Ubiquitin Conjugase UBE2K Is Essential for Normal Rat Development and Spermatogenesis

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2016-04-14

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Chaudhary, Jaideep

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Animal models allow us to investigate questions about human physiology using in vivo systems. Following the advent of gene targeting by homologous recombination in mouse pluripotent embryonic stem cells during the early 1980's [Reviewd by (Hamra, 2010)], the laboratory mouse has emerged as the most widely published animal model in science (NCBI, PubMed). Interestingly, prior to the year 2000, annual publications in the laboratory rat outnumbered annual publications in the mouse by greater than 2-3 fold in almost every field of science spanning each decade in the 20th century (NCBI, PubMed). Popularity of the laboratory rat as a model organism in science derived from its many attributes for modeling human physiology and disease in a laboratory scale mammal. However, genetic tools to effectively manipulate the rat genome lagged behind that of the mouse for almost 4 decades up until ~2009 due to inabilities to maintain pluripotent rat cells in culture, and inefficient methods for micro-manipulating rat early embryos. Now, several laboratories have made large strides to advance technologies to genetically engineer rats (Geurts et al., 2009; Tong et al., 2011), including our laboratory, which succeeded in applying stem cell-based technologies to the rat's "genomic toolbox" by using donor stem spermatogonia as vectors to directly genetically modify the rat's germline (Chapman et al., 2015; Hamra et al., 2002; Izsvak et al., 2010). For this dissertation, I focused on phenotyping the growth and reproduction defects in the UBE2K knockout rat strain, which happens to represent the first knockout rat strain reported using genetically selected germline stem cells form culture (Izsvak et al., 2010). I reasoned that phenotyping UBE2K rats in more detail would allow me to formulate testable hypotheses on the cellular and molecular mechanisms by which UBE2K functioned to regulate rat body growth and reproduction. UBE2K is an ubiquitin ligase, and knocking its gene (Ube2k) out in rats resulted in stunted growth, compromised motor capacity of hind legs and infertility. I found infertility in male UBE2K-deficient rats to be caused by an arrest during meiosis-I that prevented the zygotene to pachytene spermatocyte transition. Based on known interactions of UBE2K, and the biochemical function of UBE2K as ubiquitin ligase, I hypothesized that UBE2K is a component of the PRC1 complex that ubiquitinates H2A to prevent transcription as a mechanism necessary for germ cells to undergo meiosis. I showed using immunostaining, that there is an inverse correlation between H2A ubiquitin staining and UBE2K expression that is disrupted in UBE2K-deficient rats, supporting my hypothesis. However, future work will need to address whether UBE2K directly or indirectly associates with the PRC1 complex. Based on my research presented in this Dissertation, I propose a novel function for the UBE2K ubiquitin conjugase in meiotic transcriptional control during spermatogenesis.

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