Molecular cloning of porcine estrogen receptor-beta complementary DNAs and developmental expression in periimplantation embryos.
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-beta) mRNA by reverse transcription-polymerase chain reaction (RT-PCR) using specific primer sets designed initially within conserved regions of human and bovine ER-beta mRNAs, and subsequently within regions of identified porcine ER-beta cDNA sequences. The ER-beta 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-beta proteins. Northern and Western blot analyses using porcine filamentous embryos from Day 12 of pregnancy demonstrated the presence of multiple ER-beta mRNA transcripts of approximately 9.5, 4.9, and 3.5 kilobases, and a similar 64-kDa protein corresponding in size to human ovarian granulosa cell ER-beta, respectively. In Day 12 filamentous embryos, ER-beta expression was immunolocalized to trophoblastic cell nuclei, coincident with that of proliferative cell nuclear antigen (PCNA). The developmental ontogeny of ER-beta mRNA was evaluated in embryos of different morphologies (spherical, tubular, and filamentous) by semiquantitative RT-PCR, along with those for other steroid hormone receptors (ER-alpha and progesterone receptor) and known embryonic genes associated with cell differentiation (cytochrome P450 aromatase type III) and growth (cyclin D1). ER-beta 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-alpha gene was barely detectable and appeared constitutive for all developmental stages examined. Estradiol-17 beta treatment of Day 12 filamentous embryos in culture up-regulated ER-beta 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-beta likely mediates the autocrine functions of estrogens in the dynamic regulation of embryonic growth and development at periimplantation.
Kowalski
Andrés A
AA
Interdisciplinary Concentration in Animal Molecular and Cell Biology, Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910, USA.
Graddy
Logan G
LG
Vale-Cruz
Dustin S
DS
Choi
Inho
I
Katzenellenbogen
Benita S
BS
Simmen
Frank A
FA
Simmen
Rosalia C M
RC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D047108:Embryonic Development; D004717:Endometrium / Q000737:chemistry; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013552:Swine / Q000196:embryology* / Q000235:genetics
D018076:DNA, Complementary; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D004958:Estradiol