| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The relationship between P4 action and the EGF-R system during pregnancy has not been extensively explored. After ovulation and during pregnancy, P4 becomes the quintessential uterotropic hormone required for decidualization [12, 13], expression of P4 receptors (PR) and ER [14, 15], and stromal cell cycle progression [16]. The EGF/EGF-R system is also important for decidualization [17–20]. Piva and coworkers [21] demonstrated that either EGF or basic fibroblast growth factor was required for cell division in cultured stromal cells; however, neither growth factor was mitogenic in the absence of P4. Furthermore, EGF can substitute for estrogen in initiating implantation in ovariectomized (Ovx) rats if P4 is also present [22]. One explanation for P4 dependency may be that the steroid is required for expression of EGF-R. This study demonstrates that the EGF-R is P4-dependent and is most abundant in early pregnancy during proliferation and decidualization of mesometrial stromal cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adult female Holtzman rats weighing 225–250 g were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and bred in the laboratory animal care facility of our institution. All animal care and use were conducted in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and a protocol approved by the Medical College of Georgia Committee on Animal Use in Research and Education. Pregnancy (Day 1) was identified by the presence of vaginal sperm following overnight exposure to a fertile male. Rats were killed at 0800–0900 h between Days 8 and 21 of pregnancy (term is on Day 22) or as mentioned for specific experiments.
P4 and Estradiol-17ß (E2) Treatment Regimens
In some experiments, pregnant animals were Ovx on Day 8 and Day 12 of pregnancy and treated immediately with the following hormonal regimens: 1) Ovx + Vehicle: this group received 0.2 ml corn oil daily, s.c.. 2) Ovx + E2: this group received 2.0 µg E2 dissolved in 0.2 ml corn oil, s.c. 3) Ovx + P4: a 25-mg pellet of crystalline P4 was placed i.p. via the dorsal incision made for ovariectomy to maintain pregnancy levels of serum P4. A single injection of P4 (0.5 mg, saline/20% ethanol, i.p.) was also given immediately after surgery to ensure continuous high levels of P4 immediately following ovariectomy. Corn oil vehicle was given daily s.c. (0.2 ml). 4) Ovx + P4 and E2: this group received P4 and E2 as described above. Ovx + Vehicle and Ovx + E2 groups were Ovx at 0800–0900 h on Day 9 (instead of Day 8) because tissue harvest was very limited due to extensive degeneration in absence of P4. Because vehicle and E2 treatments have shown similar effects on the PR and histology of the decidua basalis (DB), we used the Ovx + E2 treatment group as the control group for administration of P4 and P4 + E2 [15, 16, 23]. We have previously reported that the serum levels of P4 produced by the pellet implant are comparable with those found in normal pregnancy; however, 2 µg of estradiol-17ß produced serum levels of E2 about 3-fold higher than in normal pregnancy on Day 10 [14, 23].
Antiprogestin Treatment
Twenty-five-milligram pellets of crystalline RU-486 (Mifepristone; 11ß-(4-dimethylaminophenyl)-17ß-hydroxy-17
(prop-1-ynyl)-estra-9,9-dien-3-one) were placed i.p. in otherwise normal pregnant rats at 0830–0900 h on Day 9. Rats also received 0.5 mg RU-486 in saline/40% ethanol i.p. immediately after implantation of a pellet to ensure rapid onset of antiprogestin action. Animals were killed 3, 6, 12, and 24 h later. This regimen of treatment does not alter serum P4 [23]. Although RU-486 is most potent as an antiprogestin, it has significant antiglucocorticoid activity [24]. However, the RU-486 effects described herein are most likely due to blockade of P4 action since they were similar to those induced by ovariectomy and reversed by P4 replacement ([15, 16, 23] and this study). Furthermore, glucocorticoids do not appear to be involved in decidualization [25].
Immunohistochemistry
For immunolocalization of EGF-R, tissues were fixed in buffered formaldehyde, embedded in paraffin wax, sectioned at 6 µm, and mounted on Probe-Plus slides (Fisher Scientific, Pittsburgh, PA). Subsequent procedures were as previously described [26]. Briefly, staining procedures used Vectastain Elite ABC kits and diaminobenzidine-nickel chloride as substrate for horseradish peroxidase (Vector Laboratories, Burlingame, CA). EGF-R was localized with a polyclonal IgG antibody made in sheep against a recombinant human EGF-R fusion protein at a 1:150 dilution (6.7 µg/ml) (Upstate Biotechnology Inc., Lake Placid, NY). Specificity of staining was checked by incubating sections with antibody preincubated with 40-fold excess antigen peptide. Tissue sections from 3 to 5 pregnant animals at each stage of pregnancy and for each treatment group were prepared as described and subjectively evaluated for distribution of immunoreactive EGF-R by two investigators without regard to treatment.
Tissue Preparation for Immunoprecipitation and Immunoblotting
Uteri from Day 8 to Day 17 pregnant rats were rapidly excised and placed in ice-cold saline until dissected. Uteri were carefully laid on a glass plate and placed on the stage of a dissecting microscope. The plate was held at 0–4°C by an underlayer of ice. DB were isolated by gently separating the placenta and myometrial regions with 23-gauge needles. In early pregnancy, the placenta and decidua are at an early stage of differentiation and cannot be reliably separated. For this reason, DB dissected from animals between Days 8 and 10 of pregnancy contain some chorioallantoic cells, but antimesometrial decidua, choriovitelline tissues, fetus, and myometrium were removed. Additionally, the DB begins to regress on Day 14 and becomes too thin to reliably dissect after Day 17. Other details are as described previously [14]. After dissection, DB were snap-frozen in liquid nitrogen and stored at -70°C to -80°C until homogenized. Histologic preparations of DB dissected from rats after Day 10 have shown them to be free of trophoblastic cells. Tissues from a single litter were pooled together; the number of individual DB comprising each pool ranged from 3 to 30 (depending on stage of development and treatment) to yield 0.1–1.0 mg DNA/ml of homogenate.
Tissues were homogenized in a hand-driven glass homogenizer according to the procedures of Cao and colleagues [27] with aprotinin (2 µg/ml) and leupeptin (0.5 µg/ml) added just before use. A 200-µl aliquot of each homogenate was taken for DNA determinations [28]. The homogenate was centrifuged at 700 x g for 10 min, and the supernatant was divided into two equal aliquots. Both were centrifuged at 38 000 x g for 15 min. The resulting pellet from one aliquot was dissolved in 1 ml Laemmli buffer [29] and stored at -20°C until applied to a gel for determination of total EGF-R. The second pellet was resuspended in 1 ml of TEN buffer (50 mM Tris-HCl, 1 mM EDTA, and 0.1% Nonidet P-40 containing 150 mM NaCl, gelatin 0.25%, and sodium azide 0.02%) and incubated with rat IgG (1 µg/ml) and protein-A agarose (10 µl/ml) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h and centrifuged at 1500 rpm. The supernatant was further incubated with antibody to phosphotyrosine residues (0.4 mg/ml; Santa Cruz Biotechnology, Inc.) for 1 h and then with protein-A agarose (10 µl/ml) for 4 h. The pellets were then resuspended in 1 ml Laemmli buffer and stored at -20°C after centrifugation at 600 x g. This fraction was used to determine the relative amount of the tyrosine-phosphorylated form of the EGF-R.
Immunoblot Analysis
A tissue equivalent of 40 µg DNA was applied to SDS polyacrylamide gels and electrophoresed. After electrophoresis, the gel was electroblotted to a nitrocellulose hybridization transfer membrane (Hybond-ECL; Amersham Life Science, Inc., Buckinghamshire, UK). The blots were incubated for 18 h at 4°C in the presence of rabbit (anti-human EGF-R) polyclonal IgG (Santa Cruz) at 2.5 µg/ml. Blots were then washed, incubated with second antibody (1 µl/5 ml), and exposed to detection substrates as described previously [15, 26]. The membranes were exposed to x-ray film for 3 min to 1 h, and the autoradiographs were scanned and quantified by densitometry using Intelligent Quantifier software (Bio Image Systems Corp., Ann Arbor, MI).
Molecular sizes of immunoreactive bands were determined by the comigration of biotinylated SDS-PAGE molecular weight standards (Bio-Rad Laboratories, Hercules, CA) applied to a lane in each gel. Prestained SDS-PAGE standards were also applied to gels to assess transfer efficiency of samples. The relative abundance of EGF-R protein was determined by comparing immunostained bands from experimental samples to the corresponding immunostained bands from a pool of DB (Day 10) similarly prepared and applied to each gel. We have found that this pooled reference preparation is the most reliable way to correct for variations in gel loading, blotting efficiency, antibody efficacy, etc. [14, 26].
Expression of Data and Statistical Analysis
Each value is derived from a pool of DB tissues, usually from a single litter. However, to attain the required number of DB for analysis, occasionally it was necessary to combine DB from more than one litter (e.g., as many as 30 DB were required per pool in early pregnancy and for some treatments, whereas 3 DB per pool were sufficient for others). Observations are reported as means ± SEM (n = 4–6 tissue pools). Statistical analysis was made by one-way ANOVA. When ANOVA indicated significant treatment effects, the Student-Neuman-Keuls multirange test was employed to compare only those pairs of treatment means within a given dependent variable.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Immunohistochemistry Intensity of immunoreactive staining for EGF-R in mesometrial stromal cells varied with extent of decidualization and stage of pregnancy (Fig. 1). On Day 8, mature decidualized cells were restricted to the region surrounding the invasive trophectoderm. These cells exhibited relatively weak staining (Fig. 1A, upper right corner), whereas many of the fibroblast-like stromal cells near the myometrium, not yet decidualized, exhibited strong nuclear and cytoplasmic staining (Fig. 1A, lower left corner). Almost all stromal cells exhibited intense cytoplasmic and nuclear staining by Day 10 (Fig. 1B).
|
By Day 12, stromal cells exemplified the mature decidualized phenotype (Fig. 1C) relative to Day 10 by exhibiting a large increase in cell size, rounding of cell nuclei, and a substantial decrease in extracellular space (reviewed in [30]). Marked zonation of EGF-R expression within the DB was also noted, i.e., strong staining of cytoplasm and cell membranes in the region bordering trophoblast giant cells of the junctional zone (JZ; Fig. 1C). Staining became more diffuse and progressively less intense in cells lying more mesometrially; in fact, lack of cytoplasmic staining produced a clear "halo" around nuclei in many of the more distal cells. Similar zonation was also noted on Day 14 (Fig. 1D), but the stromal cells consistently exhibited a lower level of immunostaining throughout the DB as compared to Day 12. On Days 17 and 21, significant staining of stromal cell membranes and cytoplasm was essentially lost; a few stromal cells were faintly positive for EGF-R, but most were negative (Fig. 1, E and F). A few cells of the metrial gland were weakly positive for EGF-R on Day 17, but this staining disappeared by Day 21 (Fig. 1, E and F, respectively). By Days 17 and 21, the DB had regressed sufficiently for the entire DB to be viewed in cross section (Fig. 1, E and F).
Immunoblots The DB proliferates rapidly in the first half of pregnancy, reaching maximal size at Day 12. It then begins to regress at Day 14, becoming reduced to a relatively thin layer of stromal cells by the end of pregnancy [16]. We hypothesized that the tyrosine-phosphorylated form of EGF-R, the bioactive form [31, 32], would be most abundant during stromal cell proliferation and decline thereafter. Figure 2, A and B, are representative Western blots showing changes in total (tyrosine-phosphorylated and tyrosine-unphosphorylated EGF-R) and tyrosine-phosphorylated (pEGF-R) receptor proteins with the progression of pregnancy. EGF-R and pEGF-R were expressed as doublets of approximately 170 kDa and 172–5 kDa in early pregnancy (as late as Day 12), whereas the more mobile form predominated thereafter. Summary analysis (Fig. 3) showed that total EGF-R was abundantly expressed from Days 8 to 14, roughly reflecting the intensity of in situ immunostaining (Fig. 1). However, by Day 17, EGF-R protein had declined to only 11% of the Day 12 level (p < 0.05). The pEGF-R form gradually increased from Day 8, reaching a peak on Days 10 and 12, and falling to about 20% of the Day 10 level on Days 14 and 17 (p < 0.05).
|
|
P4 and Estrogen Regulation of EGF-R Expression
Immunohistochemistry We next examined the roles of P4 and E2 in regulation of EGF-R after ovariectomy on Day 8 or 9 of pregnancy. Rats were given hormone replacement therapy immediately after ovariectomy for 48 h (P4 or P4 + E2 treatment groups) or for 24 h (E2 only treatment group) before being killed on Day 10. Figure 4 shows representative photomicrographs of the effects of P4 and E2. Treatment with E2 alone resulted in loss of histological integrity and an absence of detectable EGF-R expression (Fig. 4A). The darkly stained cells are primarily invasive granular and agranular leukocytes. On the other hand, P4 was able to maintain normal histological organization of stromal cells and strong expression of cytoplasmic and nuclear reaction product (Fig. 4B) similar to that exhibited in Day 10 untreated control animals (Fig. 1B). However, treatment with P4 + E2 resulted in some strongly positive staining of stromal cells for EGF-R, but many were either weakly stained or negative for EGF-R (Fig. 4C).
|
Immunoblots Pellets of the antiprogestin RU-486 were implanted in the next experiments as described in Materials and Methods to ascertain more directly the role of P4 in maintaining EGF-R expression without the stress of surgery or the removal of estrogens. Representative immunoblots of total EGF-R protein (Fig. 5A) and pEGF-R (Fig. 5B) show the effects of RU-486 treatment. Day 9 and Day 10 pregnant animals served as controls: Day 9 represents the level of receptor expression in DB at the onset of RU-486 treatment, and the Day 10 group shows the expected receptor protein expression in absence of 24 h of RU-486 exposure. Figure 6 summarizes the results of Western analysis. RU-486-induced down-regulation of total EGF-R was evident within 6 h of RU-486 exposure, and by 12 h pEGF-R was also reduced (p < 0.05); 24-h exposure reduced total EGF-R and pEGF-R to 13% and 9% of the Day 10 control level, respectively.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although maximum growth and development of DB has been attained by Day 12 [34], this period represents a transitional stage in the life cycle of mesometrial decidual cells, bridging the period of active decidualization and proliferation (Day 10) with that of DB regression (Day 14). Protein synthesis and stromal cell proliferation are substantially decreased on Day 12 as compared to Day 10, but Day 12 levels are higher than those found on Day 14 [16, 34]. The activity of the EGF/EGF-R system also appeared to be in a state of transition on Day 12 in that 21% of total EGF-R was in the bioactive form (i.e., pEGF-R), whereas on Day 10, 68% was bioactive as compared with only 8% on Day 14. EGF-R immunostaining was weak and diffuse throughout the cytoplasm on Day 12 and strongly expressed only on cell membranes of stromal cells in the region of the placental junctional zone (Fig. 1C). Regression on Day 14 was marked by a 75% decline in pEGF-R (Fig. 3), and immunostaining was restricted to stromal cell surface membranes immediately underlying the junctional zone (Fig. 1D). Extensive thinning of the DB had occurred by Days 17 and 21, and few cells were positive for EGF-R (Fig. 1, E and F, and Fig. 3).
The cytoplasmic localization of EGF-R noted in this study has been described by other investigators for a variety of cell types and probably relates to activation of EGF-R by ligand binding, which in turn leads to internalization and accumulation in the cytoplasm of cells [3, 33, 35–37]. Functional EGF-R have also been specifically localized to the nucleus, where they may exert additional mitogenic actions [27, 38, 39]. Our observation of immunopositive nuclei, particularly, on Days 8 and 10 of pregnancy support this notion (Fig. 1, A and B, and Fig. 4B). By mid-pregnancy (Day 14) immunostaining was restricted to the cell membranes of stromal cells, suggestive of a relative lack of ligand activation [40] and consistent with the low-to-undetectable level of pEGF-R expression on Days 14 and 17. Thus, high levels of pEGF-R expression corresponded to stromal cell cycle progression, whereas declining expression of this form was associated with DB regression and withdrawal of P4 action (Figs. 3 and 5).
EGF-R expression was down-regulated by antiprogestin (Fig. 6) but could be maintained by P4 after ovariectomy (Fig. 4B). However, P4 is apparently unable to maintain EGF-R expression during DB regression (Days 14 and 17) because EGF-R expression fell markedly from Day 12 levels (Fig. 3) in spite of the fact that serum P4 is still elevated [34, 41]. We have observed similar changes in the ability of P4 action to maintain a variety of stromal cell functions in late pregnancy, which we attribute to the specific down-regulation of the transactivationally important PR-A and PR-B isoforms of the PR [42, 43] that occurs on Day 14 [26]. Expression of these isoforms is also rapidly lost after ovariectomy or treatment with RU-486 on Day 10 [15]. However, PR-C, a transactivationally inactive isoform [44], continues to be abundantly expressed throughout pregnancy and after ovariectomy and RU-486 treatment [15, 26]. We hypothesize that these changes in PR expression may be ultimately responsible for triggering DB regression by withdrawing trophic signals and allowing activation of latent cell death signaling pathways in spite of high levels of circulating P4 [45, 46]. The present findings suggest that EGF-R is a P4-dependent protein associated with decidualization and proliferation of the DB in early pregnancy and that loss of EGF-R expression after Day 12 may be due to altered P4 action.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: T.F. Ogle, 1120 15th St., Medical College of Georgia, Augusta, GA 30912-3000. FAX: 706 721 7299; togle{at}mail.mcg.edu
3 Current address: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912.
Accepted: March 2, 1999.
Received: December 10, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  H.-S. Byun, G.-S. Lee, B.-M. Lee, S.-H. Hyun, K.-C. Choi, and E.-B. Jeung Implantation-Related Expression of Epidermal Growth Factor Family Molecules and Their Regulation by Progesterone in the Pregnant Rat Reproductive Sciences, September 1, 2008; 15(7): 678 - 689. [Abstract] [PDF]
|
|
|
|
 |
|
 J. H. C. Kengni, I. St-Louis, S. Parent, V. Leblanc, C. Shooner, and E. Asselin Regulation of prostaglandin D synthase and prostacyclin synthase in the endometrium of cyclic, pregnant, and pseudopregnant rats and their regulation by sex steroids J. Endocrinol., November 1, 2007; 195(2): 301 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Shao, B. Weijdegard, K. Ljungstrom, A. Friberg, C. Zhu, X. Wang, Y. Zhu, J. Fernandez-Rodriguez, E. Egecioglu, E. Rung, et al. Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E59 - E72. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-Y. Nie, Y. Li, H. Minoura, J. K. Findlay, and L. A. Salamonsen Specific and Transient Up-Regulation of Proprotein Convertase 6 at the Site of Embryo Implantation and Identification of a Unique Transcript in Mouse Uterus During Early Pregnancy Biol Reprod, February 1, 2003; 68(2): 439 - 447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Liu and T. F. Ogle Signal Transducer and Activator of Transcription 3 Is Expressed in the Decidualized Mesometrium of Pregnancy and Associates with the Progesterone Receptor Through Protein-Protein Interactions Biol Reprod, July 1, 2002; 67(1): 114 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Varayoud, J. G. Ramos, P. P. Joazeiro, G. S. Montes, M. M. Munoz de Toro, and E. H. Luque Characterization of Fibroblastic Cell Plasticity in the Lamina Propria of the Rat Uterine Cervix at Term Biol Reprod, August 1, 2001; 65(2): 375 - 383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Dai, B. C. Moulton, and T. F. Ogle Regression of the Decidualized Mesometrium and Decidual Cell Apoptosis Are Associated with a Shift in Expression of Bcl2 Family Members Biol Reprod, July 1, 2000; 63(1): 188 - 195. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. R. Jones, B. F. Kimler, W. M. Justice, and V. Rider Transit of Normal Rat Uterine Stromal Cells through G1 Phase of the Cell Cycle Requires Progesterone-Growth Factor Interactions Endocrinology, February 1, 2000; 141(2): 637 - 648. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
