GASZ Is Essential for Male Meiosis and Suppression of Retrotransposon Expression in the Male Germline Lang Ma1., Gregory M. Buchold1,2., Michael P. Greenbaum1, Angshumoy Roy1, Kathleen H. Burns3, Huifeng Zhu 4 , Derek Y. Han1, R. Alan Harris5,6, Cristian Coarfa6, Preethi H. Gunaratne1,4", Wei Yan1,7", Martin M. Matzuk1,2,3* 1 Department of Pathology, Baylor College of Medicine, Houston, Texas, United States of America, 2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America, 3 Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America, 4 Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America, 5 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 6 Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States of America, 7 Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States of America Abstract Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI mice. In Gasz testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in nuage. Citation: Ma L, Buchold GM, Greenbaum MP, Roy A, Burns KH, et al. (2009) GASZ Is Essential for Male Meiosis and Suppression of Retrotransposon Expression in the Male Germline. PLoS Genet 5(9): e1000635. doi:10.1371/journal.pgen.1000635 Editor: Michael T. McManus, University of California San Francisco, United States of America Received March 23, 2009; Accepted August 6, 2009; Published September 4, 2009 Copyright: 2009 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were supported in part by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreements U54-HD07495 (to MMM) as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research, HD050281 (to WY), K08-CA134746 and Burroughs Wellcome Fund Career Award for Medical Scientists grant (to KHB), and T32-HD07165 (to GMB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mmatzuk@tmc.bcm.edu . These authors contributed equally to the work. " These authors also contributed equally to the work. Introduction The differentiation program of the germline is distinct from somatic cells in that resetting of the epigenome by demethylation of DNA and histones must take place for proper post-fertilization development of the embryo [1]. DNA demethylation occurs during primordial germ cell (PGC) migration as part of their normal development [2]. Different elements within the genome are remethylated at distinct time windows in a sex-specific fashion. Remethylation of retrotransposons occurs in the male germline at embryonic day 17.5 (E17.5) and in the female germline during postnatal oocyte maturation [1]. The resetting of the epigenetic state of the germline followed by the acquisition of male-specific methylation imprints, while a necessary component for post- fertilization development, exposes the germline to potential risk from retrotransposon mobilization [3]. Insects and mammals resolve this problem through the action of several classes of small RNAs including piRNAs (,27 nt PIWI family-interacting RNAs) [4–9]. Two classes of piRNAs, repeat-associated piRNAs and non- repeat-associated piRNAs based on their similarity to retro- transposons, are present in the germline of animals as primitive as sponges [10]. Repeat-associated piRNAs limit expression of retrotransposons at the post-transcriptional level and through epigenetic silencing by the recruitment of DNA methyltransferases including DNMT3A and DNMT3L [11–15]. In the absence of these small RNAs, retrotransposon expression is dramatically increased in the germline, leading to DNA damage and cell death. Regulation of retrotransposon repression is coordinated by proteins in spatially specialized compartments of ribonucleopro- tein-rich structures called nuage. According to the nomenclature proposed by Chuma et al. [16], embryonic prospermatogonia, postnatal spermatogonia and spermatocytes possess a form of nuage appearing as perinuclear granules transiently associated with mitochondria and thus termed intermitochondrial cement. In contrast, a single large granule of nuage, present in post-meiotic spermatids is called the chromatoid body [17,18]. Multiple proteins have been localized by electron microscopy to both intermitochondrial cement and the chromatoid body including PLoS Genetics | www.plosgenetics.org 1 September 2009 | Volume 5 | Issue 9 | e1000635