Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.

AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline.

The four mammalian Argonaute family members are thought to share redundant functions in the microRNA pathway, yet only AGO2 possesses the catalytic "slicer" function required for RNAi. Whether AGO1, AGO3, or AGO4 possesses specialized functions remains unclear. Here we show that AGO4 localizes to spermatocyte nuclei during meiotic prophase I, specifically at sites of asynapsis and the transcriptionally silenced XY subdomain, the sex body. We generated Ago4 knockout mice and show that Ago4(-/-) spermatogonia initiate meiosis early, resulting from premature induction of retinoic acid-response genes. During prophase I, the sex body assembles incorrectly in Ago4(-/-) mice, leading to disrupted meiotic sex chromosome inactivation (MSCI). This is associated with a dramatic loss of microRNAs, >20% of which arises from the X chromosome. Thus, AGO4 regulates meiotic entry and MSCI in mammalian germ cells, implicating small RNA pathways in these processes.

Small RNAs and RNAi pathways in meiotic prophase I.

RNA interference is involved in many aspects of cell biology, and the recent identification of germ-cell specific small RNAs has led to speculation that RNAi might also be involved in gametogenesis. Work in yeast indicates that RNAi is involved in establishing and maintaining heterochromatin at centromeres, an important component of yeast and mammalian meiosis. Here we review developments in the field of RNAi and relate these to possible roles in mammalian gametogenesis.