Masculine Epigenetic Sex Marks of the CYP19A1/Aromatase Promoter in Genetically Male Chicken Embryonic Gonads Are Resistant to Estrogen-Induced Phenotypic Sex Conversion1

Abstract

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

Differential Incubation Temperatures Result in Dimorphic DNA Methylation Patterning of the SOX9 and Aromatase Promoters in Gonads of Alligator (Alligator mississippiensis) Embryos1

“…Interestingly, in both fish species examined, administration of exogenous estrogens did not significantly affect methylation patterns within gonads, suggesting that methylation patterning of the fish cyp19a promoter is independent of estrogen signaling. In the chicken, the CYP19A1 promoter is also elevated in embryonic testes relative to that in ovaries [33], but in contrast to fish, treating chromosomal male chicken embryos with a synthetic estrogen is sufficient to partially reduce methylation levels in the CYP19A1 promoter. However, it should be noted that whereas treatment with ethinyl estradiol appeared to completely feminize the embryonic gonad at the histological level, DNA methylation levels in the CYP19A1 promoter was only partially feminized and remained significantly elevated compared to that in normal females [33].…”

“…In the chicken, the CYP19A1 promoter is also elevated in embryonic testes relative to that in ovaries [33], but in contrast to fish, treating chromosomal male chicken embryos with a synthetic estrogen is sufficient to partially reduce methylation levels in the CYP19A1 promoter. However, it should be noted that whereas treatment with ethinyl estradiol appeared to completely feminize the embryonic gonad at the histological level, DNA methylation levels in the CYP19A1 promoter was only partially feminized and remained significantly elevated compared to that in normal females [33]. The origin of sexually dimorphic methylation patterns within the alligator CYP19A1 promoter reported in this study is currently unknown.…”

“…In the chicken, the methylation status of the CYP19A1 promoter is hypermethylated in males relative to that in females [33]. Chickens also exhibit another interesting sexually dimorphic methylation pattern referred to as the chicken male hypermethylated region (cMHM) on the Z chromosome.…”

Cardio-respiratory development in bird embryos: new insights from a venerable animal model

“…Curiously, although avian embryos have been used extensively in many types of developmental biology studies, they have been far less exploited in either intraor transgenerational epigenetic studies. Certainly, DNA methylation and histone modification patterns have been investigated as a function of development in avian embryos and hatchlings (Ellis et al, 2012;Li et al, 2015), and in some cases these molecular findings have been related to the respiratory transition from allantoic to pulmonary gas exchange (Gryzinska et al, 2014). Perhaps the biggest contributions to date from studies of avian embryos attempts to answer the question What is the role of "maternal provisioning" in epigenetic inheritance?…”

Problems of Birds Sex Determination

“…Gonads of birds on stage injection EE2-3 -4 days incubation contain somatic cells and PGC of future gonads of both types. At the same time estrogen affects only somatic cells but does not affect predecessor of germ one [29]. So the control of aromatase activity through its methylation in chickens and the impact on estrogen metabolites can significantly reduce the activity of at least the main male sex determining genes and cause temporary inversion of gender different from the complete inversion of fish or reptiles.…”