A critical time window of Sry action in gonadal sex determination in mice

In mammals, the Y-linked sex-determining gene Sry cell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9), leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However, how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects

Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage

Expression of ARX, 3β-HSD, LHX9, and AD4BP/SF-1 in interstitial cells of fetal testes.

<p>Expression of ARX (red), AD4BP/SF-1 (blue), and 3β-HSD (green) in fetal testis at E13.0 was examined. Merged images of ARX and 3β-HSD (upper panels of A–E), and ARX and AD4BP/SF-1 (lower panels of A–E) are shown. The arrowhead in A indicates an ARX-strongly positive, AD4BP/SF-1-negative, and 3β-HSD-negative cell. The arrowhead in B indicates an ARX-weakly positive, AD4BP/SF-1-weakly positive, and 3β-HSD-negative cell. The arrowhead in C indicates an ARX-weakly positive, AD4BP/SF-1-weakly positive, and 3β-HSD-weakly positive cell. The arrowhead in D indicates an ARX-weakly positive, AD4BP/SF-1-strongly positive, and 3β-HSD-modestly positive cell. The arrowhead in E indicates an ARX-negative, AD4BP/SF-1-strongly positive, and 3β-HSD-strongly positive cell. Cells showing expression of the marker proteins above are illustrated (F). Expression of LHX9 (green) and ARX (red) in E11.5 male gonads was examined (G-I). Dashed lines in G-I indicate the gonad-mesonephros border. Expressions of MAFB (green) and AD4BP/SF-1 (red), and ARX (green) and AD4BP/SF-1 (red) in E11.5 male gonads were examined on consecutive sections (J–O, and J’–O’ for enlarged views). Arrows in J and L indicate autofluorescence of blood cells. Dashed lines in J’–O’ indicate border between MAFB or ARX-positive cells and AD4BP/SF-1-positive cells. Arrowheads in J’ and L’ indicate MAFB-positive cells just beneath AD4BP/SF-1-positive cells. Scale bars = 100 µm. g, gonad; m, mesonephros.</p

Suppressed proliferation in fetal Leydig cells.

<p>Cell proliferation was evaluated by BrdU incorporation studies with three fetal testes. BrdU labeled fetal testes at E12.5, E14.5 and E16.5 were sectioned and the sections were immunostained with antibodies for 3β-HSD (green) and BrdU (red) (left panels in A; Leydig cell), and with SOX9 (green) and BrdU (red) (right panel in A; Sertoli cell). Enclosed areas are enlarged at the top right in each panel. Arrows indicate cells double-positive for SOX9 and BrdU. Scale bars = 100 µm. Numbers of 3β-HSD and BrdU double-positive cells (closed bars) and 3β-HSD single-positive cells (open bars) (B), and numbers of SOX9 and BrdU double-positive cells (closed bars) and SOX9 single-positive cells (open bars) (C) were counted (n = 3). The numbers of these cells per unit area are plotted. The data are indicated as the mean ± SD. *, P<0.05, **, P<0.01.</p

Structural abnormalities induced in <i>Arx</i> KO mouse.

<p>Urogenital systems of wild type (Wild type XY) (A) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) (B) male mice at E18.5 are shown. Arrows indicate the testes. tes, testis; b, bladder; kid, kidney. Intratesticular testosterone levels of wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes were measured at E18.5 (C). The data are indicated as the mean ± SD. *, P<0.05. Expression of <i>Insl3</i> and <i>Vegfa</i> in wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes at E12.5 was determined by quantitative RT-PCR (D and E). The data were standardized using <i>β-actin</i> (Actb) and shown as the mean ± SD. *, P<0.05. Locations of the sections of the gonad are schematically shown with horizontal lines with numerals, 1 to 6 (F). The coelomic blood vessel is indicated with red line. Double immunofluorescent staining for LAMININ (green) and PECAM (red) was performed with the serial sections (1 to 6, corresponding to the numerals in (E)) with 100 µm interval of wild type and <i>Arx</i> KO mouse testes at E12.5 (G). Arrowheads indicate the coelomic blood vessel in wild type, while the corresponding structure could not be observed at this stage in the KO testis. Scale bars = 100 µm.</p