Manipulation of Estrogen Synthesis Alters MIR202* Expression in Embryonic Chicken Gonads1

Abstract

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.

The potential role of microRNAs in regulating gonadal sex differentiation in the chicken embryo

“…In the chicken embryo, we have shown that miR-202-5p is expressed with a male bias during embryonic gonad development (Bannister et al 2009(Bannister et al , 2011. MiR-202-5p expression is reduced in male and female embryos treated with oestrogen (estradiol-17β) and increased in female embryos treated with oestrogen inhibitors (Bannister et al 2011). This suggests that miR-202-5p may play a role downstream of oestrogen signalling, perhaps in maintaining the testis phenotype.…”

“…We have previously shown miR-202-5p to be up-regulated in males from the point of differentiation and that its expression is altered by oestrogen levels (Bannister et al 2011). With this in consideration, miR-202-5p may act to modulate TGF-β signalling in differentiated testis.…”

“…Percentages represent miRNAs in the gonads of males (blue) or females (pink) with greater than twofold expression relative to the opposite sex is however the only gonadal miRNA-mRNA interaction that has been clearly demonstrated. In the chicken embryo, we have shown that miR-202-5p is expressed with a male bias during embryonic gonad development (Bannister et al 2009(Bannister et al , 2011. MiR-202-5p expression is reduced in male and female embryos treated with oestrogen (estradiol-17β) and increased in female embryos treated with oestrogen inhibitors (Bannister et al 2011).…”

“…Error bars show standard error of the mean. MiR-202-5p expression data from Bannister et al (2011), used here with permission function to modulate the activity of TGF-β pathway inhibitors, allowing factors such as AMH to operate. Similarly, in females, modulation of TGF-β pathway repressors may allow TGF-β family members required for ovarian development to function, such as activins, inhibins, follistatin, and BMPs.…”

The role of non-coding RNAs in male sex determination and differentiation

“…In both species, they are expressed in the key Sertoli cell lineage. In chickens, experimental male-to-female sex reversal with oestrogen causes a decline in chicken miR-202-3p expression (Bannister et al 2011). Interestingly, miR-202-3p also was found to be one of the main miRNAs depleted in the sterile gonads of Xenopus hybrids, suggesting it is important for gametogenesis (Michalak & Malone 2008).…”