UCLA

Infertility is a broad disorder with numerous causes including physical, genetic and environmental. While techniques are currently in use to address certain causes of infertility, such as in vitro fertilization and hormonal therapies, there is currently no treatment option for those who are either unable to make or no longer possess viable gametes. Recently, advances have been made in the development of in vitro gametogenesis which, if perfected, promises an option for gametes to be derived from a patient’s own tissue. In order to bring this technique to fruition, further research is needed into the mechanics directing the specification and epigenetic reprogramming of the earliest stage of the germline, the primordial germ cells (PGCs).Mammalian PGCs are specified early in embryonic development and give rise to the entire adult germline. Following specification, PGCs undergo epigenetic reprogramming in order to establish a permissive epigenetic landscape for proper gametogenesis prior to differentiation into either oocyte or spermatogonial progenitors. Any errors in either of these processes can result in the complete loss of the germline and infertility. In order to better understand the mechanisms underlying PGC development, we utilized the in vitro PGC-like cell (PGCLC) differentiation to study human PGC specification and PGC-specific conditional knockout mice to assess epigenetic remodeling. In our studies into specification, we further characterized the differences between mouse and human PGC specification mechanisms. Using CRISPR/Cas9 gene editing we identified that EOMES directs human PGCLC specification, whereas in the mouse this role is accomplished by T. Our exploration into epigenetic reprogramming utilized a Cre/lox driven PGC-specific conditional knockout mouse to assess the role of epigenetic regulatory proteins during PGC differentiation. We used two knockouts, the first being UHRF1 which interacts with DNMT1 to promote DNA methylation maintenance and the second being EED, a key component of PRC2 which adds the repressive H3K27me3. Through this we identified that while UHRF1 appears to play no role in regulating the PGC stage of germline development, it is necessary for the viability of the spermatogonial stem cell population within the adult testes. In the case of EED, we identified that PRC2 is essential for regulating the timing of sex-specific differentiation in PGCs as well as a novel role for H3K27me3 in X chromosome decompensation within the embryonic testis. Finally, we identified a dual enrichment of H3K27me3 and DNA methylation within the promoters of gametogenesis genes at the time of PGC specification from the mouse epiblast. This provides an exciting glimpse into the complex interactions between the epigenetic regulatory networks that direct PGC differentiation. Further work will need to be conducted to identify the extent of these epigenetic regulator interactions in human PGCs and to apply these findings into developing better methods to more accurately recapitulate human PGC differentiation in vitro.