Catechol estrogen formation by pig blastocysts during the preimplantation period: Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity

Judith S. Mondschein, Roscoe M. Hersey, Sudhansu K. Dey, Duane L. Davis, Judith Weisz

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Abstract

Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blas-tocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up t o 30 min and with blastocyst protein concentrations of up to 100 ng in a reaction volume of 150 μl. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: Maximu m velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein -30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 μ m, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein-30 min), increased to a maximum on days 12 and 13 (628 ± 153 and 516 ± 227 pmol 2-OHE2/mg protein 30 min, respectively), and declined by day 14 (63.2 ± 32.9 pmol 2-OHE2/mg protein-30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.

Original languageEnglish (US)
Pages (from-to)2339-2346
Number of pages8
JournalEndocrinology
Volume117
Issue number6
DOIs
StatePublished - Jan 1 1985

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Catechol Estrogens
Aromatase
Blastocyst
Swine
Pregnancy
Proteins
Estradiol
Niacinamide
Polysorbates
estrogen 2-hydroxylase
NADP
Detergents
NAD
Estrogens

All Science Journal Classification (ASJC) codes

  • Endocrinology

Cite this

@article{7e7a85fa9b684cd491c04a6b65330f3c,
title = "Catechol estrogen formation by pig blastocysts during the preimplantation period: Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity",
abstract = "Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blas-tocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up t o 30 min and with blastocyst protein concentrations of up to 100 ng in a reaction volume of 150 μl. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: Maximu m velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein -30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 μ m, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein-30 min), increased to a maximum on days 12 and 13 (628 ± 153 and 516 ± 227 pmol 2-OHE2/mg protein 30 min, respectively), and declined by day 14 (63.2 ± 32.9 pmol 2-OHE2/mg protein-30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.",
author = "Mondschein, {Judith S.} and Hersey, {Roscoe M.} and Dey, {Sudhansu K.} and Davis, {Duane L.} and Judith Weisz",
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pages = "2339--2346",
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Catechol estrogen formation by pig blastocysts during the preimplantation period : Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity. / Mondschein, Judith S.; Hersey, Roscoe M.; Dey, Sudhansu K.; Davis, Duane L.; Weisz, Judith.

In: Endocrinology, Vol. 117, No. 6, 01.01.1985, p. 2339-2346.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Catechol estrogen formation by pig blastocysts during the preimplantation period

T2 - Biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity

AU - Mondschein, Judith S.

AU - Hersey, Roscoe M.

AU - Dey, Sudhansu K.

AU - Davis, Duane L.

AU - Weisz, Judith

PY - 1985/1/1

Y1 - 1985/1/1

N2 - Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blas-tocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up t o 30 min and with blastocyst protein concentrations of up to 100 ng in a reaction volume of 150 μl. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: Maximu m velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein -30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 μ m, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein-30 min), increased to a maximum on days 12 and 13 (628 ± 153 and 516 ± 227 pmol 2-OHE2/mg protein 30 min, respectively), and declined by day 14 (63.2 ± 32.9 pmol 2-OHE2/mg protein-30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.

AB - Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blas-tocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up t o 30 min and with blastocyst protein concentrations of up to 100 ng in a reaction volume of 150 μl. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E-2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: Maximu m velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein -30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 μ m, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein-30 min), increased to a maximum on days 12 and 13 (628 ± 153 and 516 ± 227 pmol 2-OHE2/mg protein 30 min, respectively), and declined by day 14 (63.2 ± 32.9 pmol 2-OHE2/mg protein-30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig.

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