Spermatogonial stem cell regulation and maintenance of male fertility

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Project Summary Infertility affects 15% of couples and in about half of these cases the male partner contributes to the failure to conceive. One cause of male infertility is an inability to produce adequate amounts of sperm. Continuous an robust sperm production is dependent on the spermatogonial stem cells (SSCs), which have the capacity to divide to produce more SSCs as well as produce cells that differentiate into sperm. The regulation of SSC proliferation and differentiation is controlled by a complex regulatory environment called the stem cell niche, which is comprised of several testicular cell types including, Sertoli, Leydig, myoid, and macrophages. Furthermore, SSCs are positioned near the vasculature suggesting that circulating factors also contribute to the SSC niche. How this complex niche manages the SSCs and how the SSCs incorporate the multitude of signals provided by the niche is not understood. To investigate regulatory mechanisms governing SSC maintenance, this study utilizes the genetic and experimental attributes of the zebrafish model. This study is aimed at understanding two processes found to be important for SSCs. Aim1 will investigate SSC maintenance by post-transcriptional RNA regulation. Our preliminary work identified the Adad1 RNA binding protein as essential for either SSC establishment or maintenance. We will test the hypothesis that RNA regulation by the Adad1 RNA binding protein is critical for SSC regulation and sperm production. First, to understand how RNA regulation controls SSC function, the basic processes regulated by Adad1 in SSCs will be defined: proliferation, differentiation, quiescence, or apoptosis. Next, to discover mechanisms subject to post- transcriptional RNA regulation in SSCs, we will identify Adad1 target RNAs and ask how stability of target RNAs is affected by Adad1. Aim2 will investigate how a moderate tissue stressor, high sperm demand from frequent breeding, promotes SSC activation and robust sperm production. Our previous work demonstrated that the Cdk21 cell-cycle regulator is necessary to maintain the germline in breeding males but not in non- breeding males. We hypothesize that the SSCs respond to moderate stressors, such as high sperm demand, by invoking a proliferative response. Furthermore, we propose that Cdk21 participates in this process through activation of the quiescent stem cell (QSC) population. We will test if the wild-type testis invokes a proliferative response to frequent breeding and if this response involves SSCs. Furthermore, we will test if cdk21 is required for this proliferative response and specifically if it regulates exit from quiescence in QSCs. The proposed experiments will reveal insights as to how RNA-based regulation promotes continued fertility through SSC regulation. In addition, this work may reveal a tissue-wide response to high sperm demand for maintenance of testis homeostasis and robust fertility. Knowledge gained from this study can be applied to a better understanding of human reproductive health and to improved methods for diagnosis and treatment of infertility, assisted reproductive technologies, and development of male-targeted family planning methods.