The male sterility and histoincompatibility (mshi) mutation in mice is a natural variant of microtubule-associated protein 7 (Mtap7)

Abstract

Males homozygous for the mouse male sterility and histoincompatibility (mshi) mutation exhibit small testes and produce no sperm. In addition, mshi generates an "antigen-loss" histoincompatibility barrier, such that homozygous mutants reject skin grafts from wild type co-isogenic BALB/cByJ donors. To facilitate the molecular characterization of the pleiotropic mshi mutation, we genetically mapped mshi into a 0.68 megabasepair region which contains fewer than 10 candidate genes. Complementation testing showed that one of these, Mtap7, is disrupted in mshi mice. Sequence analysis has revealed a 13 kilobasepair deletion in BALB/cByJ-mshi/J mice that begins in Intron 10-11 of Mtap7, and ends less than 2000 base pairs downstream of the wild type gene. Analysis of the mutant cDNA predicts that Mtap7(mshi) encodes a 457 amino acid protein, the first 423 of which are identical to wild type, and the last 34 of which are due to aberrant mRNA splicing with two cryptic exons in the Mtap7 to P04Rik intergenic region. This molecular assignment for the mshi mutation further supports an essential role for microtubule stabilization in spermatogenesis and indicates a new role in allograft transplantation.

Fig. 1

Genetic and physical mapping of the mouse mshi locus. (A) Genetic map based on a 402-member (C57BL/6J × BALB/cByJ-mshi/J)F₁ × BALB/c-mshi/J backcross, typed for 6 regional microsatellite DNA markers (Rule et al. 1999), and 3 single-nucleotide polymorphisms. The number of recombinants out of 402 total backcross mice, and the percent recombination (± 1 standard error) are shown in each interval. D10Csu1 and D10Mit213 are aliases for IL20RA and Pde7b, and define the maximal location of mshi. (B) The known genes in the critical region between D10Csu1 and D10Mit213 (Ensembl Mouse 2008) are drawn on a 0.1 Mb scale. Three of these candidates, Mtap7, P04Rik, and Bclaf1 were investigated further (see text). D10Csu2 and D10Csu3 are aliases for Bclaf1 and P04Rik, respectively. (C) Genomic structure of the Mtap7 gene. Solid black boxes indicate the 18 exons that comprise Mtap7, an asterisk indicates the location of the Mtap7^k.o. βgal/neo insertion in Intron 1–2 (Komada et al. 2000), and a dotted bracket indicates the extent of the 13,080 bp deletion that defines the mutant mshi allele. The number below each exon is the number of basepairs each contains. In the mutant, Exon 10 is spliced to two downstream cryptic exons, designated Exon 11M and 12M. The number at the right end of the final exon in the wild and mutant transcript is the length of the mRNA in nucleotides.

Fig. 2

Testis size displayed by three-month-old males with various Mtap7 genotypes. (A) Testes testes from heterozygous control (mshi/+) and mutant (mshi/Mtap7^k.o.) mice. (B) Mass of testes from 3-month old heterozygous controls and homozygous mutants from 3 different test crosses: mshi/mshi × mshi/+ (left), Mtap7^k.o./Mtap7^k.o. × Mtap7^k.o./+ (center), and mshi/mshi × Mtap7^k.o./+ (right). The average (combined) testicular mass is shown, ±1 standard error. The n number in each category is shown at the base of each bar.

Fig. 3

Hematoxylin and eosin stained cross sections of testicular tubules of three-month-old heterozygous control (mshi/+, A), and mutant (mshi/mshi, B; mshi/Mtap7^k.o., C; and Mtap7^k.o./Mtap7^k.o., D) mice. White bars at the lower left measure 100 μm. (E) Diameter of testicular tubules from heterozygous controls and homozygous mutants from 3 different test crosses: mshi/mshi × mshi/+ (left), Mtap7^k.o./Mtap7^k.o. × Mtap7^k.o./+ (center), and mshi/mshi × Mtap7^k.o./+ (right). The average tubule diameter is shown, ±1 standard error. The number of individual testicular cross sections examined (n) in each category is shown at the base of each bar; four different tubules from each testicular cross section were measured.

Fig. 4

Morphology of microtubules in three-month-old heterozygous control (mshi/+, A), and mutant (mshi/mshi, B; mshi/Mtap7^k.o., C; and Mtap7^k.o./Mtap7^k.o., D) mice. Testicular sections were stained with anti-β-tubulin antibody (red), nuclei were costained with Hoechst’s reagent (blue). Basement membranes of the seminiferous tubules are indicated by broken lines. White bars at the lower right measure 50 μm.

Fig. 5

Sequence analysis of Mtap7alleles. The predicted amino acid sequence for the wild type (A) and mshi mutant allele of Mtap7 are shown (B). The wild type sequence was based on the C57BL/6J and the BALB/cByJ inbred strains of mice. Black bars indicate the exon boundaries; the red arrow indicates the position of the βgal/neo insertion in Mtap7^k.o. (Komada et al. 2000); single-nucleotide differences between C57BL/6J and BALB/cByJ (in Exons 4, and 9, and 10) are highlighted in yellow (although all of these third-positions variants are silent, see Fig. S1). The section of the wild-type protein that is boxed in red is deleted in the mutant mshi allele, and is replaced with the green-boxed sequence in part B due to splicing of Exon 10 to two exons, designated 11M and 12M, that are downstream of the deletion breakpoint. The DNA sequence that defines the mshi deletion is shown in Fig. S2. The DNA sequence of mutant Exons 11M and 12M are shown in Fig. S3.