Puberty onset is a complex trait regulated by multiple genetic and environmental factors. In this study, we narrowed a puberty-related QTL region down to a 1.7 Mb region on chromosome X in female mice and inferred miR-505-3p as the functional gene. We conducted ectopic expression of miR-505-3p in the hypothalamus of prepubertal female mice through lentivirus-mediated orthotopic injection. The impact of miR-505-3p on female puberty was evaluated by the measurement of pubertal/reproduction events and histological analysis. The results showed that female mice with overexpression of miR-505-3p 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. We also constructed miR-505-3p-knockout mice by CRISPR/Cas9 technology. miR-505-3p-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 miR-505-3p 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 miR-505-3p/SRSF1/RP could play a role in the sexual maturation regulation of mammals.
Table S1. Primers for target genes used in the qRT-PCR
Table S2. Comparison of ages at VO among eight ISCS and the control strain C3.
Table S3. Sensible variations of 5 consecutive SNPs existed near 5’ upstream region of miR-505 gene between B6 and C3
Fig S1. The overexpression of miR-5-5-3p in pGT1-7 cells. (****: P<0.0001)
Fig S2. The miR-505-3p knockout strategy by CRISPR/Cas9 technology. A: the sequence of two sgRNAs targeting miR-505 cDNA region were given. In the coding sequence of mir-505 cDNA, the targeting region of the two sgRNAs were high-lighted in yellow shadow. The sequence of mature miR-505-5p is in the red box, and the sequence of mature miR-505-3p is in the green box. B: The DNA sequences for the two mice with large deletion in the cDNA region of mir-505, and they were used as founders of the knockout mice for further study.
Fig S3. The expression level of miR-505-3p in the hypothalamus of DKO, SKO and WT mice at different ages.
Fig S4. Luciferase assay for the target genes of miR-505-3p (**: P<0.01)
Fig S5. Pathway analysis of differentially expressed genes between GT1-7 and pGT1-7 cell.