A Window of MHM Demethylation Correlates with Key Events in Gonadal Differentiation in the Chicken

Abstract

The MHM (male hypermethylated) region on the chicken Z chromosome is of interest in terms of sex determination both because hypermethylation of the promoter in males results in female-specific expression, and in females the non-coding transcripts accumulate in the region of a locus that encodes a key candidate gene for testis differentiation in birds - DMRT1. However, it has yet to be established whether MHM plays a role in either sex determination or gonadal development in chicken. Here, we sought to establish the MHM methylation pattern during chicken gonadal development and determine how this pattern correlates with the expression profiles of genes involved in sex determination and gonadal differentiation. As expected, we found that MHM methylation levels were higher in male than in female gonads at most stages of development. We also showed that there was no significant difference in the methylation patterns between the right and left gonad and that the pattern of methylation in female gonads was not affected by sex reversal. However, we did find an unexpected window where MHM promoter methylation levels were very low in both males and females. This developmental window coincided with a stage of gonad development where there is a dramatic increase in expression of key testis genes in males and of key ovary genes in females. The current study raises the possibility that the non-coding RNA transcripts of MHM may affect testis and ovary differentiation by influencing sex-specific gene expression.

Experimental Sex Inversion of Chicken Embryos at Aromatase Inhibition, Estrogen Receptor Modulation, DNA Demethylation and Progesterone Treatment

“…Some inconsistency was noted regarding the time of maximum expression of MHM-segment in embryogenesis from the first day of incubation to 8 and even 14. In a recent study was found that MHM-amplicon shows maximum demethylation at day 8 of incubation in the developing testes that is coincident with both the lowest methylation levels found in the developing ovary and with a dramatic increase in expression of key genes required for gonadal differentiation such as DMRT1, SOX9, AMH, P450arom, FOXL2 and ERα [27].…”

Non-Coding RNAs: lncRNAs, miRNAs, and piRNAs in Sexual Development

“…Functional studies have shown that misexpression of MHM induces asymmetric ovarian development in females and altered gonadal expression of the avian testis-determining gene DMRT1, indicating that MHM plays an important role in chicken sex differentiation [Roeszler et al, 2012]. Interestingly, a window of MHM demethylation was found to coincide in time with key events in gonadal differentiation in the chicken [Yang et al, 2016]. The authors suggested that MHM transcripts may affect both testis and ovary differentiation by influencing sex-specific gene expression.…”

Sex determination and gonadal sex differentiation in the chicken model

“…The likely avian testis-determinant DMRT1 (see below), is located very close to the site of MHM binding, and indeed both are located on the same loop in lampbrush preparations of the Z chromosome (Teranishi et al, 2001). This had led to the suggestion that MHM could play a role in gonadal sex differentiation by contributing to epigenetic repression of DMRT1 expression female gonadal cells (Caetano et al, 2014, Roeszler et al, 2012, Yang et al, 2016 Hirst et al "house-keeping" functions (Handley et al, 2004). There are 884 protein-coding sequences and some 348 non-coding genes (mi-croRNAs and long non-coding sequences) (Bellott et al, 2010, Ellegren, 2011.…”