Transcriptional landscape of mouse-aged ovaries reveals a unique set of non-coding RNAs associated with physiological and environmental ovarian dysfunctions

Abstract

The progressive and physiological decline in ovarian function depends on the rate of follicular loss by atresia, contributing to the reduction in ovarian reserve. Genetics and environmental factors play important roles in ovarian senescence and in the onset of ovarian dysfunctions such as diminished ovarian reserve. A better understanding of the mechanisms underlying ovarian aging and their regulation by genetic and environmental factors is needed to evaluate ovarian reserve and to predict fertility potential by identification of more accurate and less invasive markers. We report transcriptomic data (i) implicating novel (e.g. EIF2 signalling) and well-known pathways (e.g. TGFβ signalling), and (ii) defining a unique set of non-coding RNA (ncRNA), both associated with ovarian function. The latter includes miRNAs (e.g. Mir143 and Mir145), snoRNAs (e.g. Snord16a and Snora34), and one lncRNA (Gas5), which are differentially expressed in middle-aged ovaries (12 months) vs young-aged (3 months) from CD1 mice. Experimental analysis confirms that ovary lifespan varies across genetic backgrounds in mice and, genetics influences the response to environmental perturbations such as diet. Moreover, the identified ncRNAs were verified in a model of reproductive dysfunction promoted by the environmental toxicant ethylenthiourea. We also report the increase of miRNA143 and miRNA145 in follicular fluid of women with diminished ovarian reserve. Their levels inversely correlate with the hormonal profile and with the number of the oocytes recruited upon hormonal stimulation. Overall, we report a transcriptomic signature for ovarian dysfunction in vivo that provides a valuable resource for translational research in human reproductive aging.

Fig. 1. Gene expression analysis identifies an OAGS in female CD1 mice.

A  Hierarchical clustering of differentially expressed genes in middle-aged (M, n = 9) and young (Y, n = 9) groups. Microarray was conducted in three samples, each including three animals. B qRT-PCR of established markers of ovarian aging. C, D qRT-PCR of OAGS consisting of a lncRNA, snoRNAs and miRNAs differentially expressed genes in the two age categories. E  Mir143 and Mir145 and cognate targets Fshr and Acvr1b, mRNA levels in ovaries of M and Y mice. Data are reported as the ratio between mRNA/miRNA content in M and Y groups normalized to β-actin/Rnu6. qRT-PCR data are mean ± s.d. with 5–10 animals per group. Significant differences are indicated with *P < 0.05; ** P < 0.01

Fig. 2. Networks of putative target genes regulated by differentially expressed miRNAs.

Validated target genes (grey nodes) mapped to pathway annotations derived from literature and gene ontology using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com) A  Mir143 regulatory network B and Mir145 regulatory network. The solid lines connecting molecules represent a direct relation and dotted lines an indirect relation. IPA constructs networks that optimize for both interconnectivity and number of Focus Genes (the grey nodes) under the constraint of a maximal network size. White nodes are added by the algorithm to build a highly connected molecular network between Focus Genes

Fig. 3. OAGS changes in C57BL/6J and FVB/NJ mice fed different diets.

A Dietary effects on ovarian reserve were determined by qRT-PCR of established ovarian marker Amh, Gdf9, Bmp15 in both strains fed with different diets from weaning to necropsy. B OAGS downregulated genes were verified by qRT-PCR in both strains. Data are reported as the ratio between mRNA content exposed and control groups normalized to β-actin. Data are mean ± s.d. with five animals per group. Significant differences are indicated with *P < 0.05; ** P < 0.01

Fig. 4. miRNAs changes in C57BL/6J and FVB/NJ mice fed different diets.

Dietary effects on OAGS miRNAs were determined by qRT-PCR in C57BL/6J and FVB/NJ mice fed with different diet from weaning to necropsy. A Mir143 and Mir145 expression levels were verified in both strains. B Mir681 and Mir692-1 expression levels were verified in both strains. Data are reported as the ratio between miRNA content exposed and control groups normalized to Rnu6. Data are mean ± s.d. with five animals per group. Significant differences are indicated with *P < 0.05; ** P < 0.01

Fig. 5. OAGS changes in CD1 mice exposed to ETU since the conception.

A ETU effects on ovarian reserve were determined by qRT-PCR of established ovarian marker Amh, Gdf9, Bmp15, in mice exposed from the GD0 to PND180 (Fig S1, Supplementary information). B ETU-induced ovarian dysfunction was assessed by testing the OAGS across all doses of ETU by qRT-PCR. C miRNAs included into the OAGS were tested by qRT-PCR in all groups. D, E Mir143/Mir145 and cognate targets expression levels were determined by qRT-PCR. Data are reported as the ratio between mRNA/miRNA content in exposed and control groups normalized to β-actin/Rnu6 and, are mean ± s.d. with five animals per group. Significant differences are indicated with *P < 0.05; ** P < 0.01

Fig. 6. miRNAs aging-related expression in FF of women with DOR.

A MIR143 and MIR145 levels were tested by qRT-PCR in FF from DOR-affected and healthy women. FSHR and ACVRIB, respectively, were evaluated by qRT-PCR in GCs isolated from FF of women with DOR and healthy women. Data are reported as the ratio between mRNA/miRNA content exposed and control groups normalized to β-ACTIN/RNU6. Data are mean ± s.d. with 5 pz per control group and 7 pz DOR-affected. Significant differences are indicated with *P < 0.05; ** P < 0.01. B Scatterplots of relative expression show the correlation between MIR143 and MIR145 and DOR diagnosis and C AMH serum levels. D Spearman’s rank correlation illustrated as corrplot. Brown circles indicate positive correlation. Yellow circles illustrate negative correlation. The colours are linked to values between −1 (yellow) and 1 (brown) using the scale on the side. Size of the circles corresponds to absolute value of the correlation coefficients