Epigenetic regulation of 5α reductase-1 underlies adaptive plasticity of reproductive function and pubertal timing

Abstract

Background: Women facing increased energetic demands in childhood commonly have altered adult ovarian activity and shorter reproductive lifespan, possibly comprising a strategy to optimize reproductive success. Here, we sought to understand the mechanisms of early-life programming of reproductive function, by integrating analysis of reproductive tissues in an appropriate mouse model with methylation analysis of proxy tissue DNA in a well-characterized population of Bangladeshi migrants in the UK. Bangladeshi women whose childhood was in Bangladesh were found to have later pubertal onset and lower age-matched ovarian reserve than Bangladeshi women who grew-up in England. Subsequently, we aimed to explore the potential relevance to the altered reproductive phenotype of one of the genes that emerged from the screens.

Results: Of the genes associated with differential methylation in the Bangladeshi women whose childhood was in Bangladesh as compared to Bangladeshi women who grew up in the UK, 13 correlated with altered expression of the orthologous gene in the mouse model ovaries. These mice had delayed pubertal onset and a smaller ovarian reserve compared to controls. The most relevant of these genes for reproductive function appeared to be SRD5A1, which encodes the steroidogenic enzyme 5α reductase-1. SRD5A1 was more methylated at the same transcriptional enhancer in mice ovaries as in the women's buccal DNA, and its expression was lower in the hypothalamus of the mice as well, suggesting a possible role in the central control of reproduction. The expression of Kiss1 and Gnrh was also lower in these mice compared to controls, and inhibition of 5α reductase-1 reduced Kiss1 and Gnrh mRNA levels and blocked GnRH release in GnRH neuronal cell cultures. Crucially, we show that inhibition of this enzyme in female mice in vivo delayed pubertal onset.

Conclusions: SRD5A1/5α reductase-1 responds epigenetically to the environment and its downregulation appears to alter the reproductive phenotype. These findings help to explain diversity in reproductive characteristics and how they are shaped by early-life environment and reveal novel pathways that might be targeted to mitigate health issues caused by life-history trade-offs.

Keywords: 5α reductase-1; Aging; Colitis; Hypothalamus; Menopause; Mice; Ovary; Puberty; Reproduction; Women.

Fig. 1

Pre-pubertal colitis in mice affects pubertal timing and ovarian function. A Age of vaginal opening (VO), indicating onset of puberty. ***P < 0.001; n = 47, 59. B Age of VO in second generation, relative to that in off-spring of parent littermate controls. P > 0.05; n = 20, 30. C Ovulatory rates (average number of successful pregnancies for each mouse) and D litter sizes in DSS-treated (n = 14 litters) and littermate control mice (n = 20 litters) from 5 mice in each treatment group after four encounters with the same male; *P = 0.024 (Mann-Whitney t-test). E Circulating AMH (ng/ml) in control [n = 11] and DSS-treated [n = 14] mice, **P = 0.008. F–I H&E stained ovarian histological sections from F, G control and H, I DSS-treated groups. Some atretic follicles (yellow asterisks) and zona pellucida remnants (green arrows) are marked. J Follicle counts from sections of mice ovaries (n = 4, n = 6), compared by t-test. K Volcano plot and L heat map of differentially expressed ovarian genes at Padj < 0.05 from RNA-seq analysis. M Signaling pathway to follicle activation, showing some of the differentially expressed genes (DEGs; P < 0.05). Red boxes signify upregulated genes; green boxes signify downregulated genes

Fig. 2

Srd5a1 is downregulated and hypermethylated in mice and women following early-life challenge. A Differentially methylated regions associated with three genes (Illumina EpicMethylation sites) in buccal DNA of Bangladeshi women who grew up in Bangladesh (n = 16) or UK (n = 13); mean ± SEM. B The mRNA levels of the same three genes in control (n = 16) and DSS-treated (n = 14) mice ovaries from the RNA-seq analysis; ***Padj < 0.001; mean ± SEM. C Confirmatory qPCR analysis of the Srd5a1 mRNA levels (n = 5), **P = 0.007; showing means with individual data points. D Levels of CpG methylation in the 5’region of the Srd5a1 first intron (corresponds with first site in Fig. 2A: see Fig S4), in control (n = 24) and DSS-treated (n = 29) mice ovaries, mean ± SEM shown relative to controls; **P = 0.015 (Mann-Whitney t-test). E Correlation between the levels of Srd5a1 mRNA (from RNA-seq analysis) and methylation measured in the same samples; P = 0.0085

Fig. 3

The upregulation of Srd5a1 by estradiol is blunted by anti-inflammatory cytokines. A Srd5a1 mRNA levels in ovaries of mice of various ages (n = 3–7). Groups sharing same letter: P > 0.05 (ANOVA, Tukey-Kramer t-test). B–D In ovarian KK-1 cells: B Srd5a1 mRNA levels (n = 3) after exposure to estradiol (E2), progesterone (P4), dihydrotestosterone (DHT), or dexamethasone (Dex). For E2-treatments, #P < 0.05; ##P < 0.01 compared to controls, otherwise P > 0.05. C Srd5a1 mRNA levels after cytokine exposure in KK-1 cells (n = 3). D Also in KK-1 cells, Srd5a1 (n = 6) and Cyp19a (n = 3) mRNA levels after E2 alone (10 nM) or with cytokine (100 ng/ml); #P < 0.025, ##P < 0.01, ### P < 0.001 vs control; **P < 0.02 vs E2; where not marked P > 0.05. All graphs show mean with individual data points

Fig. 4

5α reductase-1 regulates the central control of reproduction and pubertal timing. Srd5a1 mRNA levels were measured in A whole hypothalamus (n = 14), prefrontal cortex (n = 15, 14) and pituitary (n = 7) of control and DSS-treated mice or B preoptic area (POA: n = 6, 5) and arcuate nucleus (ARC: n = 6, 5) of the hypothalamus, and the cerebellum (CB: n = 6). C Srd5a1 mRNA levels in the ovary (n = 8) and whole hypothalamus (n = 4, 5) of female off-spring of DSS-treated mice and their littermate controls. D The mRNA levels of genes encoding reproductive regulatory factors were measured in the whole hypothalamus of the DSS-treated and control mice, with Fkbp5 as an indicator for stress (n = 7 or 8). E, F The effect of 5α reductase inhibitor, dutasteride in GT1-7 GnRH neuronal cells on E gene expression (n = 4 or 3) and F GnRH release, in which some cells were also exposed to the GABA_AR agonist, muscimol, alone (n = 2), with dutasteride (n = 7) or together with allopregnanolone (AP) (n = 3), all in the presence of AP precursor, P4 (for NT n = 7, for dutasteride alone n = 3). G Age of VO following dutasteride: female mice and their control littermates were given dutasteride (or vehicle) in the diet each day after weaning, and checked daily for VO (n = 6). For all, significant differences are shown for comparisons with control mice or untreated cells, otherwise P > 0.05