National Centers for Translational Research in Reproduction and Infertility

Elucidating the Roles of Mammalian Argonautes (AG03 & 4) in Meiotic Prophase 1

Cornell University

PI: P.E. Cohen

The four mammalian Argonaute family members are thought to share redundant functions in the microRNA pathway, yet only AG02 possesses the catalytic “slicer” function required for RNA interference. Whether AG01, AG03, or AG04 possess specialized functions remains unclear, but both Ago4 and, to a lesser extent, Ago3 are expressed highly in mouse spermatocytes. AG04 localizes to spermatocyte nuclei during meiotic prophase I, specifically at sites of asynapsis and in the transcriptionally silenced XY sub-domain, the sex body, a result that is exciting given the usually cytoplasmic location of the Argonaute proteins. The PI’s laboratory has recently generated Ago4 knockout mice and showed that during prophase I, the sex body assembles incorrectly in Ago4”’ mice, leading to disrupted meiotic sex chromosome inactivation (MSCI) associated with a dramatic loss of microRNAs from meiotic cells. Loss of MSCI results in apoptosis due to expression of Y-linked genes that are toxic to spermatocytes. However, expression analysis reveals that Ago3 may at least partially compensate for the loss of Ago4, and the localization of AG03 in spermatocytes is dramatically upregulated in Ago4”’ mice. This has led to the hypothesis thatAG03 and AG04 co-operate to regulate key events in genome silencing during mammalian meiosis. The goal of these studies is to further elucidate these roles in in meiotic silencing, and to understand further the redundancy that exists between these two Argonautes in this process. The three specific aims are: (1) To generate and characterize the germ cell phenotype of Ago3.Ago4 mutant mice and to compare germ cell phenotypes between single and double mutant animals as they pertain to meiotic silencing, (2) To identify which small RNA pathways are involved in AG04-related events in the sex body during prophase I, and (3) To isolate and define the AG04-related complexes present specifically within the mammalian sex body in order to elucidate the mechanism of silencing of this structure during prophase I. Taken together, these studies will provide the first fully integrated mechanistic view of how different Argonautes function within mammalian germ cells to promote proper meiotic entry and to ensure meiotic silencing during prophase I.