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Taken together, we identified a panel of potentially candidate male development transcripts during early chicken embryogenesis some might be regulated by sex hormones. The interaction effects of sex by formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). Treatment with an aromatase inhibitor formestane at E4 affected the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), similar to what was observed for the doublesex and mab-3 related transcription factor 1 gene.

Whole mount and frozen cross-section in situ hybridization, as well as Western blotting analysis further corroborated that riboflavin kinase ( RFK ), WD repeat domain 36 ( WDR36), and EY505808 transcripts RFK and WDR36 protein products were predominantly expressed in E7 male gonads. At 72 wk of age, 20 and 4 transcripts were expressed at higher levels in the testes and brains of male than in the ovaries and brains of female chickens (P < 0.05), respectively. Twelve of these transcripts mapped to the Z chromosome.

Of 35 selected transcripts, quantitative reverse transcription-polymerase chain reaction validated that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). A total of 89 and 127 candidate male development transcripts that represented 83 known and 119 unknown non-redundant sequences, respectively, were characterized in an embryonic day 3 (E3 Hamburger and Hamilton Stage 20: HH20) male-subtract-female complementary DNA library. Early transcripts related to male development in chicken embryos and their expression profiles were examined.

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