(Scale bars, 5 m
(Scale bars, 5 m.) We generated a polyclonal antibody against MAPS protein. late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general. Meiosis is a conserved and specialized form of cell division that produces haploid gametes for sexual reproduction. Meiosis prophase I is a relatively long period during which a series of orderly events occur. Programmed DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like Pozanicline protein SPO11 are formed at leptonema (1). The DSBs then undergo 3 end resection to generate 3 single-stranded DNA overhangs that are protected by the heterotrimeric RPA (replication protein A) complex. This event is followed by loading of the RAD51 recombinase and DNA meiotic recombinase 1 along with other proteins to enable strand invasion into duplex DNA for homolog pairing and recombination intermediate formation (2C4). All DSBs on autosomes are repaired, but only a subset leads to crossovers (5). When autosomes complete Pozanicline synapsis at pachynema, the X and Y chromosomes remain largely unsynapsed and compartmentalized into a nuclear subdomain called the XY body or sex body (6). In response to asynapsis, the X and Y chromosomes are subjected to chromatin modifications leading to transient transcriptional silencing in a process termed meiotic sex chromosome inactivation (MSCI) (6, 7). Over the past decades, the underlying molecular aspects of meiosis prophase I have been widely studied. Various mutant mouse models have been developed for studying the key events during prophase I progression through the pachytene stage (for reviews, see refs. 8C10). However, the machineries regulating this progression are still not fully understood (11, Pozanicline 12). Transcriptomes during meiosis in males have been reported, but the regulatory factors remain poorly described (13, 14). Nevertheless, epigenetic regulation is thought to be of great importance for the progression of meiosis prophase I, and the role of ubiquitination represents a topic of interest (15C17). For instance, a recent study reported that ubiquitinated proteins localized on the chromosome axis might regulate the stabilization and degradation of recombination factors in order to enable proper synapsis, DSB repair, and crossover formation (16). Wu et al. (18) have reported that the gene is specifically expressed in mouse testes, and the knockout male mice were infertile. To identify novel genes regulating meiosis prophase I progression in spermatocytes, we previously performed RNA sequencing of spermatogonia, leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids isolated by bovine serum albumin gradient sedimentation (19, 20). A number of genes were found to be highly expressed in spermatocytes relative to spermatogonia and somatic tissues, including genes, such as (male meiosis recombination regulator) and (speedy/RINGO cell cycle regulator family, member A), both of which we had further studied (21, 22). In this report, we selected for detailed analyses and independently concluded that it is a male germline-specific protein. We found that its RNA and protein were primarily expressed in high levels in pachytene spermatocytes. We named this protein MAPS (male pachynema-specific) protein because it is indispensable for the progression of pachynema. This protein is of 252 amino acids but has no GP9 predicted functional domain through analyses by an online protein domain predictor, ThreaDom (23). To investigate its function, we independently knocked out this gene and generated polyclonal antibodies. Indirect immunofluorescence (IF).