Abstract
Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 DG/DG mice compared to Rxfp2 +/DG and Rxfp2 +/+ mice. Testicular histology of the Rxfp2 DG/DG mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 −) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 DG/−) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 +/− littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7–9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.
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Acknowledgments
The authors thank the laboratory of Dr. David Beier for the SNP analysis. This work was supported by the Northwestern University Genomics Core and a Cancer Center Support Grant (NCI CA060553). This work was supported by the National Institutes of Health grants U01 HD043425-01(JLJ) and R01HD03706 (AIA).
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The authors have no conflicts of interest or financial ties to disclose.
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Harris, R.M., Finlayson, C., Weiss, J. et al. A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism. Mamm Genome 21, 442–449 (2010). https://doi.org/10.1007/s00335-010-9291-5
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DOI: https://doi.org/10.1007/s00335-010-9291-5