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

ABSTRACT: 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.

ORGANISM(S): Rattus rattus

PROVIDER: GSE182438 | GEO | 2022/03/15

REPOSITORIES: GEO

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Project description:Spermatogonial stem cells undergo both self-renewal to maintain the stem cell population and differentiation to produce mature sperm. These processes are controlled by both stem cell-intrinsic and external niche factors. DOT1L, the sole H3K79 methyltransferase, is dispensable for mouse embryonic stem cell self-renewal but instead functions as a barrier to somatic cell reprogramming. Here we show that DOT1L is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L in the germ cells show a failure in the maintenance of spermatogonial stem cells without a block in spermatogenic cell differentiation and thus a progressive loss of germ cells, leading to a Sertoli-cell-only syndrome. Chemical inhibition of DOT1L in cultured stem cells reduces the spermatogonial stem cell activity after transplantation. RNA-seq analysis reveals downregulation of Hoxc cluster genes in DOT1L-inhibited spermatogonia stem cells. Single cell RNA-seq analysis demonstrates that inhibition of DOT1L sequesters spermatogonial stem cells in a primitive state and prevents them from transitioning to a progenitor state. These results identify a new function for DOT1L in adult stem cells and provides a paradigm for regulation of spermatogonial stem cell self-renewal. Self-renewal of spermatogonial stem cells is vital to life-long production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli-cell-only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation, prevents spermatogonial stem cells from transitioning to a progenitor state, and sequesters them in a primitive state. Furthermore, DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

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

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