Pregnancy Without Progesterone

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 355-388-Sex Hormone Receptor Action & Reaction
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-363
C. Jo Corbin*1, Elizabeth Scholtz1, Barry Ball2, Karen McDowell2, Shweta Krishnan3, Donald Patrick McDonnell*3 and Alan James Conley1
1University of California, Davis, CA, 2University of Kentucky, Lexington, KY, 3Duke University School of Medicine, Durham, NC
Progesterone is undetectable in the second half of pregnancy in mares, but is progressively replaced by 5α-dihydroprogesterone (DHP) and a variety of other 5α-reduced pregnanes of unknown bioactivity. DHP binds equine endometrial and mammary cytosol with affinity comparable to progesterone, as it does in elephant and rock hyrax cytosols, but the bioactivity of DHP at the progesterone receptor (PR) has not been confirmed in any species. Previous data demonstrated that DHP stimulates endometrial growth and maintains pregnancy to day 27 in mares after luteolysis is induced on day 14, but estimating relative efficacy and potency requires an in vitro bioassay. The equine PR was cloned and sequenced from an endometrial cDNA library and genomic DNA, and sub-cloned into pcDNA3.1. The coding sequence of equine PR is highly conserved but has S717C and G722A substitutions (relative to human PR) in the ligand binding domain (LBD), the latter consistent with the elephant but not rock hyrax LBD. Immunoblot analysis of transiently transfected cells indicated that recombinant equine PR expression was represented by a single protein comparable in size to human PR-B. Chinese hamster ovarian (CHO) cells were transfected, geneticin-resistant cells stably expressing equine PR were isolated, and transfected transiently with a mouse mammary tumor virus (MMTV) luciferase reporter expression plasmid. Ligand activation of equine PR and subsequent stimulation of MMTV-driven luciferase activity was measured after a 24h incubation with increasing concentrations of progesterone and DHP (1, 3, 10, 30 and 100nM). MMTV-luciferase induction was saturated at 30nM for both ligands but DHP stimulated nearly twice the maximum luciferase expression as progesterone, suggesting almost twice the efficacy of equine PR activation at saturation. DHP (1mM) did not stimulate reporter expression in CHO cells transfected with human PR and MMTV-luciferase reporter plasmids. These data are the first to demonstrate  directly that DHP activates the equine PR with similar potency to progesterone, and suggest it may even be a super-agonist in horses. Residues responsible for DHP binding  to the PR have yet to be defined. The axiom that mammalian pregnancies rely on progesterone alone needs revision and alternative endogenous progestogens sought in some species at least.

Nothing to Disclose: CJC, ES, BB, KM, SK, DPM, AJC

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Sources of Research Support: Center for Equine Health, School of Veterinary Medicine, University of California, Davis CA 95616John P. Hughes Endowment, University of California, Davis CA