The Role of the Transcription Factor RUNX3 in Germline Development and Steady-State Spermatogenesis

Kassie Marie Stadler

doi:10.7273/000006380

Spermatogenesis is a highly complex process in which male germ cells undergo both proliferation and differentiation to become specialized haploid sperm. The progression of spermatogenesis involves the regulation of thousands of genes. However, the factors that regulate these changes in gene expression are not known. Within the male testes is the active site of spermatogenesis called the seminiferous tubules. Spermatogenesis is comprised of several cell types: spermatogonia stem cells (SSCs), undifferentiated spermatogonia, differentiating spermatogonia, spermatocyte, spermatids, and spermatozoa. Recent studies by our lab suggest that runt-related transcription factors (RUNX) may be important for spermatogenesis, but the role of RUNX proteins in the germline has not been explored. The RUNX family consists of three TFs, RUNX1, RUNX2, and RUNX3, that are known to be key regulators of developmental processes throughout the body. Preliminary enrichment analysis through single-cell RNA-seq shows Runx3 being upregulated in cell population with different predicted fates. We hypothesized that RUNX3 regulates gene networks associated with proper spermatogenesis and completion of meiosis. Using immunohistochemistry of mouse testes, we determined that RUNX3 was in subsets of both the undifferentiated and differentiated pools. Utilizing Stra8-CreTg to ablate Runx3 in the male germline, we discovered tubules that contain spermatogonia only and disrupted advance germ cell development. However, the Stra8-Runx3 knockout models only targeted the differentiating spermatogonia population , thus we are generated knockouts utilizing Nanos2-Cre to target cell types earlier in germline development. Nanos2-Runx3 cKO revealed similar results to the Stra8-Cre cKO with some tubules containing spermatogonia only and others with irregularities in advanced germ cells. Our data indicates the potential for RUNX3 as a fate regulator within the germline, however further studies and analysis are needed to determine the precise function. In the next chapter, we looked to understand the relationship between histone regulation and steady-state spermatogenesis maintenance. Histones are protein complexes that interact with DNA and form the complex chromatin structure. A more relaxed histone generally increases chromatin accessibility, opening the genome for transcription, while tight histones decrease chromatin accessibility. We studied the regulation of Histone 3 lysine 27 (H3K27) as H3K27 trimethylation (H3K27me3) function in the germline is still to be elucidated. We hypothesize increased H3K27me3 would cause disruptions in spermatogenesis and germline maintenance. KDM6A/6B are two main demethylases of H3K27me3, thus we utilized the inhibitor GSK-J4 to inhibit KDM6A/6B within the germline. We developed an intraperitoneal injection series using a range of doses, 10-100 mg/kg, over the course of 9 days. Histological examination of the 10 and 25 mg/kg treatment groups indicated a function in germline maintenance as some tubules were completely devoid of germ cells. Further experiments revealed negligible results as no germline disruptions were present in the high dose series. Overall, these studies suggest increased H3K27me3 does not affect germline maintenance, however additional experiments are needed to confirm these results. Overall, the work in this dissertation describes the potential function of RUNX3 within developmental spermatogenesis, and the negligible effect increased H3K27me3 has on the germline.

The Role of the Transcription Factor RUNX3 in Germline Development and Steady-State Spermatogenesis

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