Mouse Strains Used and Genotyping

All animal experimentation was approved by the Animal Welfare and Ethical Review Body at MRC Harwell and conducted in accordance with the Society for the Study of Reproduction's specific guidelines and standards. Mice used in this study were bred by license under the Animals (Scientific Procedures) Act with approval from the U.K. Home Office (PPL 30/2877). Mice were housed in individually ventilated cages in a specific opportunistic pathogen-free environment. Further details of micro- and macro-environmental conditions are available on request. Adult mice were humanely euthanized by dislocation of the neck, confirmed by palpation, and embryos were decapitated in ice-cold, phosphate-buffered saline solution.

Adult mice and embryos were sexed by a PCR assay that simultaneously amplifies the Ube1y1 and Ube1x genes, using the following primer pair: 5′-TGGATGGTGTGGCCAATG-3′ and 5′-CACCTGCACGTTGCCCTT-3′ [17]. Mice harboring targeted null alleles for Map2k3^tm1Flv [18] and Map2k6^tm1Flv [11] were purchased from Jackson Laboratory. Both these null alleles encode proteins lacking catalytic domains essential for activity. Mice were maintained on the C57BL/6J (B6) background. The Map3k4^tm1Flv targeted allele and detection of the Y^AKR chromosome have been previously described [4].

Generation of Embryos and Expression Analyses

Noon on the day of the copulatory plug was counted as 0.5 dpc. Embryos were staged accurately based on the number of tail somites (ts) or limb and gonad morphology. Wholemount in situ hybridization (WMISH) analysis of embryonic tissues was performed as previously described [4]. Probes for Sox9 [19], Sry [20], and Stra8 [4, 21] have been previously described. WMISH was performed on at least three independent gonad samples from each embryonic class. Probes for Map2k3 and Map2k6 were generated by amplification of the 3′ untranslated region (3′UTR) and coding sequences from gonadal cDNA, using the following forward and reverse primers: Map2k3 3′UTR (5′-TCTGCCAGCATCCATACATC-3′ and 5′-GGTGGTAACCAGAGGCTGTC-3′); Map2k3 coding (5′-GGATATCCTGCGTGTCCAAG-3′ and 5′-AGCCAGCATCCATTGTCTTT-3′); Map2k6 3′UTR (5′-TGGTCGACCCTACTGTGGAT-3′ and 5′-CAGCCATGAGAGCTGCAAG-3′); Map2k6 coding (5′-AACAGCCTCAGACCAGTTCC-3′ and 5′-AGGAGCCATGTATGGTTTGC-3′). These probes gave identical results in respect to both genes.

Quantitative Reverse Transcription Polymerase Chain Reaction

Quantitative RT-PCR analyses of gene expression were performed as previously described [5]. Briefly, total RNA was extracted from gonadal tissue using an RNeasy plus micro kit (Qiagen). Two-hundred and fifty nanograms of RNA was reverse transcribed, and quantitative RT-PCR was performed using a 7500 Fast Real-Time PCR system (Applied Biosystem). At least three samples for each genotype were analyzed. Primer sequences are available on request.

Immunohistochemistry

Antibodies to the following proteins were utilized in this study: FOXL2 (a kind gift from Dagmar Wilhelm and Peter Koopman [22]), AMH (sc28912; Santa Cruz), and SRY (a kind gift from Makoto Tachibana [23]). Immunostaining was performed on sectioned, paraffin wax-embedded tissue from two independent gonad samples using the above primary antibodies (1:100) and Alexa Fluor 594 (AMH) or 488 (FOXL2, SRY) conjugated secondary antibodies (1:200). Images were captured using a Zeiss 710 multiphoton microscope.

Map2k3 and Map2k6 Are Expressed in the Embryonic Gonad

Expression of Map2k3 and Map2k6 has previously been reported in the somatic supporting cell lineage of XX and XY gonads at 11.5 dpc after microarray expression profiling [24]. We examined the embryonic gonadal expression of Map2k3 and Map2k6 using WMISH at the sex-determining stage of 11.5 dpc (17–18 ts stage) (Fig. 1). Map2k3 expression was detected in the gonad and mesonephros; expression in the mesonephros was higher in the Wolffian duct (Fig. 1A). Map2k6 expression was detected in the gonad and mesonephros at 11.5 dpc, with levels appearing higher in the mesonephros (Fig. 1B). Quantitative RT-PCR analyses of subdissected gonadal tissue at 11.5 dpc revealed expression of Map2k3 and Map2k6 in XX and XY wild-type embryos (Fig. 1C); Map2k3 expression in XX gonads is higher than XY gonads by a small, but significant, amount. The expression of both genes was confirmed by comparison of wild-type and mutant gonadal tissue, revealing absence of expression of both genes in mutant gonads (Fig. 1, D and E).

FIG. 1

Map2k3 and Map2k6 are expressed in the developing mouse gonad at the sex-determining stage. A) Wholemount in situ hybridisation (WMISH) at 11.5 dpc showing Map2k3 expression in the gonad (area within the white dotted lines) and, to a lesser extent, the adjacent mesonephros, with prominent expression in the Wolffian duct. No sexual dimorphism is evident. Bar = 500 μm. B) WMISH at 11.5 dpc showing widespread expression of Map2k6 in the developing gonad and mesonephros, with stronger signal detected in the mesonephros. C) Comparison of expression of Map2k6 and Map2k3 in XY and XX wild-type gonads. Map2k3 expression in XX gonads is higher than XY gonads by a small, but significant, amount. Error bars throughout show standard error of the mean; *P < 0.05. D) Quantitative RT-PCR was performed using primers that amplify exonic sequences that are deleted in the Map2k3 null allele. Expression detectable in wild-type gonads is all but absent in gonads from MAP2K3-deficient embryos, validating the specificity of the expression measurement. E) Quantitative RT-PCR was performed using primers that amplify exonic sequences that are deleted in the Map2k6 null allele. Expression detectable in wild-type gonads is absent in gonads from MAP2K6-deficient embryos. RNA expression levels were normalized to those of Hprt1 using the ΔΔCt method. Error bars show standard error of the mean.

Analyses of Mutant Embryos on the C57BL/6J (B6) Background Reveal Subtle Defects in Map2k6-Deficient Gonads

In order to test whether MAP2K3 and MAP2K6 function in mouse testis determination, we examined sexual development in B6 XY embryos homozygous for a Map2k3-targeted null allele (Map2k3^tm1Flv, henceforth Map2k3⁻) or a Map2k6 null (Map2k6^tm1Flv, henceforth Map2k6⁻). Morphological examination and marker analysis at 14.5 dpc with the Sertoli cell marker Sox9 and the ovary-specific meiotic germ cell marker Stra8 revealed no abnormalities of gonad development in embryos lacking Map2k3 (Fig. 2A). Embryos lacking Map2k6 exhibited no overt abnormalities apart from a very small patch of Stra8-positive cells at one pole of the mutant gonad that is not present in XY wild-type controls (Fig. 2B). This abnormality was subtle but consistent. It may reflect a very brief delay in initiating testis determination in Map2k6-deficient embryos because, in other contexts, delayed testis determination, at least in part due to delayed Sry expression, is associated with Stra8-positive cells at the poles of XY gonads at 14.5 dpc [5]. Consistent with such a delay, Map2k6-deficient XY gonads at 14.5 dpc also contained a number of FOXL2-positive cells at one pole exhibiting nuclear staining (Fig. 2C). FOXL2 normally marks ovarian granulosa cells at this stage [22], suggesting that a small number of somatic cells in these XY mutant gonads have undergone sex reversal.

FIG. 2

Characterization of gonadal phenotypes at 14.5 dpc in B6 embryos lacking MAP2K3 and MAP2K6. A) WMISH of Sox9 and Stra8 in gonads from MAP2K3-deficient embryos reveals no overt abnormalities. Bar = 500 μm. B) WMISH of Sox9 and Stra8 in gonads from MAP2K6-deficient embryos reveals a small but consistent patch of Stra8 expression (arrow) at one pole of the mutant gonad. Bar = 500 μm. C) Immunostaining of gonadal tissue sections comparing MAP2K6-deficient B6 XY embryos and wild-type controls with the Sertoli cell marker AMH (red) and granulosa cell marker FOXL2 (green) reveals a small cluster of FOXL2-positive cells at the caudal pole of the mutant (2k6^−/−) gonad (white box) exhibiting nuclear staining (see higher magnification image in inset). A large number of fluorescent cells in the interstitium of the mutant gonad are blood cells. The occasional interstitial cell can also be seen exhibiting nuclear staining, but these rare cells are also observed in wild-type control gonads and are of unknown significance. The dotted line indicates the border between the gonad (right) and mesonephros (left). Bar = 500 μm. D) Compound mutant embryos (Map2k3^+/−, Map2k6^−/−) have gonads exhibiting a larger area of Stra8 expression at one pole, indicating some redundancy between MAP2K3 and MAP2K6 function during testis determination. Bar = 500 μm. E) WMISH reveals more extensive regions of Stra8 expression at the gonadal poles in compound mutants (Map2k6^−/−, Map3k4^+/−) when compared to littermates only lacking Map2k6 (Map2k6^−/−). Bar = 500 μm.

In order to address whether Map2k3 compensates for the absence of Map2k6, we generated XY embryos lacking both copies of Map2k6 and one copy of Map2k3 (Map2k3^+/−, Map2k6^−/−) (Fig. 2D). These compound mutants exhibited pronounced abnormalities of testis development at 14.5 dpc. Testis cord morphology was noticeably less well defined in comparison to littermate controls, and clumps of Stra8-positive cells, sometimes quite large, were detected at one or both poles of compound mutant gonads (Fig. 2D). These data suggest disruption to testis determination by a further delay in its initiation and propagation to the gonadal poles caused by the additional loss of one copy of Map2k3.

A previous study of the role of GADD45γ and MAP3K4 in testis determination showed that B6 XY embryos heterozygous for a Map3k4 null allele form normal testes at 14.5 dpc, apart from a very small patch of Stra8 expression at one pole (see Fig. 4 of [6]), reminiscent of Map2k6-deficient gonads at the same stage. We generated embryos combining these two genotypes (Map2k6^−/−, Map3k4^+/−). At 14.5 dpc, gonads of this genotype had regions of Stra8 expression at their poles that were much more extensive than either Map2k6^−/− or Map3k4^+/− gonads alone (Fig. 2E). These data confirm a role for Map2k6 in testis determination and also reveal genetic interaction between Map2k6 and Map3k4 consistent with a role for these two genes in the same testis-determining pathway.

Loss of MAP2K6 on the Highly Sensitized B6.Y^AKR Background Causes XY Gonadal Sex Reversal

We predicted that doubly homozygous (Map2k3^−/−, Map2k6^−/−) embryos would exhibit much more severe disruptions to testis determination, but examination of testis development in these double homozygotes was precluded by lethality at around 11.5 dpc [25]. In order to examine and compare the distinct contributions made by Map2k3 and Map2k6 to testis determination on the same genetic background, we bred the null alleles on to B6.Y^AKR, a background that is highly sensitized to disruptions to testis determination [26]. Whilst B6.Y^AKR mice develop as normal males, embryonic gonad development is characterized by delayed testis determination and transient ovotestis formation as a consequence. This is associated, at least in part, with a delay in peak expression of the Mus domesticus Sry allele, Sry^AKR, on the B6 background at the onset of testis determination [5, 27].

Embryos lacking Map2k3 on the B6.Y^AKR background had ovotestes at 14.5 dpc (Fig. 3A). Sox9 expression is detectable in central cords with an irregular morphology, but is lacking from one or both poles, whilst Stra8 expression is prominent in the polar regions of these ovotestes (Fig. 3A). Control B6.Y^AKR gonads also exhibit polar Stra8 expression as expected, although there was a tendency for the extent of Stra8 expression to be greater in Map2k3-deficient mutants than in controls (Fig. 3A and data not shown), and the most severely affected gonads were from homozygous mutant embryos. Thus, the disruptive impact of Map2k3 deletion on testis determination is difficult to quantify but is significant.

FIG. 3

Gonadal sex reversal phenotype at 14.5 dpc in MAP2K6-deficient embryos on the B6.Y^AKR background. A) WMISH with Sox9 and Stra8 probes showing loss of Sox9 signal, abnormal cord morphology, and extensive expression of Stra8 in Map2k3 null mutant gonads compared to wild-type controls. Control XY gonads (center, lower panel) exhibit small Stra8-positive regions due to transient ovotestis formation associated with the reduced functionality of the Sry^AKR allele on B6. Bar = 500 μm. B) WMISH with Sox9 and Stra8 probes reveals complete gonadal sex reversal in Map2k6-deficient gonads and compound mutant gonads (Map2k3^+/−, Map2k6^−/−), associated with overt ovarian morphology, loss of Sox9, and ectopic Stra8 expression throughout. Heterozygous mutant gonads (Map2k6^+/−) may also exhibit large Stra8-positive regions and reduced Sox9 expression at the poles and abnormal cord morphology, in contrast to wild-type controls. Bar = 500 μm. C) Immunostaining of gonadal tissue sections comparing MAP2K6-deficient embryos and wild-type controls. AMH (red) is detected in testis cords of control gonads but is absent from mutant gonads. The ovarian granulosa cell marker FOXL2 (green) is detected throughout the XY mutant gonad, as it is in XX controls. Some polar FOXL2 is detected in the B6.Y^AKR wild-type control for reasons explained above. Bar = 500 μm. D) WMISH at 11.25 dpc with an Sry probe shows clear expression throughout the gonad (area within white dotted lines; limits of expression indicated by arrowheads) in two B6.Y^AKR wild-type (+/+) controls; but signal is negligible and restricted to central regions (indicated by arrowheads) in two MAP2K6-deficient gonads (2k6^−/−) on the same background at exactly the same stage (16 ts). Bar = 200 μm. E) Immunostaining with an anti-SRY antibody at 17 ts reveals a large number of SRY-positive cells in the control (+/+) gonad (area within white dotted line), but few SRY-positive cells in a stage-matched mutant (2k6^−/−) gonad. Bar = 200 μm.

In contrast to this, B6.Y^AKR embryos lacking Map2k6 exhibited complete gonadal sex reversal, as evidenced by gonadal morphology, absence of Sox9 expression (Fig. 3B), and high levels of Stra8 (Fig. 3B) and FOXL2 (Fig. 3C) throughout the gonad at 14.5 dpc. Complete sex reversal was also seen in Map2k3^+/−, Map2k6^−/− compound mutants (Fig. 3B). Interestingly, B6.Y^AKR embryos lacking just a single copy of Map2k6 occasionally developed ovotestes with substantial amounts of Stra8-positive tissue (Fig. 3B and data not shown). These data clearly indicate a requirement for Map2k6 function in testis determination; additionally, a minor role for Map2k3 is suggested by these data. Such a minor role is also supported by the subtle increase in severity of gonadal phenotype in Map2k3^+/−, Map2k6^−/− embryos on the B6 background when compared to Map2k6^−/− (Fig. 2D).

Sry Expression Is Disrupted in Map2k6^−/− Gonads on B6.Y^AKR

Given the link between Map3k4 expression and Sry expression in both loss- and gain-of-function mouse models [4, 5], and the positive link between p38 MAPK activity and Sry [6, 28], we examined Sry expression in embryonic gonads lacking Map2k6 on the B6.Y^AKR background at the early stage of 11.25 dpc (16 ts) using WMISH (Fig. 3D). A clear reduction in Sry signal was observed in mutant gonads when compared to stage-matched wild-type controls, consistent with the observed XY gonadal sex reversal in this strain. Similarly, detection of SRY protein in wild-type and mutant gonads at the 17 ts stage using immunostaining revealed a greatly reduced number of SRY-positive cells in mutant gonads and lower levels of SRY protein in those cells (Fig. 3E).