A role for TRPC3 in mammalian testis development

Abstract

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Keywords: DSD; SOX9; TRP; TRPC3; sertoli cells; sex determination; testis.

FIGURE 1

Expression of Trpc3/TRPC3 in the developing mouse testis. (A) qRT-PCR analysis confirming the downregulation of Trpc3 in E13.5 mouse Sox9 knockout gonads. n = 5 for XY wildtype (XY WT) and XY Sox9 conditional knockout (XY Sox9 KO), n = 3 for XX wildtype (XX WT). Transcript expression level of the Trpc3 gene was normalized to Tbp and presented as relative expression. Mean ± SEM. One-way ANOVA test. ****p < 0.0001. (B) Trpc3 RNA expression levels in developing male and female gonads from E11.5 to E13.5. The figure is generated based on the microarray dataset from Jameson et al. (2012). (C, D) Co-immunofluorescence staining of TRPC3 (green) with somatic cell nuclear marker GATA4 (red) and Sertoli cell cytoplasmic marker AMH (red) in the mouse testis from E11.5 to E15.5. Nuclei are visualized with the nuclear marker DAPI (blue). Scale bar = 50 μm.

FIGURE 2

TRPC3 inhibition leads to a reduction in germ cell numbers and proliferation in cultured XY mouse gonads. (A) Immunofluorescence staining of male gonads after exposure to either the vehicle control DMSO or the TRPC3 inhibitor Pyr3 during ex vivo culture for 24, 48, and 72 h. The sections are stained for germ cell marker DDX4 (red) and Sertoli cell marker SOX9 (green). Nuclei are visualized with the nuclear marker DAPI (blue). Dashed lines outline gonads. Scale bar = 50 μm. (B) Quantification of germ cells and Sertoli cells in XY DMSO and XY Pyr3-treated gonads at 24-, 48- and 72-h post-culture. n = 2–4; sections counted = 2–10. Mean ± SEM. Unpaired Student’s t-test. *p < 0.05, ***p < 0.001; ns, not significant. (C) Immunofluorescence staining for the germ cell marker DDX4 (red) and cell proliferation marker phospho-histone H3 (PH3) (green). White arrows indicate examples of proliferating germ cells. (D) Quantification of proliferating germ cells is performed for PH3+ germ cells relative to the total germ cell population. n = 2–4; sections counted = 4–9. Mean ± SEM. Unpaired Student’s t-test. ***p < 0.001, ****p < 0.0001; ns, not significant.

FIGURE 3

TRPC3 inhibition disrupts the coelomic blood vessel after 72 h of culture. (A) Immunofluorescence staining of GATA4 (green) and PECAM1 (red) in control (XY DMSO) and Pyr3-treated (XY Pyr3) gonads cultured ex vivo for 48 and 72 h. White arrows indicate the coelomic blood vessel. (B) Immunofluorescence staining of the cell apoptosis marker cleaved Caspase-3 (CC3) (green) and PECAM1 (red) in XY DMSO and XY Pyr3 gonads cultured ex vivo for 48 and 72 h. Arrowheads indicate examples of apoptotic endothelial cells. Nuclei are visualized with the nuclear marker DAPI (blue). Dashed lines outline gonads. Scale bar = 50 μm. (C) Quantification of apoptotic endothelial cells is performed for the number of CC3+ signals per 100 μm of the coelomic blood vessel. n = 3–4; sections counted = 6–8. Mean ± SEM. Unpaired Student’s t-test. *p < 0.05.

FIGURE 4

TRPC3 stimulates proliferation and controls cell morphology in NT2/D1 cells. (A, B) Cell proliferation (5–72 h) and cell adhesion (0–5 h) curves were generated from the xCELLigence system. Electrical impedance was measured and reported as cell index. The rate of cell proliferation or adhesion was calculated as the slope (1/hr). Mean ± SEM values were taken from three independent biological experiments. One-way ANOVA test. **p < 0.01, ***p < 0.001; ns, not significant. (C) qRT-PCR analysis showing the expression levels of TRPC3 in control (siCON) and siTRPC3 groups. Data are presented as Mean ± SEM, with statistical significance determined by unpaired Student’s t-test. **p < 0.01, n = 4. (D–J) HoloMonitor measurements of cell morphology include cell area, volume, eccentricity, irregularity, thickness, count, and confluence. Mean ± SEM values represent the average of two independent experiments. Unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. (K) Holographic phase images depict cells from siCON and siTRPC3 groups at 0, 12 and 24 h. Scale bar = 50 μm.