Insulin-Like 3 Exposure of the Fetal Rat Gubernaculum Modulates Expression of Genes Involved in Neural Pathways1

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.

What Signals Testis Descent?

“…As summarized in Figure 1 As expected, genes involved in the cAMP pathway were identified; mRNA levels of Crem (cAMP-responsive element modulator) and three phosphodiesterases (Pde10a, Pde8a, and Pde4d) were all upregulated following INSL3 exposure [2]. CREM is a transcription factor induced by cAMP, whereas phosphodiesterases are enzymes that degrade cAMP and, thus, regulate intracellular cAMP gradients and responses.…”

“…In addition to the cAMP pathway, Johnson et al successfully identified several genes involved in neurogenesis as well as genes belonging to the bone morphogenetic protein (BMP) and wingless/Int1 (WNT) signaling pathways that were upregulated by INSL3 [2]. Genes involved in neuronal development include Pnoc (codes for the secreted neuropeptide nociceptin) and Pcsk2 (encodes a prohormone convertase that processes prepronociceptin) [2].…”

“…What pathways are activated by this hormone? In this issue of Biology of Reproduction, Johnson et al [2] have achieved an important advance in our understandings of testis descent by identifying several pathways activated by INSL3 in the gubernaculum.…”

Loss of Wnt5a Disrupts Primordial Germ Cell Migration and Male Sexual Development in Mice1

“…However, because no change in Insl3 expression was observed at 13.5 dpc, we surmise that Wnt5a acts either independently or downstream of Insl3 during this process. The latter option is perhaps the most likely, because ex vivo culture experiments on early postnatal rat gubernacula have shown that exogenous INSL3 can induce endogenous Wnt5a expression [57].…”

Effects of Nociceptin and Nocistatin on Uterine Contraction

“…The insulin-like 3 protein has been suggested to control testis descent during development ( Johnson et al, 2010). An analysis with murine Sertoli cells and testes has demonstrated that both PNOC mRNA and N/OFQ are induced by FSH.…”