microRNA 92b-3p regulates primordial follicle assembly by targeting TSC1 in neonatal mouse ovaries

Abstract

The primordial follicle pool, providing all oocytes available to a female throughout her reproductive life, is established perinatally. The formation of primordial follicle pool is regulated by precise transcriptional and post-transcriptional mechanisms. Recent studies have identified several microRNAs as post-transcriptional regulatory factors in the process of primordial follicle assembly. Here, we showed that miR-92b-3p was significantly upregulated in the stage of primordial follicle assembly in newborn mouse ovaries. Inhibiting miR-92b-3p suppressed the formation of primordial follicles, while overexpression of miR-92b-3p accelerated the processes of cyst breakdown and the following primordial follicle assembly. Accordingly, the expression of follicular development-related genes was reduced upon inhibiting of miR-92b-3p and increased under miR-92b-3p overexpression. Mechanistic studies identified TSC1 as a direct target of miR-92b-3p. miR-92b-3p could activate mTOR/Rps6 signaling through targeting and inhibiting TSC1 expression. In addition, knockdown of TSC1 showed an identical phenotype with that of miR-92b-3p overexpression in accelerating processes of cyst breakdown and primordial follicle formation. Thus, our work demonstrates that miR-92b-3p is a novel regulator of primordial follicle assembly by negatively regulating TSC1 in mTOR/Rps6 signaling.

Keywords: Mir-92b-3p; TSC1; mTOR; primordial follicle formation.

Figure 1.

MiR-92b-3p expression in the developing mouse ovary. (a) Expression of miR-92b-3p in human organs from an integrated miRNA-seq database YM500v2. (b) Real-time PCR analysis of miR-92b-3p expression in tissues of adult mice. (c) Real-time PCR analysis of miR-92b-3p expression in developing mouse ovaries. Data were obtained from at least six ovaries and are presented as mean ± SED. MiR-92b-3p was normalized to miR-U6 expression. *p < 0.05 was considered statistically different.

Figure 2.

Validation of efficiency of ovarian transfection of microRNA in vitro. (a) Ovaries were collected after incubation with miR-92b-3p labeled with Cy3 and evaluated by confocal microscopy. Strong red fluorescence was observed after 96 h of transfection. DAPI was used for staining nuclei. Bar = 50 μm. (b, c) Real-time PCR analysis of miR-92b-3p expression in ovaries transfected with miR-92b-3p inhibitor, miR-92b-3p mimics, or their respective controls. *p < 0.05.

Figure 3.

miR-92b-3p promotes primordial follicle assembly in neonatal mouse ovaries. The cultured ovaries after transfection at 0.5 dpp were subjected to immunohistochemistrical analysis with VASA antibody (dark brown) to help visualize the germ cells in the primordial follicles. Follicles were detected and counted in sections of mouse ovaries 1, 2, 3, and 4 days after transfection with (a) Inhibitor NC or miR-92b-3p inhibitor, (b) Mimics NC or miR-92b-3p mimics. Percentage of germ cells in cysts, primordial follicles was shown on the right panel. Data were obtained from at least six ovaries and are presented as mean ± SED. Dashed red circles represent cysts. Black circles represent primordial follicles. *p < 0.05.

Figure 4.

miR-92b-3p activates the expression of the genes related to growing oocytes and granulosa cells. (a) Real-time PCR analysis of genes in control or miR-92b-3p inhibitor-treated ovaries. The levels of mRNA expression in miR-92b-3p inhibitor-treated groups were normalized to the control groups. (b) Real-time PCR analysis of genes in control or miR-92b-3p mimics treated ovaries. The levels of mRNA expression in miR-92b-3p mimics treated groups were normalized to the control groups. *p < 0.05.

Figure 5.

miR-92b-3p targets TSC1 to activate Rps6 during primordial follicle assembly. (a) Vein diagram showing the potential 258 miR-92b-3p targeted mRNAs from two computational prediction algorithms miRDB and Targetscan. (b) FunRich database analysis of the 258 miR-92b-3p targets showing TSC1 to be an important target. (c) The sequence alignment of mouse miR-92b-3p and the 3′-UTR of TSC1. Mutations of the 3′-UTR of TSC1 (underlined CACGUUAU) was used to create the mutant luciferase reporter construct. The WT and Mut-TSC1 3ʹUTR were separately cloned into the modified pGL3 luciferase reporter vector. (d) Luciferase reporter assay showing that the activity of WT-TSC1 3ʹUTR, but not Mut-TSC1 3ʹUTR, was repressed by miR-92b-3p overexpression. (e) TSC1 mRNA expression was detected by Real-time PCR analysis after miR-92b-3p inhibitor or miR-92b-3p mimics transfection. (f) TSC1 protein expression was detected by western blotting after miR-92b-3p inhibitor or miR-92b-3p mimics transfection. P-Rps6 and total Rps6 were also tested. β-tubulin was used as a loading control. (g) Rps6 mRNA expression was detected by Real-time PCR analysis after miR-92b-3p inhibitor or miR-92b-3p mimics transfection. (h) The relative expression levels of p-Rps6 and total Rps6 proteins were analyzed by ImageJ software. *P < 0.05.

Figure 6.

TSC1 negatively regulates primordial follicle assembly in the mouse ovary. Real-time PCR (a) and western blot (b) analyses of TSC1 in 0.5dpp ovaries transfected with TSC1 siRNA or respective controls and cultured for 4 days. (c, d) The 0.5dpp ovaries were transfected with TSC1 NC or TSC1 siRNA and then collected 4 days later to count the germ cells in cyst (dashed red circle) and primordial follicles (black circle). The representative images were shown in (c), and percentage of germ cells in cysts, primordial follicles was shown in (d). (e) Real-time PCR analysis of growth-related genes in TSC1 NC or TSC1 siRNA treated ovaries. The levels of mRNA expression in TSC1 siRNA treated group were normalized to the control groups. * p < 0.05.