Expression of the epidermal growth factor receptor gene is regulated in mouse blastocysts during delayed implantation.
The delayed implantation model was used to study epidermal growth factor receptor(s) (EGF-R) in the mouse blastocyst. Delayed implantation and blastocyst dormancy were induced by ovariectomy on day 4 of pregnancy and were maintained by daily (days 5-7) injections of progesterone (P4). Blastocyst activation and implantation were initiated by coinjection of estradiol-17 beta (E2) with P4 on the 3rd day of delay. Immunostaining of EGF-R, autoradiographic detection of 125I-labeled EGF binding, and measurement of EGF-inducible subcellular protein tyrosine phosphorylation demonstrated the loss of EGF-R from blastocysts (dormant) recovered 24 h after ovariectomy or on the 3rd day of P4-maintained delay. However, increased EGF-R levels were detected in blastocysts (activated) recovered 12 or 24 h after E2 injection. Blastocyst EGF-R mRNA levels were quantitated by reverse transcriptase (RT)-PCR and distribution of this mRNA was examined by in situ hybridization. To provide a homologous probe for these studies, a mouse EGF-R partial cDNA was cloned and used as the template for synthesis of antisense- and sense-strand EGF-R RNA. Quantitative RT-PCR demonstrated an 8- to 10-fold reduction in EGF-R mRNA copies per cell in dormant blastocysts. In contrast, an 8-fold increase in EGF-R mRNA copies per cell was detected in activated blastocysts 8 h after injection of E2. In situ hybridization detected EGF-R mRNA in most cells of normal day 4 blastocysts but not in those of dormant blastocysts. These studies establish that expression of the EGF-R gene in mouse blastocysts is tightly regulated by maternal steroid hormonal status.
Paria
B C
BC
Department of Obstetrics-Gynecology, University of Kansas Medical Center 66160-7338.
Das
S K
SK
Andrews
G K
GK
Dey
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D010064:Embryo Implantation / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D007457:Iodine Radioisotopes; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010052:Ovariectomy; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D007457:Iodine Radioisotopes; D009838:Oligodeoxyribonucleotides; D009842:Oligopeptides; D012333:RNA, Messenger; D004958:Estradiol; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases