. In human, a new classification was proposed for SPG compartment based on protein markers such as, MAGEA4, GFRA1, KIT, etc. Even though, the kinetic of monkey SPG has been previously investigated, less is known about the expression profile of spermatogonial protein markers. Recently, sc-RNAseq data attempt to clarify the relationship between different cell subsets in SPG compartment in monkey. Aims: Here we tested the hypothesis that SPG marker expression and progression in human and non-human primates is conserved. To this end, we analyzed the expression of known human markers and novel sc-RNAseq identified markers in Macaca fascicularis SPG and their kinetics during the stages of the seminiferous epithelium cycle. Material and methods: Intact seminiferous tubules from Macaca fascicularis testis were stained by whole mount immunofluorescence as previously described. Stained seminiferous tubules were analyzed by confocal microscopy with 40× oil immersion objective. For each staining, 10 to 15 fields (250×250 μm) were randomly selected from different seminiferous tubules. For each field, confocal z-stacks were acquired (at 1 μm increments between z-slices). The mean fluorescence intensity was quantified using LAS AF Software. Results and Conclusions: We found that SPG marker progression is conserved between human and non-human primate with some quantitative differences: while the high heterogeneity of undifferentiated SPG seems to be conserved, the relative percentage of undifferentiated versus differentiating SPG in Macaca fascicularis that differ from what has been found in human SPG. Moreover, a novel population of KIT positive SPG has been found, characterized by a transient phenotype between undifferentiated and differentiating SPG, providing evidence that, as in the mouse spermatogenesis, the first generation of differentiating spermatogonia arise early during the cycle challenging the current model for monkey SPG expansion.