Molecular cloning of porcine estrogen receptor-β complementary DNAs and developmental expression in periimplantation embryos

Andrés A. Kowalski, Logan G. Graddy, Dustin S. Vale-Cruz, Inho Choi, Benita S Katzenellenbogen, Frank A. Simmen, Rosalia C.M. Simmen

Research output: Contribution to journalArticle

Abstract

In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90% identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RTPCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.

Original languageEnglish (US)
Pages (from-to)760-769
Number of pages10
JournalBiology of reproduction
Volume66
Issue number3
DOIs
StatePublished - Jan 1 2002
Externally publishedYes

Fingerprint

Estrogen Receptor beta
Molecular Cloning
Estrogen Receptors
Swine
Embryonic Structures
Complementary DNA
Messenger RNA
Aromatase
Cyclin D1
Nuclear Antigens
Estrogens
Progestins
Progesterone Receptors
Granulosa Cells
Steroid Receptors
Endometrium
Cell Nucleus
Growth and Development
Northern Blotting
Cytochrome P-450 Enzyme System

Keywords

  • Embryo
  • Estradiol receptor
  • Implantation
  • Pregnancy
  • Steroid hormone receptors

ASJC Scopus subject areas

  • Reproductive Medicine
  • Cell Biology

Cite this

Molecular cloning of porcine estrogen receptor-β complementary DNAs and developmental expression in periimplantation embryos. / Kowalski, Andrés A.; Graddy, Logan G.; Vale-Cruz, Dustin S.; Choi, Inho; Katzenellenbogen, Benita S; Simmen, Frank A.; Simmen, Rosalia C.M.

In: Biology of reproduction, Vol. 66, No. 3, 01.01.2002, p. 760-769.

Research output: Contribution to journalArticle

Kowalski, Andrés A. ; Graddy, Logan G. ; Vale-Cruz, Dustin S. ; Choi, Inho ; Katzenellenbogen, Benita S ; Simmen, Frank A. ; Simmen, Rosalia C.M. / Molecular cloning of porcine estrogen receptor-β complementary DNAs and developmental expression in periimplantation embryos. In: Biology of reproduction. 2002 ; Vol. 66, No. 3. pp. 760-769.
@article{899f3542cbfb44938216227db9fb0a89,
title = "Molecular cloning of porcine estrogen receptor-β complementary DNAs and developmental expression in periimplantation embryos",
abstract = "In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90{\%} identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RTPCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.",
keywords = "Embryo, Estradiol receptor, Implantation, Pregnancy, Steroid hormone receptors",
author = "Kowalski, {Andr{\'e}s A.} and Graddy, {Logan G.} and Vale-Cruz, {Dustin S.} and Inho Choi and Katzenellenbogen, {Benita S} and Simmen, {Frank A.} and Simmen, {Rosalia C.M.}",
year = "2002",
month = "1",
day = "1",
doi = "10.1095/biolreprod66.3.760",
language = "English (US)",
volume = "66",
pages = "760--769",
journal = "Biology of Reproduction",
issn = "0006-3363",
publisher = "Society for the Study of Reproduction",
number = "3",

}

TY - JOUR

T1 - Molecular cloning of porcine estrogen receptor-β complementary DNAs and developmental expression in periimplantation embryos

AU - Kowalski, Andrés A.

AU - Graddy, Logan G.

AU - Vale-Cruz, Dustin S.

AU - Choi, Inho

AU - Katzenellenbogen, Benita S

AU - Simmen, Frank A.

AU - Simmen, Rosalia C.M.

PY - 2002/1/1

Y1 - 2002/1/1

N2 - In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90% identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RTPCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.

AB - In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estrogens and progestins are, however, unknown. Here we report the cloning of porcine embryonic estrogen receptor-beta (ER-β) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-β mRNAs, and subsequently within regions of identified porcine ER-β cDNA sequences. The ER-β mRNA has an open reading frame of 1578 nucleotides and encodes a 526 amino acid polypeptide that displays greater than 90% identity with other mammalian ER-β proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-β mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a ∼64-kDa protein corresponding in size to human ovarian granulosa cell ER-β, respectively. In Day 12 filamentous embryos, ER-β expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-β mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RTPCR, along with those for other steroid hormone receptors (ER-α and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-β mRNA levels varied with embryo morphology (filamentous maximum at Day 12), coincident with that of cyclin D1. Progesterone receptor mRNA levels were maximal in tubular embryos, similar to that of P450 aromatase, whereas the expression of the ER-α gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17β treatment of Day 12 filamentous embryos in culture up-regulated ER-β and P450 aromatase (type III) mRNA levels, respectively, but decreased those of PCNA, and had no effect on cyclin D1 mRNA levels. These studies taken together suggest that embryonic ER-β likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.

KW - Embryo

KW - Estradiol receptor

KW - Implantation

KW - Pregnancy

KW - Steroid hormone receptors

UR - http://www.scopus.com/inward/record.url?scp=0036190764&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036190764&partnerID=8YFLogxK

U2 - 10.1095/biolreprod66.3.760

DO - 10.1095/biolreprod66.3.760

M3 - Article

C2 - 11870084

AN - SCOPUS:0036190764

VL - 66

SP - 760

EP - 769

JO - Biology of Reproduction

JF - Biology of Reproduction

SN - 0006-3363

IS - 3

ER -