miR-505-3p is a repressor of puberty onset in female mice

in Journal of Endocrinology
Correspondence should be addressed to J Xiao: xiaojunhua@dhu.edu.cn
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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.

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  • Supporting Information
  • 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.

 

      Society for Endocrinology

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    The evidence of miR-505-3p being a candidate gene in the puberty onset-related QTL region on chromosome X. (A) Schematic representation of the ISCSs and control strains. The genotype on chromosome X for each of the eight congenic strains is represented by the horizontal bars. Green portions indicate a known homozygous B6 segment, blank portions represent a known homozygous C3H segment, and hatched regions depict an area where a crossover between C3H and B6 occurs. The dotted lines indicate the boundary of the refined QTL region. (B) The expression situation of six protein-coding genes in the QTL region in HPG axis in B6 and C3H strains. H, hypothalamus; O, ovary; P, pituitary. (C) The expression difference of miR-505-3p in HPG axis between B6 and C3H mice. (D) The spatial-specific expression of miR-505-3p in different time point of B6 mice. (E) The temporal-specific expression of miR-505-3p in different tissues of B6 mice. (F) The expression level of puberty-related genes in GT1-7 and pGT1-7cells. Bars are means and vertical bars represent SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

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    The HE-stained ovarian sections at various time points. Arrows in the graph point to different kinds of follicles before ovulation and CL after ovulation. (A and B) 5 days after injection, untreated, LV-treated, respectively. (C and D) 20 days after injection, untreated, LV-treated, respectively. (E and F) 30 days after injection, untreated, LV-treated, respectively. (G and H) 40 days after injection, untreated, LV-treated, respectively.

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    The influence of miR-505-3p ectopic expression in the hypothalamus on the body weight, VO timing and fertility of the tested mice. (A) The growth curve of the mice; (B) the VO time of the tested mice; (C) the interval time between paring with male mice and delivery (LV-treated = 25.83 ± 1.144 days, saline-treated = 24.33 ± 1.247 days, untreated = 22.92 ± 0.8307, *P < 0.05); (D) the death rate of offspring before weaning of the tested mice (newborn offspring: LV-treated = 6.100 ± 0.794, saline-treated = 7.111 ± 0.309, untreated = 7.949 ± 0.179; dead offspring: LV-treated = 2.111 ± 0.626, saline-treated = 0.111 ± 0.111, untreated = 0.256 ± 0.108. *P < 0.05, **P < 0.01).

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    Fertility analysis of the miR-505-3p-overexpressed mice and in situ hybridization of their brain section. (A) Pie chart shows the percentage of female mice which had different number of dead pups per litter and the rate of abortion and infertility among the three groups; (B) Images of coronal sections showing the fluorescence signals of hypothalamic miR-505-3p detected by in situ hybridization among one sterile, one with reduced fertility, one normal, saline-treated and untreated mice using U6, miR-505-3p and scrambled LNA probes, respectively. The white arrows indicate the location of injection with most intensive florescence signals.

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    The phenotype of miR-505-3p-knockout mice. (A) The growth curve of the DKO, SKO and WT mice; (B) the percentage of female mice attaining VO at different time point; (C) the mass of reproductive system at PND45; (D) the serum LH and FSH of the DKO and WT mice; and (E) HE-stained ovarian sections of the tested mice. (*P < 0.05, **P < 0.01).

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    The expression level of Srsf1, Kiss1 and GnRH in the hypothalamus of mir-505-knockout mice at PND15, PND25 and PND45.

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    The regulatory role of miR-505-3p on Srsf1 gene expression in GT1-7 cells. (A) The translation of SRSF1 was inhibited by miR-505-3p overexpression in pGT1-7 cell line. (B) The mRNA expression level of Gnrh and Kiss1 varied along with the expression of Srsf1 gene in GT1-7 and pGT1-7 cells. ‘Srsf1-pGT1-7’ column refers to the pGT1-7 cells transfected by a plasmid carrying a Srsf1 expression cassette. ‘shRNA-Srsf1-GT1-7’ column refers to the GT1-7 cells transfected by shRNA targeting Srsf1 gene. Bars are means and vertical bars represent s.e.m. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no statistical significance).

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