Sites of Estrogen Receptor and Aromatase Expression in the Chicken Embryo Jane E. Andrews, Craig A. Smith, and Andrew H. Sinclair 1 Department of Paediatrics and Centre for Hormone Research, The University of Melbourne, Royal Children’s Hospital, Parkville, Victoria 3052, Australia Accepted July 17, 1997 Estrogens have been implicated in sexual differentiation of both the gonads and the genitalia of birds. In chicken embryos, the gonads are steroidogenically active from an early age, and the aromatase gene, (cAROM), necessary for estrogen synthesis, is expressed only in females at the time of gonadal sex differentiation. However, no studies have directly demonstrated the distribution of estrogen receptor (cER) transcripts or proteins in the embryonic avian reproductive system. Whole-mount in situ hybrid- ization and immunohistochemistry were used here to identify sites of estrogen receptor expression in the embryonic chicken urogenital system. Estrogen receptor mRNA was observed in both male and female gonads prior to morphological differentiation, at Stage 26 (4.5 days of incubation), and continued until after sexual dimorphism at Stage 32 (7.5 days). Transcripts of cER were also detected in the Mu ¨llerian ducts and developing external genitalia of both sexes. Estrogen receptor pro- tein was analysed in the embryonic gonads by immuno- histochemistry and found to be most abundant in the cortex of the left ovary, although it was also present in the medulla of both female gonads. No significant cER protein expression was detected in the male gonad by immunohistochemistry. In contrast, the aromatase gene was expressed in the gonads of female embryos from the onset of sexual dimorphism but was not detectable in male gonads at any stage examined. These findings suggest that estrogen involvement in both gonadogenesis and genital development in chickens is mediated by the estrogen receptor.  1997 Academic Press Sex is determined genetically in birds; the female is the heterogametic sex (ZW) and the male is homoga- metic (ZZ). A genetic switch determines whether the indifferent gonad develops into an ovary or a testis (reviewed in Thorne, 1995). In female birds, the left ovary is functional in the adult and the right regresses. The hormones released from the ovary or testis regulate the development of secondary sexual characteristics such as the external genitalia, syrinx, and feathering (Taber, 1964). Estrogens are hormones, synthesized by the ovary, which are involved in the development of female secondary sexual characteristics, sexual differentiation of the nervous system, and vitellogenesis (Norris, 1985; van Tienhoven, 1983). In birds, as in other vertebrates, aromatase is the key enzyme responsible for estrogen synthesis. The aromatase gene is expressed in female, but not male, chicken gonads at the time of gonadal sex differentiation (Smith et al., 1997; Yoshida et al., 1996). The physiological effects of estrogen are mediated by estrogen receptors, specific nuclear recep- tor proteins, which bind estrogen and can act as transcription factors (reviewed in Katzenellenbogen, 1996). Only one estrogen receptor gene has been identi- fied in birds, but two estrogen receptor subtypes ( and 1 To whom correspondence should be addressed. Fax: 61 3 9345 6000. E-mail: sinclair@cryptic.rch.unimelb.edu.au. General and Comparative Endocrinology 108, 182–190 (1997) Article No. GC976978 182 0016-6480/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.