Supplemental materials for Keiko Shioda et al. “Transcriptomal and epigenetic preservation of genetic sex identity in estrogen-feminized male chicken embryonic gonads”

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within one year. To identify the feminization-resistant “memory” of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomal and DNA methylome analyses. Protein-coding genes were classified based on their relative mRNA expression levels across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (e.g., DAZL or DDX4/VASA) and anti-apoptotic gene CBL was stronger in FemZZs than normal ZZ-testes or ZW-ovaries, suggesting that an increased population of germline cells may be present in FemZZs. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal gonads and showed feminization-induced hypermethylation at the transcription start sites of representative meiosis genes STRA8, ASZ1, and MEI1. Taken together, our study provides evidence that morphologically feminized FemZZs still maintain significant transcriptomal and epigenetic memories of genetic sex.