Insulin-like 3 exposure of the fetal rat gubernaculum modulates expression of genes involved in neural pathways

Abstract

Insulin-like 3 (INSL3) signaling directs fetal gubernacular development and testis descent, but the actions of INSL3 in the gubernaculum are poorly understood. Using microarray gene expression profiling of fetal male rat gubernaculum explants exposed to 10 or 100 nM INSL3, significant changes in expression were identified for approximately 900 genes. Several of the genes showing the largest inductions regulate neuronal development or activity, including Pnoc (34-fold), Nptx2 (9-fold), Nfasc (4-fold), Gfra3 (3-fold), Unc5d (3-fold), and Crlf1 (3-fold). Bioinformatics analysis revealed BMP and WNT signaling pathways and several gene ontologies related to neurogenesis were altered by INSL3. Promoter response elements significantly enriched in the INSL3-regulated gene list included consensus sequences for MYB, REL, ATF2, and TEF transcription factors. Comparing in vivo gene expression profiles of male and female rat fetal gubernaculum showed expression of the Bmp, Wnt, and neurodevelopmental genes induced by INSL3 was higher in males. Using quantitative RT-PCR, the microarray data were confirmed, and the induction of Bmp3, Chrdl2, Crlf1, Nptx2, Pnoc, Wnt4, and Wnt5a mRNA levels were examined over a range of INSL3 concentrations (0.1-100 nM) in male and female gubernaculum. In both sexes, an increasing gene expression response was observed between 0.1 and 10 nM INSL3. These data suggest that INSL3 signaling in the fetal gubernaculum induces morphogenetic programs, including BMP and WNT signaling, and support other rodent data suggesting a role for these pathways in development of the gubernaculum.

FIG. 1.

After incubation for 24 h in the presence of INSL3 concentrations ranging from 0 (control) to 100 nM, male fetal gubernacular bulb gene expression was examined by Taqman-based qRT-PCR. Each graph shows mRNA levels, with the gene identified above the graph. Below each graph, the fold-change (FC) is given for each gene after 24-h exposure to 10 or 100 nM INSL3 as determined by microarray expression analysis. Data are presented as the mean ± SD (n = 7–11). An asterisk indicates a significant difference from control (P < 0.05) as determined by one-way ANOVA and Dunnett post-hoc test.

FIG. 2.

After incubation for 6 h in the presence of INSL3 concentrations ranging from 0 (control) to 100 nM, male fetal gubernacular bulb gene expression was examined by Taqman-based qRT-PCR. Each graph shows mRNA levels, with the gene identified above the graph. Data are presented as the mean ± SD (n = 5–6). An asterisk indicates a significant difference from control (P < 0.05) as determined by one-way ANOVA and Dunnett post-hoc test.

FIG. 3.

After incubation for 24 h in the presence of INSL3 concentrations ranging from 0 (control) to 100 nM, female fetal gubernacular bulb gene expression was examined by Taqman-based qRT-PCR. Each graph shows mRNA levels, with the gene identified above the graph. Data are presented as the mean ± SD (n = 6–7). An asterisk indicates a significant difference from control (P < 0.05) as determined by one-way ANOVA and Dunnett post-hoc test.