Experiment
Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, the authors narrowed a puberty-related QTL region (Pubt1; MGI:3811896) down to a 1.7Mb region on chromosome X in female mice and inferred miR-505-3p (Mir505; MGI:3629955) as the functional gene.
In the authors' previous studies, they isolated a QTL region on chromosome X affecting the vaginal opening in female mice (Pubt1; Zhu et al. 2008). In this study, they narrowed this QTL region down to a 1.7 Mb region by constructing eight interval-specific congenic strains (ISCSs) between C57/BL6 and C3H/He mice. The mice of the strain carrying the QTL region were backcrossed with C3H/He mice. The resulting male F2 mice holding at least one recombination in the interval of interest were chosen and then backcrossed with female C3H/He to obtain N2 generation. The female N2 continued to mate with male C3H/He to generate N3 generation. N3 male mice holding only one recombination at the target interval were selected and continued to backcross with female C3H/He to generate an N4 generation. N4 mice siblings were crossed until N7 generation. Finally, the age at vaginal opening of all female mice of N7 progenies was then recorded.
Among the genes in this region, Mir505 was assumed to be the potential candidate due to the variation in its flanking sequence and gene expression differences between C3H and B6 mice, as well as its functional annotation.
The authors conducted ectopic expression of Mir505 in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of Mir505 on female puberty was evaluated by the measurement of pubertal/reproduction events and histological analysis. The results showed that female mice with overexpression of Mir505 in the hypothalamus manifested later puberty onset timing both in vaginal opening and ovary maturation, followed by weaker fertility lying in the longer interval time between mating and delivery, higher abortion rate and smaller litter size. They also constructed Mir505-knockout mice by CRISPR/Cas9 technology. Mir505-knockout female mice showed earlier vaginal opening timing, higher serum gonadotrophin and higher expression of puberty-related gene in the hypothalamus than their WT littermates. Srsf1 proved to be the target gene of Mir505 that played the major role in this process. The results of RNA immunoprecipitation sequencing showed that SRSF1 (or SF2), the protein product of Srsf1 gene, mainly bound to ribosome protein (RP) mRNAs in GT1-7 cells. The collective evidence implied that Mir505/SRSF1/RP could play a role in the sexual maturation regulation of mammals.
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