Re-establishment of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells reveals an important impact in differentiation capacity

Treatment of cancer in children is increasingly successful but leaves many prepubertal boys suffering from infertility or subfertility later in life. A current strategy to preserve fertility in these boys is to cryopreserve a testicular biopsy prior to treatment with the expectation of future technologies allowing for the reintroduction of stem cells and restoration of spermatogenesis. Spermatogonial stem cells form the basis of male reproduction, differentiating into all germ cell types, including mature spermatozoa and can regenerate spermatogenesis following transplantation into an infertile testis. Here we demonstrate for the first time that rat spermatogonial stem cells frozen for more than 20 years can be transplanted into recipient mice and produce all differentiating germ cell types. However, compared with freshly-isolated cells or those frozen for a short period of time, long frozen cells do not colonize efficiently and showed reduced production of spermatids. Single cell RNA sequencing reveled similar profiles of gene expression changes between short and long frozen cells as compared with fresh immediately after thawing. Conversely, following transplantation, long frozen samples showed enhanced stem cell signaling in the undifferentiated spermatogonia compartment, consistent with self-renewal and a lack of differentiation. In addition, long frozen samples showed fewer round spermatids with detectable protamine expression, suggesting a partial block of spermatogenesis after meiosis resulting in a lack of elongating spermatids. These findings strongly suggest that prolonged cryopreservation can impact the success of transplantation to produce spermatogenesis, which may not be revealed by analysis of the cells immediately after thawing. Single-cell RNA analysis of 56 samples across three treatments. Freezing state, 3 levels - fresh, short frozen, long frozen. Transplantation status, 2 levels - untransplanted and transplanted. Selection, 2 levels - unselected and EpCAM+ cells. Each treatment contains 3-5 biological replicates.