Germ Cell-Intrinsic and -Extrinsic Factors Govern Meiotic Initiation in Mouse Embryos

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Science  12 Dec 2008:
Vol. 322, Issue 5908, pp. 1685-1687
DOI: 10.1126/science.1166340


Retinoic acid (RA) is an essential extrinsic inducer of meiotic initiation in mammalian germ cells. However, RA acts too widely in mammalian development to account, by itself, for the cell-type and temporal specificity of meiotic initiation. We considered parallels to yeast, in which extrinsic and intrinsic factors combine to restrict meiotic initiation. We demonstrate that, in mouse embryos, extrinsic and intrinsic factors together regulate meiotic initiation. The mouse RNA-binding protein DAZL, which is expressed by postmigratory germ cells, is a key intrinsic factor, enabling those cells to initiate meiosis in response to RA. Within a brief developmental window, Dazl-expressing germ cells in both XX and XY embryos actively acquire the ability to interpret RA as a meiosis-inducing signal.

Diploid eukaryotes generate haploid cells via meiosis, a program of two successive cell divisions preceded by one round of DNA replication. The onset of this program is referred to as meiotic initiation. In mammals, debate has focused on whether meiotic initiation is promoted by factors extrinsic or intrinsic to germline cells (16). Meiotic initiation in female mice, commencing at embryonic day 12.5 (E12.5) (7, 8), is induced by an extrinsic factor, retinoic acid (RA) (810), but RA alone cannot account for the exquisite temporal and cell-type specificity of meiotic initiation. Although diverse somatic cell types are exposed and respond to RA during mammalian development (11), meiotic initiation is limited to the germ line. Indeed, embryonic germ cells do not respond specifically to RA until their migration ends, at the developing gonad. Does meiotic initiation in mammals also require an intrinsic competence factor expressed in germ cells? Consider the yeast Saccharomyces cereviseae, in which meiosis is induced by a nutrient-depleted environment (12). For an S. cereviseae cell to be competent to initiate meiosis in response to this extrinsic cue, the cell must express the a/α mating-type heterodimer (13). We wondered whether an analogous interplay of extrinsic and intrinsic factors governs meiotic initiation in mammals.

We considered the possibility that the Dazl (Deleted in azoospermia-like) gene might be an intrinsic meiotic competence factor, given the location and timing of its expression. In both XX and XY mouse embryos, germ cells begin to express Dazl at about the time of their arrival at the gonad, between E10.5 and E11.5 (14). No somatic lineage has been shown to express Dazl (15). Furthermore, Dazl-deficient mice are infertile because of germ cell–differentiation defects (1619). These defects are more consistent and pronounced in inbred C57BL/6 mice (19) than in noninbred mice (1618). Accordingly, we analyzed Dazl function in inbred C57BL/6 animals.

We began by testing whether germ cells survive in Dazl-deficient embryonic ovaries as germ cells of Dazl-deficient C57BL/6 embryonic testes undergo apoptosis, beginning by E14.5 (19, 20). We detected two germ cell markers—endogenous alkaline phosphatase (AP) activity (21) and mouse vasa homolog (MVH) protein (22)—in the ovaries of wild-type and Dazl-deficient embryos (fig. S1, A and B). We also found MVH protein in wild-type and Dazl-deficient neonatal ovaries (fig. S1C), which indicates that Dazl-deficient ovarian germ cells survive embryonic development (fig. S1, A and B) and persist through birth (fig. S1C).

We then compared the nuclear morphology of germ cells in wild-type and Dazl-deficient ovaries at E15.5. By this stage of development, many germ cell nuclei in wild-type ovaries exhibit the chromosome condensation that characterizes early meiotic prophase (Fig. 1A). By contrast, germ cells in Dazl-deficient ovaries do not display such condensation (Fig. 1A), which suggests that Dazl function might be required for meiotic prophase to occur. We then examined the expression of Stra8, which is required for premeiotic DNA replication and the subsequent events of meiotic prophase in germ cells of embryonic ovaries (8). As expected, Stra8 is expressed abundantly in wild-type ovaries at E14.5 (Fig. 1, B and F). In Dazl-deficient ovaries, Stra8 expression is dramatically reduced if not eliminated (Fig. 1, B and F), which suggests that Dazl might have an obligatory function upstream of meiotic initiation; this would account for the absence of meiotic chromosome condensation (Fig. 1A).

Fig. 1.

Dazl is required for meiotic chromosome condensation and expression of meiotic prophase markers in C57BL/6 XX embryos. (A) Photomicrographs of hematoxylin and eosin–stained ovarian sections from wild-type and Dazl-deficient E15.5 ovaries. Arrows indicate representative germ cell nuclei. (B) Whole-mount in situ hybridization for Stra8 mRNA in wild-type and Dazl-deficient E14.5 ovaries. (C) Immunohistochemical staining for γ-H2AX protein in sections of wild-type and Dazl-deficient E15.5 ovaries. (D) Whole-mount in situ hybridization for Dmc1 mRNA in wild-type and Dazl-deficient E15.5 ovaries. (E) Immunohistochemical staining for SYCP3 protein in sections of wild-type and Dazl-deficient E15.5 ovaries. (F) Quantitative reverse transcription polymerase chain reaction analysis of Stra8, Dmc1, Spo11, Sycp3, and Rec8 mRNA levels in wild-type and Dazl-deficient E14.5 ovaries. Plotted here are average fold changes, normalized to Hprt, in three independent biological replicates. Error bars represent SD among biological replicates.

If Dazl is required for Stra8 expression and meiotic initiation in embryonic ovaries, then germ cells in Dazl-deficient ovaries should not undertake meiotic recombination. We assayed whether Dazl-deficient female germ cells form DNA double-strand breaks (DSBs), which initiate meiotic recombination. Cells respond to DNA DSB formation by phosphorylating H2AX, a histone H2A variant, to generate γ-H2AX (23). As expected, immunostaining for γ-H2AX revealed the presence of DNA DSBs in many cells of wild-type ovaries at E15.5 (Fig. 1C). In contrast, Dazl-deficient ovaries are negative for γ-H2AX, indicating that DNA DSBs have not formed (Fig. 1C). In addition, we asked whether Dazl-deficient female germ cells express Spo11 and Dmc1, which encode, respectively, a topoisomerase required to form meiotic DSBs and a recombinase functioning in meiotic DSB repair (24). In previous studies, Stra8 was shown to be required for expression of Spo11 and Dmc1 in germ cells of embryonic ovaries (8). We found that, in Dazl-deficient ovaries, expression of Dmc1 and Spo11 is markedly reduced if not eliminated (Fig. 1, D and F). The absence of H2AX phosphorylation and the absence of Spo11 and Dmc1 expression indicate that Dazl-deficient female germ cells do not engage in meiotic recombination.

These findings are consistent with Dazl being required upstream of Stra8's function in meiotic initiation. Does Dazl deficiency simply recapitulate the Stra8 null phenotype (8), or does it cause additional abnormalities? We assayed the expression in Dazl-deficient embryonic ovaries of the Sycp3 and Rec8 genes, which encode, respectively, a component of the synaptonemal complex and a meiosis-specific cohesin (25). Both SYCP3 and REC8 proteins function through their loading onto chromosomes. In Stra8-deficient female germ cells, as previously reported, Sycp3 and Rec8 are transcribed and translated, but the encoded proteins do not load onto chromosomes and therefore do not perform their meiotic functions (8). We discovered that, in germ cells of Dazl-deficient embryonic ovaries, Sycp3 function is disrupted at an even earlier step: SYCP3 protein and mRNA levels are markedly reduced as compared with those in wild-type ovaries (Fig. 1, E and F). Similarly, Rec8 exhibited little or no expression in Dazl-deficient ovaries (Fig. 1F). Thus, Dazl is required for both Stra8-mediated initiation of meiosis in female germ cells and Stra8-independent expression of Sycp3 and Rec8 there.

We propose a pathway by which embryonic germ cells advance from a primordial state to the initiation of meiosis (Fig. 2). This pathway includes a newly posited cell state, the meiosis-competent gonocyte, whose derivation from a primordial germ cell requires the germ cell–intrinsic factor Dazl and whose progression to meiotic initiation and prophase in the female germ line requires the extrinsic meiosis-inducing factor RA and Stra8. We propose that this meiosis-competent cell state exists in both male and female embryonic germ lines, despite the fact that meiosis does not initiate in male embryos. The posited meiosis-competent gonocyte contains SYCP3 protein not yet loaded onto chromosomes (Fig. 2), which is consistent with the observation (6) that both male and female embryonic germ cells express SYCP3 protein before the sexes take different paths: Female germ cells advance to meiotic prophase, where SYCP3 functions, whereas male germ cells down-regulate SYCP3 and arrest in G0.

Fig. 2.

Embryonic germ cell states: a proposed path by which primordial germ cells acquire meiotic competence (in both XX and XY embryos) and subsequently initiate meiosis (in XX embryos) or undergo G0 arrest (in XY embryos). “Low [RA]” and “high [RA]” are consequences of differential expression of RA-inactivating enzyme CYP26B1 in embryonic testes and ovaries (9, 10).

Embryonic testicular germ cells express Stra8 when exposed to exogenous RA, even though they normally express Stra8 only after birth (9, 10). Our model predicts that ectopic expression of Stra8 in RA-treated embryonic testes should require Dazl function. We dissected testes from wild-type and Dazl-deficient E12.5 embryos, cultured them in the presence of 0.7 μM RA for 48 hours, and assayed Stra8 expression by whole-mount in situ hybridization. As previously reported (9, 10), RA induced robust expression of Stra8 in wild-type testes (Fig. 3A). In contrast, in Dazl-deficient testes, no induction was observed (Fig. 3A). To confirm that this failure to induce Stra8 expression in Dazl-deficient testes was not due to germ cell apoptosis (19), we performed a control in situ hybridization for Oct4 (Mouse Genome Informatics ID Pou5f1), a gene expressed in embryonic germ cells but not in gonadal somatic cells (26). We observed abundant Oct4 expression in RA-cultured testes, both wild-type and Dazl-deficient (Fig. 3B). Thus, expression of Stra8 in response to RA requires Dazl in embryonic testis and ovary alike, confirming that Dazl is a competence factor for meiotic initiation in embryos of both sexes.

Fig. 3.

Dazl is required for RA-induced expression of Stra8 in embryonic testes. Whole-mount in situ hybridization for (A) Stra8 mRNA and (B) Oct4 mRNA in wild-type and Dazl-deficient testes dissected at E12.5 and cultured for 48 hours in the presence of 0.7 μM RA is shown.

In S. cereviseae cells, expression of the a/α mating-type heterodimer is a prerequisite to launching the meiotic initiation program in response to an extrinsic cue. Our findings demonstrate that Dazl plays an analogous role in embryonic mice. In both a unicellular eukaryote and a complex animal, meiotic initiation is governed by a cell-intrinsic competence factor and an extrinsic inducing signal.

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