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Regular article |
a Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois 60612-7313
b Institute for Primate Research, Nairobi, Kenya
c Research Laboratories of Schering AG, Berlin, Germany
ABSTRACT
Signals from the developing mammalian blastocyst rescue the corpus luteum (CL) and modulate the uterine environment in preparation for implantation and early pregnancy. Our previous studies demonstrated that both short- and long-term administration of chorionic gonadotropin (CG) markedly alters the morphology and the biochemical activity of the receptive endometrium. Because the effects of CG were superimposed on a progesterone-primed endometrium, this study was undertaken to determine if the inhibition of progesterone action by progesterone receptor antagonists (PRa) in intact and ovariectomized baboons would alter the action of CG on the endometrium at the time of uterine receptivity. In the short-term hCG-treated baboons, the PRa reduced the epithelial plaque reaction, completely inhibited
-smooth muscle actin (SMA) expression in stromal fibroblasts, and induced the reappearance of the progesterone (PR) and estrogen (ER) receptors in epithelial cells. However, this treatment protocol had no effect on the expression of glycodelin in the glandular epithelium. In contrast, glycodelin expression in addition to SMA was suppressed in the ovariectomized animals. In the long-term hCG-treated baboons, the PRa had a similar effect on both SMA, PR, and ER. In addition, this treatment also resulted in an inhibition of glycodelin expression in the glandular epithelium. These results indicate that blocking the action of progesterone on the endometrium even for a short period of time has a profound effect on the hCG-induced response in stromal fibroblasts. In contrast, for the diminution of glandular epithelial function in the presence of an ovary requires prolonged inhibition of progesterone action, suggesting a potential paracrine effect on the endometrium from the CL in response to hCG.
estradiol, estradiol receptor, hCG, implantation/early development, progesterone, uterus
INTRODUCTION
The establishment of pregnancy in pigs and ruminants requires an interaction between the embryo, uterus, and corpus luteum (CL) [1]. Signals from the trophoblast inhibit the release of prostaglandin F2, thereby extending the functional lifespan of the CL. In contrast, the primate embryo uses chorionic gonadotropin (CG) to rescue the CL and modulate the uterine environment [2]. In the endometrium, the presence of LH/CG receptors and associated G proteins have been documented, but the signal transduction pathways by which its action is modulated have not been fully elucidated [3, 4]. The physiological effects of hCG on epithelial cell responses in the endometrium are observed in the absence of detectable PR [5], while the effects on stromal cells are evident in the presence of PR [5, 6]. Therefore, this study was undertaken to determine if the endometrial response to hCG is altered if the action of progesterone was antagonized by PRa.
Human CG, in addition to other factors, is required in the morphological transformation of the endometrium that is necessary for successful implantation. Successful implantation requires a synchrony between an appropriately developed embryo and a receptive endometrium [7, 8]. Uterine receptivity has been defined as the limited period of time when the uterine luminal epithelium is favorable to blastocyst implantation [9]. Estrogen and progesterone play a critical role in establishing this receptive phase. Luteal phase administration of mifepristone (RU486) to women and monkeys induces disynchronization of the endometrium and represses glandular secretory activity and angiogenesis [10–14]. These changes in uterine function in response to a low dose of RU486 are independent of changes in normal cyclicity and ovarian steroid production [10–14]. Similar changes have been described in rhesus monkeys following treatment with ZK 137.316 [15].
Because of their varied actions on the female reproductive tract, especially during the period of uterine receptivity, antiprogestins have a potential for contraception as well as widespread gynecological and obstetrical uses in the clinical setting. These include induction of menses, once-a-month contraception, and emergency contraception. Because of their endometrial and myometrial antiproliferative effects as well as their antiglucocorticoid activity, antiprogestins have been proposed for the treatment of endometriosis, fibroids, and steroid-dependent tumors [16]. The contraceptive approaches with antiprogestins include emergency contraception with high doses for a short period of time and endometrial contraception using continuous low-dose treatment [17]. High doses have also been found to block ovulation, but low doses only impair endometrial development without affecting ovulation [18].
Based on these studies, we suggest that treatment with PRa when superimposed on an hCG-primed uterus, markedly affects the three major cell types present in the uterine endometrium, i.e., luminal and glandular epithelium and stromal fibroblasts, and may subsequently inhibit the generation of a receptive endometrium.
MATERIALS AND METHODS
Animals
All experimental procedures were approved by the Animal Care Committees of the University of Illinois at Chicago and the Institute for Primate Research in Nairobi. Cycling baboons were treated with bioactive recombinant hCG (Serono Laboratories, Norvell, MA; n = 4/group) or hCG and PRa (n = 4/group; Fig. 1A). Ovariectomized baboons (n = 2/group) were treated with steroid implants to mimic the menstrual cycle [19] and then treated with hCG or hCG and PRa (Fig. 1B). The short- and long-term hCG treatment protocols to simulate the hormonal environment of early pregnancy have been previously described [2, 5]. Briefly, in the short-term protocol, hCG was infused between Days 6 and 10 postovulation (PO) into the oviduct via a cannula attached to an Alzet minipump (group I). In the long-term protocol, hCG was administered by i.m. injections twice a day in escalating doses between Days 6 and 17 PO (group III). The PRa (ZK 137.316; Schering AG, Berlin, Germany) was dissolved in ethanol and sesame oil (1:10 v:v) and administered i.m. at a dose of 1 mg/kg body weight/day. In both the short- and long-term protocols, PRa or vehicle treatment was begun on the evening of Day 5 PO and discontinued on either Day 9 (group II) or 17 PO (group IV). Tissue was obtained at either Day 10 or 18 PO (Fig. 1A).
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To determine if the PRa antagonizes the direct effects of hCG on the endometrium, an ovariectomized, simulated pregnant model was utilized. Ovariectomized baboons were treated sequentially with silastic implants containing estradiol and progesterone to mimic the hormonal changes during the menstrual cycle [19] (Fig. 1B). Six days following the insertion of the first progesterone implant, hCG treatment was initiated using the Alzet minipump and continued for 4 days (group V). The PRa treatment was initiated on Day 5 following the insertion of the first progesterone implant and continued on a daily basis up to the evening preceding tissue collection (group VI). Endometrial tissue was harvested at the end of the 10-day treatment period. This hormonal regimen results in serum hormone levels that are comparable to those measured in intact animals [2, 19].
Estradiol and progesterone levels in peripheral serum were determined using a validated RIA [19].
Histology and Immunocytochemistry
Uterine tissues were immersion-fixed in either Bouins solution or 10% buffered formalin for 24 h at room temperature, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin [20]. For histological analyses the Bouins-fixed tissue sections (6 µm) were stained with Gomori's trichrome stain [2]. For immunocytochemistry, a monoclonal antibody to cytokeratins 8 and 18 (CAM 5.2; Becton Dickinson, San Jose, CA) at a 1:1 dilution of the manufacturer's prediluted antibody, a monoclonal antibody to -smooth muscle actin (SMA) [6] (Dako, Carpinteria, CA) at a 1:1000 dilution and a polyclonal antibody to human glycodelin [21] at 1:750 dilution, were utilized. Prior to staining with the cytokeratin antibody, the sections were pretreated with 0.5% saponin for 30 min at room temperature. The 10% formalin-fixed tissues were used in analysis of progesterone and estrogen receptor (ER) localization. The tissues were subjected to antigen retrieval in 0.5% saponin for 5 min at 37°C followed by boiling for 10 min in 0.01 M citrate buffer (pH 6) prior to steroid receptor localization. A mouse monoclonal antibody for ER alpha (ER1D5; Biogenex, San Ramon, CA) was used at a dilution of 1:50. A rat monoclonal antibody specific for progesterone receptor (PR: JZB39) was used at a dilution of 0.3 µg/µl [22]. The immunoreactive products following incubation with all primary antibodies were visualized using an ABC Vecstain kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine [6]. Controls consisted of preimmune serum at the same dilution or the omission of the primary antibody.
The intensity of the immunoreactive product was analyzed using an Image Pro-Plus 3.0 imaging processing system from Media Cybernetics (Silver Spring, MD). Microscopic images were viewed on a Nikon Eclipse E400 microscope, digitized, and stored. Three areas of equal size within the endometrium were randomly chosen and the optical density was computed on the black and white digitized image. A negative control in which primary antibody was omitted was utilized for background for each image. The optical density values of the control negative sample were subtracted from the optical density of each image. Positive histochemical staining was defined by an optical density value in excess of the mean plus one standard deviation of the optical density value of the negative control sample. The differences between the two treatment groups in the short and long hCG treatment protocol were analyzed for statistical significance using the Students paired t-test.
Explant Culture and Immunoblotting
Endometrial tissues (75 or 150 mg) from each of the six groups were cultured under serum-free conditions [23]. The explant culture media were harvested following 24 h of culture [23]. Secretory proteins in explant culture media (200 µl) were resolved by one-dimensional SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated overnight with glycodelin antibody (1:5000 dilution), and the immunoreactive product was visualized using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) [21].
Reverse Transcriptase-Polymerase Chain Reaction
The RNA from all experimental groups was reverse transcribed and subjected to polymerase chain reaction (PCR). As a control, RNA preparations that were not reverse transcribed were also subjected to PCR to ensure that no genomic contamination was being amplified. Specificity of the PCR products was further confirmed by Southern hybridization with the human glycodelin cDNA [24]. The primers were synthesized based on previously described sequences [25]. The sense primer sequence was 5'-CCC CCA GAC CAA GCA GGA CCT GGA GCT CCC-3' and the antisense primer was 5'-ATC GTC CTG CAC CAG GAC TCT GGC CAG GTA-3'. The H 3.3 histone PCR product was coamplified with glycodelin and served as an internal standard [26]. The reverse transcriptase (RT)-PCR was performed as previously described for baboon endometrial tissues [2, 26]. The PCR products were quantitated by densitometrically scanning the autoradiographs using a Molecular Dynamics Phosphoimager (Sunnyvale, CA). The level of expression of glycodelin amplicons was normalized against the internal standard.
RESULTS
Steroid Receptors
There were no significant differences in the peripheral levels of estradiol and progesterone between the control and progesterone receptor antagonist (PRa)-treated groups at either Day 10 or 18 PO (Table 1). In contrast, the localization of both ER and PR were markedly affected in the endometrium (Fig. 2). In the hCG-treated controls, ER localization was only evident in the smooth muscle cells (data not shown), while PR was localized primarily in the stromal fibroblasts (Fig. 2, A, C, and E). These data are in agreement with our previous findings [5]. Treatment with PRa however, induced the reappearance of both ER and PR in the glandular and luminal epithelium (Fig. 2, B, D, and F), and ER also was evident in the stromal fibroblasts (inset Fig. 2F). These changes are consistent with the steroid receptor distribution pattern seen during the proliferative and early luteal phases of the menstrual cycle [22].
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Morphological Changes and Protein Expression
The short-term treatment with hCG and PRa had a minimal effect on epithelial function in intact baboons. The epithelial plaque response (Fig. 3, A and B) and glycodelin localization were relatively similar (Fig. 3, E and F). In contrast, the expression of SMA in the stromal fibroblasts was completely inhibited by the PRa treatment (Fig. 3, C and D). In the ovariectomized baboons, hCG does not induce the plaque response but increases the cell height of the luminal epithelium [2]. This finding remains consistent following treatment with PRa (Fig. 4, A and B). In contrast, PRa treatment inhibited glycodelin synthesis in the glandular epithelial cells of ovariectomized baboons (Fig. 4, E and F), together with the suppression of SMA in stromal cells (Fig. 4, C and D). Treatment of long-term simulated cycling baboons with PRa also dramatically downregulated glycodelin synthesis in the glandular epithelial cells compared to the Day 10 PO animals (Fig. 5). Expression of SMA continued to be inhibited in stromal fibroblasts in the PRa-treated baboons (data not shown).
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Western Blots and RT-PCR
The secretory activity of the endometrial tissues and mRNA expression were consistent with the immunocytochemical results. In response to hCG, glycodelin expression in the baboon endometrium is transcriptionally and post-translationally modified [2]. Consistent with our previous studies, the two alternatively spliced glycodelin transcripts and post-translationally modified glycodelin protein were evident by RT-PCR (Fig. 6, A and B) and Western blot analysis (Fig. 7), respectively. Short-term treatment of intact baboons with PRa had no effect on glycodelin transcription or post-translational modification (Fig. 6, lanes 5 and 6 and Fig. 7, lanes 2 and 3). In contrast, in ovariectomized baboons treated with the short-term protocol, glycodelin inhibition was similar to that observed following the long-term treatment protocol (Fig. 6, lanes 11, 12 and 16 and Fig. 7, lanes 6 and 7 and 10 and 11).
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DISCUSSION
Progesterone is essential for the differentiation of the endometrium into a secretory phenotype that is a prerequisite for the establishment of pregnancy. In addition, our recent studies have shown that the infusion of hCG, in a manner that mimics the period of blastocyst transit and initial attachment markedly alters the morphology and function of the receptive endometrium [2]. Because these hCG-induced changes were superimposed on progesterone-primed endometrium, this study focused on determining the importance of the synergism between progesterone and hCG in the establishment of a receptive endometrium. The fact that both progesterone and hCG impart direct effects on modulating the receptive endometrium was established by using an ovariectomized, steroid-treated baboon model [2].
Administration of mifepristone (RU486) during the early luteal phase in women has been reported to be an effective method of contraception. When administered at low doses, RU486 does not alter ovarian function and has minimal effects on endometrial morphology. However, endometrial secretory activity is markedly reduced at the expected times of implantation [12, 27]. In rhesus macaques, administration of RU486 on cycle days 16–18 inhibited implantation; however, the underlying mechanism of the contraceptive effect in mated animals was not determined [14]. In addition, chronic low-dose treatment of rhesus macaques with ZK 137.316 maintained ovarian and endometrial cyclicity but inhibited pregnancy [28]. Our studies clearly demonstrate that the contraceptive effects of PRa (ZK 137.316) are a result of specific functional changes in both stromal fibroblasts and the glandular epithelium. Both short- and long-term treatment with ZK 137.316 resulted in the induction of both ER and PR in the glandular and luminal epithelium and increased staining in the stromal cells of the hCG-treated baboons. These findings are similar to that observed in rhesus macaques following RU486 and ZK 137.316 treatment in both cycling and ovariectomized, steroid-treated animals [10, 15]. These changes in steroid receptor localization within the endometrium were independent of any changes in steroid synthesis by the CL, confirming the previous studies with low-dose PRa in women and nonhuman primates [15, 28, 29].
Stromal fibroblasts directly below the luminal epithelium undergo functional differentiation in response to hCG and are characterized by the induction of SMA [2, 6]. This is a direct action of hCG because the induction pattern of SMA is similar in ovariectomized baboons treated with steroids and hCG [2]. Treatment with the PRa inhibited SMA expression in all treatment groups. We have previously suggested that the induction of SMA by hCG in stromal fibroblasts may be essential for cell proliferation and differentiation during the initial stages of pregnancy [2]. In addition, other studies have also suggested that ZK 137.316 and RU486 inhibit mitosis in the uterine endometrium [15, 30]. It is conceivable that the inhibition of SMA induction by PRa observed in our studies is a reflection of the absence of stromal cell mitosis, which is a progesterone-mediated event in the primate [31]. Thus, the inhibition of stromal cell proliferation and differentiation may be one mechanism by which implantation is blocked during early gestation.
The majority of studies on PRa effects on the endometrium have focused on the morphology and glandular secretory activity. Continuous low-dose treatment with RU486 results in an asynchronous expression of glycodelin [11], depressed lectin binding [12], and a decrease in the expression of leukemia inhibitory factor [27]. Our studies in normally cycling baboons also suggest that treatment with a low dose of the PRa, onapristone (ZK 98.299), during the luteal phase prevents the downregulation of the polymorphic mucin (Muc-1) and the lectin (DBA) at the time of uterine receptivity [32, 33]. In addition, changes in the synthesis of transforming growth factor ß, vascular endothelial growth factor, and leukemia inhibitory factor have also been reported at Day 6 of gestation in rhesus macaques following a single dose of RU486 [34]. In contrast, short-term treatment with the PRa during the luteal phase of intact hCG-treated baboons did not markedly affect the plaque response in the luminal epithelium or glycodelin expression in the glandular epithelium. The plaque response may play a central role in permitting trophoblast migration by decreasing the tight junctions between the luteal membranes of the luminal epithelium [2]. Glycodelin has been suggested to have both immunossuppresive and cellular differentiating properties [35]. Treatment with PRa for 10 days together with hCG markedly inhibited glycodelin expression. However, in the absence of an ovary, the plaque reaction was absent and glycodelin synthesis was significantly inhibited following the short-term protocol. These results suggest that an ovarian factor may need to be inhibited or absent to induce changes in the epithelial cells. In response to hCG stimulation, the primate CL secretes significantly higher concentrations of relaxin [36] and relaxin is also thought to regulate glycodelin synthesis by the glandular epithelium [37, 38]. Our preliminary studies suggest that 10 days of PRa treatment can inhibit relaxin in the baboon CL, while 3ß-hydroxysteroid dehydrogenase and PR expression are not affected (unpublished observations). Thus, we hypothesize that in intact baboons, inhibition of glycodelin synthesis requires a longer exposure to PRa. The resulting inhibition of relaxin synthesis by the CL results in the inhibition of the synergistic action of hCG and relaxin from CL on the endometrium. This hypothesis is substantiated by the fact that in the absence of an ovary, glycodelin synthesis is inhibited following short-term exposure to the PRa.
In summary, our data support the concept that low-dose PRa treatment has a direct effect on endometrial function during the receptive window. The most direct response is the inhibition of the initial phase of stromal cell differentiation and the reappearance of ER and PR expression in both epithelial and stromal cells. However, inhibition of epithelial function requires a longer treatment duration possibly due to the synergism between relaxin from the CL and the action of hCG both on the glandular epithelium and CL. Thus, the inhibition of implantation by PRa may be a result of altered stromal and epithelial cell function in response to the embryonic stimulus.
ACKNOWLEDGMENTS
We thank Serono Laboratories for their generous donations of recombinant hCG and Schering AG for the ZK 137.316 used in these studies. We are grateful to Dr. Stephen Bell for the glycodelin antibody and Dr. Geoffrey Greene for the PR antibody.
FOOTNOTES
First decision: 22 March 2000.
1 This work was supported by the National Cooperative Program for Markers of Uterine Receptivity for Blastocyst Implantation funded by the National Institutes of Health Cooperative Agreement HD 29964, TW00878, and the Ernst Schering Research Foundation (A.T.F.). 
2 Correspondence: Asgi T. Fazleabas, The University of Illinois at Chicago, Department of Obstetrics and Gynecology, 820 South Wood Street (M/C 808), Chicago, IL 60612-7313. FAX: 312 996 4238; asgi{at}uic.edu
3 Current address: Jenapharm GmbH&Co.KG, Otto-Schott-Strasse 115, 07745 Jena, Germany.
Accepted: April 24, 2000.
Received: February 17, 2000.
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