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Dual source and target of heparin-binding EGF-like growth factor during the onset of implantation in the hamster

¹ Departments of Pediatrics,
² Molecular and Integrative Physiology and
³ Obstetrics and Gynecology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338, USA
⁴ Department of Obstetrics and Gynecology and Physiology, University of Nebraska, Medical Center, Omaha, NE 68198-4515, USA

View larger version (59K): [in a new window]   Fig. 1. Northern blot hybridization of Hegfl mRNA in the periimplantation (days 1-8) hamster uterus. The expression of Hegf1 mRNA was detected in total RNA samples obtained from whole hamster uteri on days 1-4, and implant sites on day 4 afternoon and days 5-8 of pregnancy. Total RNA from day-1 mouse uterus was used as a control. An approx. 2.5 kb transcript for Hegfl was detected in the uterus. The blot was hybridized with Rpl7 to confirm integrity, equal loading and blotting of RNA samples. These experiments were repeated twice with similar results. Bar diagram above the autoradiogram shows the relative levels of Hegfl mRNA (Hegfl mRNA/Rpl7 mRNA) after densitometric scanning of the autoradiogram.

View larger version (121K): [in a new window]   Fig. 2. In situ hybridization of Hegfl mRNA in the periimplantation (days 1-8) hamster uterus. Dark-field photomicrographs of representative uterine sections on days 1-8 of pregnancy are shown. Inserts show higher magnification of Hegfl mRNA accumulation in the presumptive implantation site (D4 am; 0900 hours) and implantation site containing a blastocyst (D4 pm; 1600 hours). am, antimesometrial side; bl, blastocyst; em, embryo; le, luminal epithelium; m, mesometrial side; myo, myometrium; s, stroma.

View larger version (149K): [in a new window]   Fig. 3. In situ hybridization of Hegfl mRNA in hypophysectomized (hyx) progesterone (P4)-treated pregnant (days 4 and 5) hamster uterus. Dark-field photomicrographs of representative uterine sections are shown. Pregnant hamsters were hypophysectomized on day 2 (0900 hours) and treated with P4 (1 mg/hamster) daily from day 2. Whole uterine tissues were collected on the morning of day 4 (0900 hours). Implantation sites were collected on the afternoon of day 4 (1600 hours) and morning of day 5 (0900 hours) after an intravenous injection of blue dye as described in the Materials and Methods. Day 5 implantation sites obtained from sham operated pregnant hamsters treated with oil, were used as control. am, antimesometrial side; bl, blastocyst; le, luminal epithelium; m, mesometrial side; myo, myometrium; s, stroma.

View larger version (48K): [in a new window]   Fig. 4. Northern blot hybridization of uterine Hegfl mRNA in steroid-treated adult ovariectomized hamsters. The expression of Hegfl mRNA was detected in total RNA samples obtained from whole ovariectomized hamster uteri after various steroid treatments. Ovariectomized hamsters were given a single injection of progesterone (P4), estradiol-17ß (E2) or P4 plus E2. Control ovariectomized hamsters received an injection of oil (0 hour). Uteri were collected at the indicated hour (h) after steroid treatments. As mentioned in the Fig. 1, an approx. 2.5 kb transcript for Hegfl was detected in the uterus. The blot was hybridized with Rpl7 to confirm integrity, equal loading, and blotting of RNA samples. These experiments were repeated twice with similar results. Bar diagram above the autoradiogram shows the relative levels of Hegfl mRNA (Hegfl mRNA/Rpl7 mRNA) after densitometric scanning of this autoradiogram.

View larger version (124K): [in a new window]   Fig. 5. In situ hybridization Hegfl mRNA in uteri of ovariectomized hamster after estradiol-17ß (E2) treatment. Photomicrographs of representative uterine sections are shown at 40x magnification. Ovariectomized hamsters were given an injection of E2 (1.0 µg/hamster) and killed 2, 6, 12 and 24 hours after the treatment. (Day 1) Control unoperated uterus; (Oil) control ovariectomized hamsters received an injection of oil. le, luminal epithelium; myo: myometrium; s, stroma.

View larger version (113K): [in a new window]   Fig. 6. In situ hybridization of Hegfl mRNA in uteri of ovariectomized hamster after various doses of estradiol-17ß (E2) or E2 plus ICI 18,2780 treatments. Photomicrographs of representative uterine sections are shown. Ovariectomized hamsters were killed 6 hours after an injection of either of the three doses of E2 (0.01, 0.1 and 1.0 µg/hamster) or E2 (1 mg/hamster) plus ICI 18,2780 (1 mg/hamster). Control ovariectomized hamsters received an injection of oil. le, luminal epithelium; myo, myometrium; s, stroma.

View larger version (74K): [in a new window]   Fig. 7. Southern blot analysis of RT-PCR amplified products of Hegfl, Erbb1 and Erbb4 in preimplantation embryos and day-4 uteri of hamsters. Lanes 1-5, one-cell, two-cell, four-cell, eight-cell/morula and blastocyst stages, respectively; lane 6, day 4 uterus; lane 7, day 4 uterine RNA without RT; lane 8, primer control. These experiments were performed twice with similar results.

View larger version (32K): [in a new window]   Fig. 8. Cellular localization of HB-EGF (A) and ErbB1 and ErbB4 (B) in hamster preimplantation embryos. Various stages of hamster preimplantation embryos were fixed, permeabilized and incubated with polyclonal HB-EGF, ErbB1 or ErbB4 antibodies. Localization of antigens (red) and nuclei (blue) were visualized using TRITC-conjugated secondary antibodies and Hoechst 33,342, respectively. Indirect immunofluorescence staining of HB-EGF, ErbB1 and ErbB4 was visualized by scanning laser confocal microscopy with 6 µm optical sections. HB-EGF was mainly in the cell cytoplasm of both ICM (inner cell mass) and trophectoderm (Tr) cells of the blastocyst (A). Both ErbB1 and ErbB4 were mainly in the cell surface of two-cell, four-cell and eight-cell embryos (B). At the blastocyst stage ErbB1 was present in the cell surface of Tr cells only. However, ErbB4 was present in both the surface and the cytoplasm of blastocyst Tr cells (B).

View larger version (62K): [in a new window]   Fig. 9. HB-EGF-induced autophosphorylation of ErbB1 (A) and ErbB4 (B) in day 4 uteri and blastocysts of hamsters. Autophosphorylation of ErbBs was determined in uterine membrane and blastocyst homogenate after preincubation (10 minutes) with or without HB-EGF (4 ng/ml, human recombinant HB-EGF; a gift from Dr M. Klagsbrun, Harvard medical School, Boston, MA). Day 4 mouse uterine membranes were used as controls. The labeling reaction was initiated by the addition of [-32P]ATP for 2 minutes. After labeling, proteins were precipitated with 10% trichloroacetic acid. The precipitates were then used for immunoprecipatation with antibodies to ErbB1 and ErbB4. The immunoprecipates were separated by SDS-PAGE (6%), and phosphorylated proteins were detected by autoradiography. No growth factor was added to the samples in lanes 1, 3 and 5. HB-EGF was added to those in lanes 2, 4 and 6. Lanes 1 and 2 show hamster uterine membranes; lanes 3 and 4 show day 4 mouse uterine membranes; lanes 5 and 6 show hamster blastocyst homogenates. These experiments were performed twice.