MAINTAINING SPERMATOGONIAL STEM CELL IDENTITY AND INTEGRITY

Aileen Rice Helsel

In the mammalian testis, precise balance between spermatogonial stem cell (SSC) self-renewal and transition to progenitor state is required for continual production of gametes throughout a male’s reproductive lifespan. However, there are currently no markers distinguishing rare SSCs from their more prevalent progenitors. Thus, studying in vivo SSC function has been challenging and our limited understanding of what drives SSC fate decisions has restricted the development of culture conditions for long-term expansion of SSCs in vitro. Previously, the transcriptional regulator inhibitor of DNA binding 4 (ID4) was identified as a potential SSC marker and an Id4-eGfp transgenic mouse was generated. Here we use the Id4-eGfp mouse to demonstrate that subpopulations of undifferentiated spermatogonia are identifiable based on ID4 expression levels. Spermatogonia expressing high levels of ID4 (ID4-EGFPBright) represent a potentially pure SSC population while those expressing low levels of ID4 (ID4-EGFPDim) represent the immediate progenitors and each possess a distinct transcriptome. Furthermore, study of an ID4 overexpression mouse model revealed that down-regulation of Id4 is required for SSC to progenitor transition. These data suggest that the ID4-EGFPBright population represents the ultimate SSCs (SSCultimate) while the ID4-EGFPDim spermatogonia are the immediate progenitors. Because the SSCultimate possess high regenerative capacity, autologous SSCultimate transplantation could be used to restore fertility to male cancer patients suffering chemotherapy-induced infertility. However, because SSCultimate are rare, in vitro expansion is a prerequisite to applying this therapy. Currently, murine cultures are used as a platform for developing human SSC culture conditions but they suffer from deficiencies, including decline in SSC number and regenerative integrity over time. Furthermore, establishing cultures from adult donors is inefficient. RNA sequencing data from ID4-EGFP+ spermatogonia revealed that Enolase 1 and 2 (Eno1/2) are up-regulated, suggesting that SSCs use glycolysis as their primary energy generating process. We found that refining culture conditions to Glycolysis Optimized (GO) conditions improved SSC regenerative integrity over a 6-month period in SSC cultures established from pup donors and the efficiency of establishing GO cultures from adult donors is 100%. Thus, gaining insight into SSC function through studying the ID4-EGFP+ population has direct applications in refining SSC culture conditions.

MAINTAINING SPERMATOGONIAL STEM CELL IDENTITY AND INTEGRITY

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