Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1␤ and H3 di-and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.

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The mitochondrial electron transport chain (ETC) performs several critical biological functions, including maintaining mitochondrial membrane potential, serving as an electron sink for important metabolic pathways, and contributing to the generation of ATP via oxidative phosphorylation.The ETC is important for the survival of many eukaryotic organisms, including intracellular parasites such as the apicomplexan Toxoplasma gondii. The ETC of T. gondii and related parasites differs in several ways from the ETC of the mammalian host cells they infect, and can be targeted by anti-parasitic drugs, including the clinically used compound atovaquone. To characterize the function of novel ETC proteins found in the parasite and to identify new ETC inhibitors, a scalable assay that assesses both ETC function and non-mitochondrial parasite metabolism (e.g., glycolysis) is desirable. Here, we describe methods to measure the oxygen consumption rate (OCR) of intact T. gondii parasites and thereby assess ETC function, while simultaneously measuring the extracellular acidification rate (ECAR) as a measure of general parasite metabolism, using a Seahorse XFe96 extracellular flux analyzer. We also describe a method to pinpoint the location of ETC defects and/or the targets of inhibitors, using permeabilized T. gondii parasites. We have successfully used these methods to investigate the function of T. gondii proteins, including the apicomplexan parasite-specific protein subunit TgQCR11 of the coenzyme Q:cytochrome c oxidoreductase complex (ETC Complex III). We note that these methods are also amenable to screening compound libraries to identify candidate ETC inhibitors.