Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane

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Abstract

We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3β- hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (±SE) serum levels of P from 97.8 ± 16 to 2.7 ± 1.3 nmol/L within 18 h (P <0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H]R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (K(d)) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 ± 0.09 and 0.06 ± 0.02 fmol/μg DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

Original languageEnglish (US)
Pages (from-to)620-626
Number of pages7
JournalJournal of Clinical Endocrinology and Metabolism
Volume79
Issue number2
DOIs
StatePublished - Aug 1994
Externally publishedYes

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3-Hydroxysteroid Dehydrogenases
Radioligand Assay
Corpus Luteum
Progesterone Receptors
Primates
Assays
Promegestone
Tissue
Human Follicle Stimulating Hormone
Therapeutics
Macaca
Haplorhini
Ligands
Immunohistochemistry
trilostane
Macromolecules
Menstruation
Progesterone
Sucrose
Menstrual Cycle

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{3809c3924b124dc0b8a44e67c68044f5,
title = "Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane",
abstract = "We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3β- hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (±SE) serum levels of P from 97.8 ± 16 to 2.7 ± 1.3 nmol/L within 18 h (P <0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H]R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (K(d)) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 ± 0.09 and 0.06 ± 0.02 fmol/μg DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.",
author = "Ov Slayden and Mary Zelinski and Richard Stouffer and Brenner, {R. M.}",
year = "1994",
month = "8",
doi = "10.1210/jc.79.2.620",
language = "English (US)",
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journal = "Journal of Clinical Endocrinology and Metabolism",
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TY - JOUR

T1 - Radioligand binding assay of progesterone receptors in the primate corpus luteum after in vivo treatment with the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane

AU - Slayden, Ov

AU - Zelinski, Mary

AU - Stouffer, Richard

AU - Brenner, R. M.

PY - 1994/8

Y1 - 1994/8

N2 - We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3β- hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (±SE) serum levels of P from 97.8 ± 16 to 2.7 ± 1.3 nmol/L within 18 h (P <0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H]R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (K(d)) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 ± 0.09 and 0.06 ± 0.02 fmol/μg DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

AB - We and others have detected progesterone receptors (PR) in the primate (macaque and human) corpus luteum by immunocytochemistry. However, we have been unable to measure PR in the corpus luteum by conventional ligand binding assay, presumably because high endogenous concentrations of progesterone (P) in luteal tissue prevented specific binding of radiolabeled hormone to PR during the assays. To test this hypothesis, we treated monkeys with the 3β- hydroxysteroid dehydrogenase inhibitor, Trilostane, to reduce levels of endogenous P before conducting binding assays for PR in luteal tissue. To obtain adequate tissue for saturation analysis, rhesus monkeys (n = 6) were superovulated by treating them with hFSH beginning at menses (day 1) for 6 days, then with hFSH and hLH (days 6-9), followed by hCG (day 10). Trilostane (600 mg) was given 5 days after hCG treatment (day 15), and binding assays were conducted 18 h later. Trilostane significantly reduced mean (±SE) serum levels of P from 97.8 ± 16 to 2.7 ± 1.3 nmol/L within 18 h (P <0.001). Strong nuclear staining of PR was detected by immunocytochemistry in both Trilostane-treated and control (not Trilostane-treated) tissue, but ligand binding was measurable only in Trilostane-treated monkeys. Scatchard transformations of saturation curves revealed high affinity binding of [3H]R5020 in luteal cytosol and nuclear extracts with an approximate dissociation constant (K(d)) of 4.8 and 1.37 nmol/L, respectively. Also, the PR-specific monoclonal antibody, JZB-39, shifted a [3H]R5020-bound luteal macromolecule on sucrose gradients. Mean cytosolic and nuclear binding of [3H]R5020 were 0.31 ± 0.09 and 0.06 ± 0.02 fmol/μg DNA, respectively. Similar binding of [3H]R5020 was demonstrated in corpora lutea obtained during spontaneous menstrual cycles after Trilostane treatment (n = 3). These results show unequivocally that the PR in macaque luteal tissue can bind P with high affinity and suggest a receptor-mediated action of P in the primate corpus luteum.

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