We have examined the role of DMRT1 in spermatogonial development and differentiation by conditional gene targeting. Loss of DMRT1 in undifferentiated spermatogonia causes them to precociously exit the normal program of mitotic proliferation and differentiation, instead switching to the meiotic program and eventually completing germ cell differentiation to form spermatids. DMRT1 represses Stra8 activation by both direct transcriptional regulation and by a general inhibition of RA-dependent transcriptional activity. Repressing both Stra8 transcription and RA signaling may provide a “fail-safe” mechanism for DMRT1 to block meiosis and ensure the completion of spermatogonial proliferation and differentiation. A model for the control of spermatogonial development by DMRT1 is shown in .
How does DMRT1 limit RA-dependent transcription? In Stra8 DMRT1 binds near two promoter-proximal RA response elements and prevents expression, likely by blocking activation of Stra8 by the retinoic acid receptor transcriptional regulatory complex. DMRT1 may regulate other RA-responsive genes by this mechanism. However, we also detected changes in expression of genes involved in RA-dependent transcription that did not have promoter-proximal binding of DMRT1, including Crabp2, Tbx1, and Cyp26a1, and DMRT1 bound the promoters of the synthetic enzymes Adh1 and Aldh1a1. These observations and the misregulation of the RAREhsplacZ reporter suggest that DMRT1 also controls the accumulation and/or transcriptional potency of RA in germ cells. RA signaling involves feedback regulation, so further work will be needed to determine precisely how DMRT1 controls this signaling network.
DMRT1 levels vary in spermatogonia as they develop: DMRT1 is highest in undifferentiated spermatogonia, lower in differentiating spermatogonia, and disappears as B spermatogonia make the transition to preleptotene cells and initiate meiosis. The two decrements in DMRT1 expression correspond to steps of spermatogonial differentiation known to require RA. Since DMRT1 inhibits RA signaling, we suggest that DMRT1 may act as a transcriptional “rheostat” to modulate RA signaling during spermatogonial progression. Our previous finding that Dmrt1
tumor-suppressive activity in fetal germ cells is highly sensitive to gene dosage (Krentz et al., 2009
) is consistent with this model. A key remaining question is how DMRT1 expression is reduced to allow the switch to meiosis. Tamoxifen-induced deletion of Dmrt1
caused rapid depletion of DMRT1 in germ cells but not in Sertoli cells; this differential stability may be an important part of the regulatory mechanism.
The source(s) of the intratubular RA that promotes meiotic initiation is unknown, because genes involved in RA metabolism and storage are expressed in Sertoli cells and germ cells (meiotic and postmeiotic) (Vernet et al., 2006b
) and RA readily crosses cell membranes (Kamp et al., 1993
; Noy, 1992
). Thus control of spermatogonial development and meiotic initiation by RA might involve both the germ cell intrinsic regulation of RA signaling that we describe here and fluctuating levels of RA produced by other cell types within the tubule.
The RNA binding protein NANOS2 is a meiotic repressor in fetal male germ cells: Nanos2
mutant germ cells express STRA8 and enter meiosis around E17 (Suzuki and Saga, 2008
). The similarity with postnatal DMRT1 function raises the possibility of a mechanistic link between the two proteins. Postnatally NANOS2 is expressed only in As
spermatogonia, and fetal loss of DMRT1 does not cause inappropriate meiosis, so these proteins must have at least partially separate functions. NANOS2 does not bind Stra8
mRNA in vitro, but can bind Taf7l
, whose expression was affected by loss of DMRT1 (Barrios et al., 2010
). Thus the two proteins may control some shared target genes, albeit by different mechanisms and in incompletely overlapping germ cell types. Genome-wide identification of downstream targets for these proteins will help resolve this question.
When DMRT1 is deleted from undifferentiated spermatogonia, the mutant cells rapidly enter meiosis and produce elongated spermatids. Is the spermatogonial differentiation program therefore dispensible for male gametogenesis? In the Ngn3-cre mutant testes most germ cells lost DMRT1 as ECAD-positive undifferentiated spermatogonia. However mutant spermatogonia expressing the differentiation marker c-KIT also were present ( and data not shown), suggesting that although mutant cells bypass mitotic proliferation, they do undergo at least some spermatogonial differentiation prior to meiosis. Thus this program of gene expression may be a necessary part of male gametogenesis.
Does DMRT1 regulate female meiosis? Although DMRT1 is expressed in female germ cells during the transition from mitosis to meiosis, Dmrt1
null mutant females are fertile (Raymond et al., 2000
). Nevertheless, it will be important to ask whether meiotic initiation occurs normally in the fetal ovaries of Dmrt1
Loss of DMRT1 in germ cells uncouples meiotic initiation from the seminiferous epithelial cycle and disrupts cyclical gene expression in Sertoli cells. The effect on Sertoli cell gene expression might reflect inappropriate signaling between germ cells and Sertoli cells or a deficit in a germ cell type necessary for cyclical gene expression in Sertoli cells. Either way, our results are consistent with the idea that a germ cell-intrinsic program can influence Sertoli cells to achieve coordinated progression of the seminiferous epithelial cycle. A useful test of this idea is to ask whether transplantation of rat spermatogonia into the mouse testis causes recipient Sertoli cells to adopt the 13 day rat cycle.
DMRT1 is a DM domain transcription factor (Raymond et al., 1998
). These proteins control sexual differentiation and/or primary sex determination in varied phyla and occur throughout metazoans. It therefore will be important to ask whether DM domain proteins control the mitosis to meiosis transition outside mammals. The rise of the metazoans created a need for mitotic and meiotic cells to coexist in the same individual. DMRT1 controls the mitosis to meiosis switch (this work) and also prevents germ cells from adopting somatic cell fates (Krentz et al., 2009
). Based on these functions, we speculate that DM domain genes may have evolved in early metazoans to allow meiotic germ cells to coexist with somatic cells, and later assumed control of other reproductive functions.
The results presented here establish DMRT1 as a key regulator of spermatogonial development and differentiation that controls the mitosis versus meiosis switch in male germ cells of mammals. These findings expand the mechanistic understanding meiotic control in male mammals and suggest that in adults as in embryos the regulation of RA signaling is critical. Our results provide an entry point for further elucidation of meiotic regulation during the cycle of the seminiferous epithelium. In addition, understanding how DMRT1 functions to promote spermatogonial development and prevents meiosis may aid in the artificial manipulation of spermatogenesis for a variety of applications.