MAP3K4 kinase activity dependent control of mouse gonadal sex determination†

Abstract

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein-protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4KI/KI phenotypic females have a Y chromosome. XY Map3k4KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4KI/KI adults are smaller than either male or female Map3k4WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.

Keywords: MAP2K3/6; MAP3K4; SRY; gonadal sex determination; kinase activity; mitogen-activated protein kinase kinase kinase 4; p38 MAPK.

Figure 1

Sex reversal phenotype in XY Map3k4^KI/KI adult mice. (A) Cells isolated from Map3k4^KI/KI embryos express full-length kinase-inactive MAP3K4 but have reduced phosphorylation (p) of MAP2K3/6 and p38 MAPK. Western blots are representative images from three biologically independent experiments. (B) Phenotypic comparison of external genitalia of adult mice. Anogenital distance (AGD) of representative XY Map3k4^KI/KI mouse with sex reversal is comparable to XX Map3k4^WT/WT female. AGD is indicated using square brackets. (C) Average AGD was quantified in Map3k4^WT/WT, Map3k4^WT/KI, and Map3k4^KI/KI male and female mice. Data are displayed as box plots and each individual is plotted as one dot. (B-C) Map3k4 genotype, sex phenotype, and sex genotype are indicated below each panel. Statistical analyses were performed relative to XY Map3k4^WT/WT mice where the lowercase letters indicates statistically significant differences: c, P < 0.001; d, P < 0.0001; Student t test; n.d., not determined due to only two individuals; XY Map3k4^WT/KI mice were not significantly different from XY Map3k4^WT/WT mice.

Figure 2

Reduced body size and weight in XY Map3k4^KI/KI adult mice having a gonadal sex reversal phenotype. (A-C) Body size of three individual representative XY Map3k4^KI/KI mice with sex reversal compared to Map3k4^WT/WT female and male mice. (A) Aged 6 months. (B-C) Aged 5 months. (D, E) Reduced average body weight of XY Map3k4^KI/KI adult mice with sex reversal compared to phenotypic male and female Map3k4^WT/WT mice at 4 months (D) or 6 months (E). (D, E) Data are displayed as box plots, and each dot represents one individual. The lowercase letters indicate statistically significant differences: a, P < 0.05; b, P < 0.01; c, P < 0.001; d, P < 0.0001; Student t test.

Figure 3

Gonadal sex reversal phenotype in XY Map3k4^KI/KI adult mice. (A) Representative macroscopic images of adult reproductive tracts and enlarged images of ovaries from adult XX Map3k4^WT/WT and XY Map3k4^KI/KI mice. White arrows indicate location of primitive gonads. Black arrow indicates position of uterine structure. (B) Histological examination of H&E stained paraffin sections of XX Map3k4^WT/WT and XY Map3k4^KI/KI adult ovaries.

Figure 4

Sry expression is absent in most XY Map3k4^KI/KI embryos at E11.5. (A-E) Representative images of E11.5 embryos generated from crosses of Map3k4^WT/KI mice. White arrowhead indicates exencephaly, and white arrow indicates open posterior neuropore. (F) Relative Sry expression in E11.5 embryos generated from crosses of Map3k4^WT/KI mice. Transcript expression in E11.5 gonads measured by qPCR was normalized to Hprt1. Data are expressed as a fold change relative to XY Map3k4^WT/WT. Data shown are the mean ± SEM, and each point represents one embryo. Statistical analyses were performed relative to XY Map3k4^WT/WT embryos where the lowercase letter a indicates P < 0.05; Student t test; XY Map3k4^WT/KI embryos were not significantly different from XY Map3k4^WT/WT embryos.

Figure 5

XY Map3k4^KI/KI embryos display a gonadal sex reversal phenotype at E13.5 and E14.5. (A) Transcript expression of indicated genes in E13.5 gonads measured by qPCR was normalized to Hprt1. Data are expressed as a fold change relative to XY Map3k4^WT/WT (Sox9, Dhh) or to XX Map3k4^WT/WT (Irx3, Stra8). Data shown are the mean ± range, and each point represents one embryo. (B) Representative images of E14.5 embryos and gonads generated from crosses of Map3k4^WT/KI mice. White arrow indicates spina bifida.