Tissue-specific localization of cytochrome P450 aromatase in the equine embryo by in situ hybridization and immunocytochemistry

Estrogen production by the preimplantation equine embryo is presumed to be important in maternal-conceptus communication in the mare. The synthesis of C-18 estrogens from C-19 androgens requires cytochrome P450 aromatase (P450(arom)) in the conceptus, but little information is available on the specific tissue location or potential developmental patterns of expression for the horse. The goal of this research was to localize P450,,,, in the equine conceptus by immunocytochemistry and in situ hybridization. Intact blastocyst-stage embryos were collected by nonsurgical flush on Days 12-15 of pregnancy, fixed in 4% paraformaldehyde, and paraffin-embedded. Aromatase protein was localized using rabbit anti-human placental aromatase antiserum with a detection system utilizing peroxidase and 5-amino-9-ethylcarbazole. For in situ hybridization, tissue sections were incubated with sense or antisense [S-35]UTP-labeled cRNA probes prepared from equine aromatase cDNA. Aromatase protein and transcript were abundant in the extraembryonic trophectoderm but absent from embryonic ectoderm. No P450(arom) expression was detected in abembryonic endoderm or mesoderm. Aromatase expression was demonstrated in the endoderm beneath the disc (hypoblast), This pattern of P450(arom) expression in the equine blastocyst closely resembles that seen transiently in the porcine embryo, suggesting that regulatory mechanisms conferring tissue specificity may be conserved.