In this study, we find that the DM domain protein DMRT7 is required for male germ cells to complete meiotic prophase but is dispensable in the female germ line. In males, DMRT7 expression is highest in pachytene spermatocytes, and the protein preferentially localizes to the XY body. Consistent with this expression, we found that most mutant male germ cells arrest in pachynema and undergo apoptosis, although a small proportion can progress to diplonema and sometimes beyond. Examination of chromatin and nascent transcription in mutant cells that progressed to diplonema revealed sex chromatin abnormalities, as discussed below.
The pachytene stage of prophase involves tremendous chromosomal changes as the homologs align, synapse, and recombine. During this period, at least one pachytene surveillance system exists to monitor key events of meiotic progression. Cells in which any of these events is anomalous are efficiently eliminated by apoptosis [46
]. Another key event of pachynema in male mammals is the packaging of the sex chromosomes into the XY body and the establishment of MSCI. Examination of male meiosis in XYY mice and mice carrying a sex-chromosome-to-autosome translocation showed that cells in which a sex chromosome escapes MSCI are eliminated prior to late-pachynema [17
]. This indicates that the establishment of MSCI also is subject to surveillance.
Since the arrest and apoptosis of Dmrt7 mutant spermatocytes could result from perturbation of any of the critical pachytene events mentioned above, we tested whether they occur abnormally in the mutant cells. We found that chromosomal synapsis and recombination appear normal in Dmrt7 mutant cells. We therefore focused on the XY body, the most prominent site of DMRT7 accumulation. First, we tested whether the XY body forms and MSCI is established in Dmrt7 mutant cells. Surprisingly, we found that these cells form an XY body with normal morphology and proper accumulation of all the chromatin marks we examined. Moreover, Cot-1 hybridization and analysis of RBMY expression demonstrated that MSCI initiates normally in the XY body of mid-pachytene Dmrt7 mutant cells.
We did, however, observe three specific defects in the sex chromatin of Dmrt7 mutant germ cells that avoided arrest in pachynema and were able to enter diplonema. Normally cells accumulate H3-2meK9 and H3-3meK9 marks and HP1β protein on the sex chromatin as they progress to diplonema, but we observed mutant diplotene cells lacking these features. Thus, although a minority of Dmrt7 mutant germ cells can progress from pachynema to diplonema, there are defects in sex chromatin modification during the transition. A function in male sex chromatin can reconcile the findings that DMRT7 is required for meiosis, but only in males, and is present only in mammals. A proportion of mutant diplotene cells have apparently normal sex chromatin (for example, M); these are likely to be the cells that can progress beyond diplonema.
Because most Dmrt7 mutant germ cells are eliminated by apoptosis around the time at which we observed sex chromatin defects, a simple model is that the apoptosis is a consequence of the sex chromatin defects. The reciprocal situation (sex chromatin defects caused by apoptosis) is possible, but seems unlikely, because we observed mutant cells with sex chromatin defects but no indications of apoptosis. Alternatively, apoptosis and abnormal sex chromatin may be two independent consequences of Dmrt7 loss. This question cannot be answered definitively until we know the detailed molecular mechanism of DMRT7.
A number of other proteins have been identified that interact with the XY body, including histone variants and modified histones, a testis-specific histone methyl transferase, chromobox proteins, an orphan receptor germ-cell nuclear factor, and recombination-related proteins [60
]. A common feature of these proteins is involvement with heterochromatin and/or transcriptional repression. DMRT7 is unusual among XY body proteins in being related to highly site-specific transcriptional regulators. An attractive speculation is that DMRT7 may provide sequence specificity in recruiting other proteins, such as chromatin modifiers, to the XY body as part of the transition to PMSC. Chromatin regulation may be a common mechanism for DM domain proteins, as we find that other DM domain proteins associate with chromatin modifying complexes (M. W. Murphy, D. Zarkower, and V. J. Bardwell, unpublished data).
The finding that Dmrt7
is essential for mammalian meiosis expands the known functions of this gene family. Invertebrate DM domain genes so far have only been found to function in somatic cells. Two other DM domain genes, Dmrt1
do affect germ cell development in the mouse. Dmrt1
is required in pre-meiotic male germ cells for differentiation of gonocytes into spermatogonia, as well as in Sertoli cells, but it is not expressed in meiotic cells [31
] (S. Kim and D. Zarkower, unpublished data). The requirement for DMRT1 in pre-meiotic germ cells and DMRT7 in meiotic germ cells demonstrates that DM domain proteins act at multiple critical points of male germ cell development. Ovaries of Dmrt4
mutant females have polyovular follicles (follicles containing multiple oocytes), but it is unknown whether this reflects a defect in the germ line or the soma. It is notable that at least three mammalian DM domain genes affect gonadal development only in one sex, given the similar roles of related proteins in directing sex-specific somatic development in other phyla.
is present, not only in placental mammals, but also in marsupials and a monotreme (egg-laying mammal), the platypus, which has a clear Dmrt7
]. However, no close Dmrt7
ortholog has been reported in nonmammalian vertebrates, and our database searches did not reveal one. Thus, Dmrt7
likely arose, presumably by duplication and divergence of another Dmrt
gene, shortly before or coincident with the mammalian radiation. Monotremes have five X and five Y chromosomes, which form an extended pairing chain during meiosis and appear unrelated to the sex chromosomes of the other mammals [61
]. The presence of Dmrt7
in both lineages may support a common origin for either the sex chromosomes or the sex chromatin of monotremes and other mammals. A plausible model is that Dmrt7
evolved during the establishment of mammalian sex determination to help cope with ancestral differences in gene dosage, chromosome pairing, recombination, or other meiotic issues arising from sex chromosome heteromorphy. In this regard, we speculate that the recruitment of Dmrt7
during mammalian evolution may be analogous to the recruitment of chromatin regulatory complexes to achieve somatic dosage compensation during evolution of heteromorphic sex chromosomes in several phyla (reviewed in [62
]). It will be of interest to determine whether DMRT7 localizes to sex chromosomes during monotreme meiosis.
In summary, we have found that the mammal-specific DM domain protein DMRT7 is essential for meiotic prophase progression in males. DMRT7 localizes to the sex chromosomes after they are assembled into specialized heterochromatin, and many Dmrt7 mutant cells have epigenetic defects in the modification of the sex chromatin between pachytene and diplotene. Although Dmrt7 belongs to an ancient and conserved gene family, it is found only in mammals, and to our knowledge DMRT7 is the only example of a mammal-specific protein that is essential for meiosis. It will be important to determine the precise mechanism by which DMRT7 affects sex chromatin regulation during male meiosis.