Abstract
A genome-wide mutagenesis screen employing ENU in mice identified five mutant lines with male urogenital defects. Whole genome analysis using a highly parallel SNP assay mapped each mutation to a 20-90 Mb region. Candidate genes in these regions were identified and sequenced, allowing identification of mutations responsible for hypogonadism (Prdm9), cryptorchidism (Rxfp2) and germ cell loss (Plk4) phenotypes. Two of the mouse lines for which the causative mutations were identified (Rxfp2 and Prdm9) have known roles in male reproduction. Prdm9, a histone methyltransferase, is involved in the progression of meiotic prophase through trimethylation of lysine 4 of histone H3. Recently, PRDM9 was implicated as a key player in meiotic recombination hotspots and speciation. Rxfp2/RXFP2, a GPCR, is one of two known genes that cause cryptorchidism in both mice and humans. The mutation in mouse RXFP2 resulted in an amino acid alteration from aspartic acid to glycine at residue 294 (D294G). The D294G mutation impaired testicular descent and resulted in decreased testis weight in homozygous mice as compared to heterozygous and wild-type mice. Testicular histology of the homozygous mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell only features. Genetic complementation analysis using a Rxfp2 loss of function allele confirmed causality of the D294G mutation. The functional implication of the D294G mutation was analyzed by measuring total and cell-surface expression of mouse RXFP2 protein in transfected cells. Total expression of the mutant (D294G) protein was only slightly reduced compared to wild-type, but cell-surface expression was markedly decreased. The D294G mutation underscores the role of the RXFP2 mediated pathway in testicular descent and expands the repertoire of mutations that affect receptor trafficking and function. Another identified gene, Plk4, encodes a serine/threonine kinase, which is highly expressed in the testis. PLK4 is involved in centriole formation and cell cycle progression, however the specific role of PLK4 in spermatogenesis is unknown. The mutation in PLK4 resulted in an amino acid alteration from isoleucine to asparagine at residue 224 (I242N). Homozygous mice are embryonic lethal, but heterozygous mice are hypogonadal and have germ cell loss. This loss is not due to a secondary defect in the hypothalamic-pituitary axis, as LH, FSH and testosterone are mainly unaffected. The germ cell loss appears to result from increased apoptosis. Genetic complementation analysis using a gene-trap allele confirmed causality of the I242N mutation. Further investigation into the underlying etiology of the defect, the mechanism of PLK4 action in the testis and potential downstream effector molecules is ongoing. Fine mapping of the two remaining hypogonadal lines was completed using a custom SNP array, reducing the regions of interest to 2 Mb. High-throughput sequencing of the regions is ongoing and it is anticipated that the mutated genes in the two remaining lines will provide novel insights, as the mapped intervals do not contain genes with known roles in the testis. Funding: U01 HD043425-01