Experiment
In a previous study (J:124156), a QTL of low testis weight (Ltsw1) was mapped to a 23 Mb region of chromosome 11, using a of panel of IRCS mice that were developed at the Pasteur Institute (Paris, France) from an original cross between the SEG/Pas strain (SEG) of Mus spretus origin and the C57BL6/J (B6) Mus musculus strain. On average, each IRCS genome is composed of ~1.5% of Mus spretus genome, distributed in 1 to 8 fragments in a C57BL6/J context.
In the previous study the low testis weight QTL (Ltsw1) mapped on mouse chromosome 11 (between 3.7 Mb and 26.5 Mb) following the phenotypic analysis of the IRCS 97 C strain supposed to harbor a unique spretus fragment on MMU11. The strain exhibited a significant reduction in absolute and relative testis weight compared to the B6 control, associated with a reduction in the seminiferous tubules diameter.
To unambiguously map the Ltsw1 QTL two congenic sub-strains were created, harboring one spretus fragment, either on MMU6 or on MMU11 (called 97Crc6 and 97Crc11 respectively).
As expected, the low testis weight phenotype segregated with the MMU11 spretus fragment harbored by the 97Crc11 strain. Confirming that 97Crc11 mice exhibited the same abnormality of spermatogenesis that 97 C mice did.
To identify the genetic determinant of the Ltsw1 QTL, the MMU11 spretus fragment spreading over 23 Mb(3,539797 to 26,493225 bp) was refined to a smaller QTL region of 16 Mb (3,539797 to 19,470000 bp) by genotyping microsatellites at the fragment boundaries.
For improving the mapping resolution, 97Crc11 mice were backcrossed with B6 parents in order to generate recombination events inside the MMU11 spretus DNA segment. Three informative recombinant strains were generated at the homozygous state, 97Crc11a (14.7 Mbp-19.5 Mbp), 97Crc11b (13.4 Mbp-19.5 Mbp), 97Crc11c (9.6 Mbp-19.5 Mbp) and analyzed for the testicular phenotype.
Only 97Crc11c exhibited a significantly smaller testis weight than that of B6 with a high level of teratozoospermia (35% of abnormal spermatozoa in average). The overlapping of the spretus segments of the 3 recombinant lines narrowed down the minimal interval of the Ltsw1 QTL to a 5 Mb spretus region located between microsatellites D11Mit259 and D11Mit148 (9.6-14.7 Mb). The reduced Ltsw1 QTL region of interest contained only 11 annotated genes according to the NCBI database (Table 1).
To point out a pertinent candidate gene several screens considering expressional and functional data provided by experimental results, databases, and literature reports were applied.
All of the data collected in the literature and those obtained by the experiments done in the current study converged to present gene Fignl1 as a strong candidate for the testicular phenotype of 97 C mice. Compared to the 11 genes of the refined region, Fignl1 was the only gene which was expressed in the cell type related to the phenotype, that had a known role in a function related to the phenotype and harbored several non synonymous SNPs differentiating the spretus alleles from the musculus alleles of Fignl1.
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