The nucleoside diphosphate kinase gene Nme3 acts as quantitative trait locus promoting non-Mendelian inheritance

Abstract

The t-haplotype, a variant form of the t-complex region on mouse chromosome 17, acts as selfish genetic element and is transmitted at high frequencies (> 95%) from heterozygous (t/+) males to their offspring. This phenotype is termed transmission ratio distortion (TRD) and is caused by the interaction of the t-complex responder (Tcr) with several quantitative trait loci (QTL), the t-complex distorters (Tcd1 to Tcd4), all located within the t-haplotype region. Current data suggest that the distorters collectively impair motility of all sperm derived from t/+ males; t-sperm is rescued by the responder, whereas (+)-sperm remains partially dysfunctional. Recently we have identified two distorters as regulators of RHO small G proteins. Here we show that the nucleoside diphosphate kinase gene Nme3 acts as a QTL on TRD. Reduction of the Nme3 dosage by gene targeting of the wild-type allele enhanced the transmission rate of the t-haplotype and phenocopied distorter function. Genetic and biochemical analysis showed that the t-allele of Nme3 harbors a mutation (P89S) that compromises enzymatic activity of the protein and genetically acts as a hypomorph. Transgenic overexpression of the Nme3 t-allele reduced t-haplotype transmission, proving it to be a distorter. We propose that the NME3 protein interacts with RHO signaling cascades to impair sperm motility through hyperactivation of SMOK, the wild-type form of the responder. This deleterious effect of the distorters is counter-balanced by the responder, SMOK(Tcr), a dominant-negative protein kinase exclusively expressed in t-sperm, thus permitting selfish behaviour and preferential transmission of the t-haplotype. In addition, the previously reported association of NME family members with RHO signaling in somatic cell motility and metastasis, in conjunction with our data involving RHO signaling in sperm motility, suggests a functional conservation between mechanisms for motility control in somatic cells and spermatozoa.

Figure 1. Nme3 is a Tcd2 candidate.

(A) The position of Nme3 in the wild-type t-complex (+) and genetic mapping on t^w18 localized it to the Tcd2 region of the t-haplotype (t) (see Figure S1). Symbols of genes verified to be involved in TRD are boxed, and map positions relative to the wild-type chromosome 17 (in Mb) are indicated. Molecularly unknown Tcd loci are also listed along with Chr17 inversions and their relative orientations in both, the wild-type and t-haplotype chromosome, as well as crucial genetic markers (T, tf, H-2). (B) Northern blot analysis of Nme3 transcripts in testes from consecutive post partum stages reflecting the first round of spermatogenesis (P7–P24), and in testes of adult mice (ad). (C) In situ hybridization analysis of Nme3 on testis cryosections from an adult male. Expression is predominant in cells near the basal lamina (dotted line) representing diploid cell types. Schematic view of a seminiferous tubule. (Se) Sertoli cells; (SG) spermatogonia; (SC) spermatocytes; (RS) round spermatids; (SZ) spermatozoa. Scale bar: 50 µm. (D) Northern blot analysis of Nme3 expression in different wild-type strains and t-haplotypes. Quantification of the signals with respect to C57Bl/6 did not reveal significant differences. (E) Amino acid sequence comparison of several NME proteins encoded by Mus musculus (mmNME1 to mmNME3), the t-haplotype (mmNME3-t), Homo sapiens (hsNME3), and Drosophila melanogaster (dmAWD); a conserved proline at position 89 is altered in mmNME3-t (P89S), and a nearby proline residue was described as killer-of-prune mutation (K-pn) in the abnormal-wing-disc (awd) gene of D. melanogaster when mutated to serine (red boxed).

Figure 2. Targeted inactivation of the Nme3 gene.

(A) Gene targeting strategy: a neomycin selection cassette was used to replace the entire first and part of the second exon of Nme3. (B) Confirmation of correct homologous recombination by Southern blot analysis of XbaI digested genomic DNA derived from ES-cells or mice. (C) Loss of Nme3 transcripts in homozygous knock-out mice (−/−) as determined by expression analysis using RT-PCR confirms that the targeted allele represents a null mutation. Genomic regions in (A) are not drawn to scale. Abbr.: LP, RP: left or right external probes; DT, diphteria toxin cassette for negative selection; wt, wild-type; +, wild-type allele; −, knock-out allele.

Figure 3. The t-allele of Nme3 encodes a hypomorph.

(A) Nucleoside diphosphate kinase activities of wild-type (+) and t-allele (t)-derived proteins or mixtures of both expressed in vitro. The 1∶1 mixture reflects the relative contribution of both alleles to NME3 activity in t/+ heterozygotes, the 1∶2.6 ratio of + and t alleles corresponds to the expression of + and t alleles measured in testes from t⁶/+;Tg^t/0 males (B, C). The columns show the mean of three experiments with the standard deviation. (B) Schematic representation of the transgene construct Tg(Nme3)H17bgh (Tg^t) generated for expression of the Nme3 t-allele during spermatogenesis. (C) Expression of the Nme3 t-allele from the transgene construct determined by Northern blot analysis of testis RNA derived from a t⁶/+;Tg^t/0 male. Quantification of endogenous (t⁶, +) and transgene (Tg^t)-derived transcripts revealed a ratio of 1∶0.8. Assuming that the t⁶ allele contributes 50% of the endogenous Nme3 transcripts, the overall ratio of + to t derived Nme3 transcripts expressed in a t⁶/+;Tg^t/0 male is therefore 1∶2.6. Abbr.: Ace-prom, angiotensin converting enzyme promoter; SV40pA, Simian virus 40 polyadenylation signal fragment; ins, chicken beta globin insulator ; Ex, exon; Ndk, nucleoside diphosphate kinase.

Figure 4. Model of the role of NME3 in t-haplotype transmission ratio distortion.

Nme3^Tcd2b encodes a hypomorphic allele of Nme3 leading to reduction of NME3 activity in sperm derived from a t/+ male. NME3 may be an activator of the inhibitory Rho signaling pathway or an inhibitor of the activating pathway controlling SMOK activity, or both. NME3^Tcd2b thus might synergize with TAGAP1^Tcd1a to reduce inhibition and/or with FGD2^Tcd2a to enhance activation of SMOK in all sperm. The combined activity of all distorters leads to impairment of sperm motility, which is rescued exclusively in t-sperm by the responder encoding the dominant-negative variant SMOK^TCR, resulting in transmission ratio distortion in favor of t-sperm. Red upward pointing arrows indicate up-regulation, blue down-pointing arrows down-regulation; the green down-pointing arrow symbolizes rescued, the dark-red down-pointing arrow impaired flagellar motility.