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Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, SwedenDepartment of Neurobiology and Integrative Medicine, Shanghai Medical College of Fudan University, Shanghai, China
Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, SwedenDepartment of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, China
Reprints: Ruijin Shao, MD, PhD, Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, Medicinareg 11, PO Box 434, 413 90 Gothenburg, Sweden
The purpose of this study was to investigate clomiphene citrate (CC)–induced modulation of uterine cell function in vivo.
Prepubertal female Sprague-Dawley rats were treated intraperitoneally with CC for 6 or 24 hours or with a combination of CC and/or 17-β-estradiol (E2) for 4 days.
Chronic CC treatment induced apoptosis in a fraction of uterine stromal cells by activating the caspase-3-mediated apoptotic pathway. The damage was prevented by successive E2 treatment; however, pretreatment or concomitant treatment with E2 did not protect against CC-induced uterine apoptosis. CC decreased the protein expression of estrogen receptor α and increased its phosphorylation but did not affect estrogen receptor β expression or phosphorylation. Furthermore, changes in Hoxa11, p27, and progesterone receptor protein levels and localization were associated with CC treatment.
We provide novel mechanistic insights into cellular and molecular events by which CC regulates uterine stromal cell function and hence the implantation process and pregnancy outcome.
These observations, combined with clinical studies, have given rise to the hypothesis that CC may contribute to implantation-related complications through an unidentified regulatory process in the uterus. The goals of this study were (1) to determine whether CC treatment induces uterine cell apoptosis and (2) to investigate the molecular regulation of ERs and other potential implantation and cell cycle regulators
All experimental procedures and protocols were approved by the ethics committee at Gothenburg University. Prepubertal female Sprague-Dawley rats (20 days old) were obtained from Taconic M&B (Copenhagen, Denmark) and maintained in cages that contained wood chips under defined conditions (temperature, 21 ± 2°C; relative humidity, 45–55%; and 12-hour light/dark illumination schedule). Animals were acclimated to the animal facilities for 5 days before the initiation of the experiments. All animals had free access to tap water and were fed ad libitum with standard laboratory diet.
Three experiments were carried out (Figure 1) in 25-day-old rats. The rats were randomized to receive intraperitoneal injections of CC (Sigma-Aldrich, St. Louis, MO), E2 (Sigma-Aldrich), or both. Controls were treated with vehicle only. Bodyweight was recorded throughout the experiment. Rats were killed under light anesthesia with sodium pentobarbital (0.5 mL/kg bodyweight). Aliquots of serum were prepared from trunk blood after heart puncture and stored at –80°C until analysis. The uteri were dissected grossly with the removal of contaminating tissues (eg, adipose tissues), weighed, and immediately frozen in liquid nitrogen or fixed in formalin.
Rats received CC (1 or 10 mg/kg intraperitoneally) or an equivalent volume of vehicle (0.9% NaCl). Uteri were collected 6 and 24 hours (acute effect) after injection or on day 4 (chronic effect) after daily intraperitoneal injections (n = 8/group). For the selected doses and treatment schedule, CC is effective in rat fallopian tube and uterus in vivo.
Rats received injections of CC (10 mg/kg intraperitoneally) or vehicle once daily for 4 consecutive days. Twenty-four hours after the last injection, E2 (0.3 mg/kg in 100 μL sesame oil) or vehicle was injected for 4 consecutive days.
Rats were injected with E2 or vehicle once daily for 4 consecutive days, as in experiment 2. Twenty-four hours after the last injection, CC (10 mg/kg), CC (10 mg/kg) and E2 (0.3 mg/kg), or vehicle was injected intraperitoneally for 4 consecutive days. In experiments 2 and 3, the rats were killed, and uteri were collected 24 hours after the final injection (n = 5/group).
The primary antibodies that were used for Western blot (WB) analysis and immunofluorescence were obtained from commercial sources (anticleaved caspase 3 and anticleaved caspase 9 [Cell Signaling Technology, Beverly, MA]; anti-ERá, antiphospho-ERâ [Ser87], anti-Hoxa10, anti-Hoxa11, anti-p27, and anti-p53 [Santa Cruz Biotechnology, Santa Cruz, CA]; antiphospho-ERá [Ser118] and anti-ERâ [Upstate Biotechnology, Lake Placid, NY]; anti-pan-cytokeratin, anti-á-smooth muscle actin and anti-â-actin [Sigma-Aldrich]; and antiprogesterone receptor [PR; Novocastra Laboratories, Newcastle Upon Tyne, UK] antibodies). The secondary antibodies for WB were goat-antimouse immunoglobulin G (Sigma-Aldrich), goat-antirabbit immunoglobulin G (AC31RL; Tropix, Bedford, MA), and donkey antigoat immunoglobulin G (Santa Cruz Biotechnology), which were all conjugated with alkaline phosphatase. The Cy3-conjugated antimouse antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA).
Histologic evaluation and immunofluorescence staining
Uteri were fixed in neutral buffered 10% formalin, decalcified, dehydrated, and embedded in paraffin. Series sections (5 μm) were prepared and stained with hematoxylin/eosin to visualize cell nuclei (Histocenter, Västra Frolunda, Sweden). Uterine sections were subjected to immunofluorescence studies to test the localization of progesterone receptor A isoform, as described previously.
Tissue sections were dewaxed, protease digested, and incubated with terminal transferase mixture according to the instructions of the manufacturer, with the use of an in situ apoptosis detection kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, sections were incubated in a permeabilization solution, which contained 0.1% Triton X-100 and 0.1% sodium citrate, and then were incubated with the terminal deoxynucleotidyl transferase mediated 2′-deoxyuridine, 5′-triphosphate nick-end-labeling (TUNEL) reaction mixture, which included the enzyme solution (terminal deoxynucleotidyl transferase) and label solution (tetramethylrhodamine isothiocyanate-labeled nucleotides), in a humidified chamber for 1 hour at 37°C. After being washed with phosphate-buffered saline solution, the sections were evaluated with a confocal laser microscopy. The enzyme solution was omitted in the negative control. Sections that were treated this way remained unstained.
Protein extraction and WB analysis
Whole-cell extracts from uterine tissues were analyzed by WB, as described previously.
For the measurement of CASP-3/7 activity in whole uterine tissues, frozen tissue was homogenized in lysis buffer (100 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, and protease inhibitors), and the crude homogenate was centrifuged at 12,000g for 30 minutes at 4°C. Cellular caspase activity was determined with the use of the Caspase-Glo-3/7 assay kit (Cell Signaling Technology), as previously described.
Data were analyzed with SPSS software (version 13.0; SPSS, Inc, Chicago, IL). Two-way analysis of variance was used to assess the main effects of treatment and time and to identify interactions between them. If significant interactions between the fixed factors were observed, within-group analyses were performed with a 1-way analysis of variance followed by Bonferroni's multiple comparison test. Values are shown as means ± SEM. Significance was accepted at a probability value of < .05.
Chronic, but not acute, CC treatment induces cell type-specific apoptosis in the uterus
Rats were given 1 acute injection of CC (1 or 10 mg/kg) or vehicle, and the expression of cleaved caspase 9 and caspase 3 (Casp3) was measured in the uterus at 6 and 24 hours. Alternatively, CC was administered daily for 4 days (chronic treatment), and the expression of the same apoptotic molecules was measured in the uterus. WB analysis revealed elevated levels of cleaved caspase 9 and Casp3 in the uteri from rats that had been treated with CC for 4 days but not at 6 and 24 hours (Figure 2, A). Using the caspase-Glo 3/7 assay, we confirmed that elevated Casp3 activity was found only in uteri from rats that had been treated with CC for 4 days (Figure 2, B), which is consistent with the levels of cleaved Casp3 (Figure 2, A). Next, DNA fragmentation was detected in situ with TUNEL staining. TUNEL-positive cells were present specifically in the stromal cell layer in rats that had been treated with CC for 4 days (Figure 3, B) but not in control rats (Figure 3, A). The luminal (Figure 3, A and B) and glandular (data not shown) epithelial cells and the smooth muscle cell layers (Figure 3, A and B) in both control and CC-treated rats showed no staining. Ovarian sections (Figure 3, C and C1) were used as experimental controls.
E2 aids recovery from CC-induced uterine apoptosis but is not protective
To test whether E2 might contribute to recovery from CC-induced uterine apoptosis, rats were treated sequentially with E2 or vehicle for 4 days after chronic treatment with CC. Cleaved Casp3 levels (Figure 4, A) and activity (Figure 4, B) were lower in uteri from the E2-treated rats. Neither pretreatment nor concomitant treatment with E2 significantly affected the ability of chronic CC treatment to decrease cleaved Casp3 expression (Figure 4, C) and activity (Figure 4, D).
Chronic treatment with CC results in changes in uterine morphologic condition
As shown in the Table, although bodyweight was unaffected by acute or chronic treatment with low-dose or high-dose CC, uterine weight increased after 24-hour and 4-day treatments at both doses. In control rats, individual epithelial cells contained large round or oval basal nuclei with very little surrounding cytoplasm (Figure 5, A1). Rats that were treated with CC for 4 days had increases in epithelial height, muscular thickness, and luminal space (Figure 5, C and C1) compared with control rats (Figure 5, A and A1). Essentially, the epithelium changed in character from cuboidal to columnar cells. In CC-treated rat uteri, the nuclei were less dark, and the long axis of the nuclei of the epithelium was parallel to the basement membrane (Figure 5, C1). Although the uterine morphologic condition of E2-treated rats (Figure 5, B and B1) was quite similar to that of CC-treated rats, treatment with E2 had less of an effect on the epithelium. Moreover, clear cells in the epithelium were occasionally present in CC-treated rats. This “bubbly” appearance may indicate the lipid and/or glycogen accumulation. There was less connective tissue between stromal cells in CC-treated rats (Figure 5, C1) than in E2-treated rats (Figure 5, B1). Histologic findings also showed a lack of integrity in the epithelium of CC-treated rats after an additional 4-day vehicle treatment (Figure 5, D and D1).
TABLEEffects of clomiphene citrate (CC) on bodyweight and uterine tissue weights in rats
Chronic treatment with CC activates ER-α but not ER-β in the uterus
WB analysis revealed that chronic treatment with both low-dose and high-dose CC resulted in a significant decrease in ER-α expression (Figure 6). Because the stability and activity of ER subtypes are affected by ligand-dependent ER phosphorylation,
it is important to note that chronic treatment with CC significantly enhanced the phosphorylation of ER-α, but not ER-β (Figure 6).
Chronic treatment with CC regulates Hoxa11, p27, and the progesterone receptor A isoform in the uterus
WB analysis showed that, although the levels of Hoxa11 and p27 were decreased, neither Hoxa10 nor p53 levels were altered by chronic treatment with CC (Figure 6). Chronic treatment with low-dose or high-dose CC caused a mobility shift of pan-cytokeratin compared with controls (Figure 6, A), although the cause of this shift in the apparent molecular weight of cytokeratin remains unknown. There were no changes in the levels of α-smooth muscle actin in uteri after CC treatment (Figure 6, A). Because the PR antibody (NCL-L-PGR-312) recognizes only the progesterone receptor A (PRA) isoform in tissue sections with the immunohistochemical analysis,
only PRA expression was analyzed in successive sections of the uterus. Chronic treatment with CC (Figure 7, B) or E2 (Figure 7, C) increased the number of PRA-positive cells in the stromal cell layer compared with controls (Figure 7, A). Moreover, concomitant CC and E2 treatment for 4 days decreased the number of PRA-positive cells in the stromal cell layer (Figure 7, D) compared with E2 treatment alone (Figure 7, C).
To our knowledge, this is the first study to demonstrate that chronic treatment with CC can induce not only stromal cell apoptosis but also morphologic abnormalities in the uteri of rats in a dose-independent manner. The present study highlights the potential role of the ER-α signaling pathway in the uterus after chronic CC treatment. Furthermore, the high ovulation rates combined with low pregnancy rates in women who were treated with CC may be explained by the aberrant expression of Hoxa11, p27, and PRA because of repression of ER-α expression and consequent disruption of the implantation process.
CC is a racemic mixture of zuclomiphene (38%) and enclomiphene (62%) that provides both estrogenic and antiestrogenic activities in a variety of E2-target tissues.
Here we show that, within the uterus, the stromal cells are the main cell type to be targeted by the apoptotic effects of chronic treatment with CC. In addition, in the rat uterus, CC displays agonistic activity at low concentration, although it functions as an ER antagonist at high concentration in vivo.
These observations suggest that CC acts as an ER agonist and/or antagonist depending on the species, target tissue/cell specificity, dose range, and regimen.
We show that the effects of CC in the uterus are time dependent rather than dose dependent and that the damage can be reversed with E2, which suggests that CC, as an apoptotic inducer, blocks the antiapoptotic effect of E2 in the uterus. The clear lack of protective effects of pretreatment or concomitant with E2 could be because CC exhibits much stronger antiestrogenic activity in the presence of an E2-stimulated rat uterus.
our results suggest that aberrant apoptotic activities of uterine stromal cells after chronic CC treatment may be a mechanism whereby the implantation process is disrupted. In agreement with a previous study, Grunert et al
reported that chronic treatment with CC blocked the action of E2 only in stromal cells and not in epithelial cells. Although in vitro tissue recombinant studies have shown that stromal cells are able to regulate epithelial cell proliferation in response to E2 stimulation,
our finding of massive damage to the epithelial cell layer in rats that are treated with CC when E2 is absent (Figure 5, D) indicates that stromal cells may fail to support and maintain their epithelial structure in response to CC when antagonizing E2 in vivo. Indeed, the addition of E2 after CC treatment in women has been shown to improve cervical mucus that results in increased endometrial thickness and conception rates.
Collectively, our results suggest that negative effects of CC on the transformation of stromal cells (direct) and the disintegration of stromal-epithelial interactions that lead to epithelial cell destruction (indirect) may inhibit implantation in the uterus. Although chronic CC treatment increases endogenous E2 levels in rats,
The molecular mechanism of E2 supplementation in the uterus of CC-treated rats suggests that estrogen status of patients who are appropriate candidates for CC therapy should be taken into consideration to increase efficacy. A better understanding of the CC-induced apoptotic mechanism in the uterus could reveal new therapeutic strategies for the improvement of implantation rates and reduction of the risk of miscarriage in humans who are treated with CC. For example, additional treatment with the antioxidant N-acetylated cysteine (an antiapoptotic agent) significantly improves endometrial function and leads to increases in ovulation and pregnancy rates, compared with CC treatment alone, in women with polycystic ovary syndrome.
the cellular response to CC is dependent on ER-α expression and phosphorylation in the rat uterus in vivo, which indicates that ER-α and ER-β are not functionally equivalent in the response to CC treatment. Both ER subtypes are expressed in rodent uteri and human endometrium,
which suggests that the expression and activation of these genes are essential to the implantation process in vivo. In the mouse uterus, treatment with E2 increases ER-α expression in stromal cells but not in epithelial cells.
which provides evidence for a vital and distinctive role of the 2 ER subtypes in the uterus. Moreover, studies that have used ER-α knockout mice in vivo and uterine tissue recombinants in vitro have revealed that the estrogenic regulation of PR expression requires stromal ER-α but is independent of epithelial ER-α.
Previous studies, however, could not distinguish fully which stromal PR isoform was responsive to E2 stimulation. Although PRA is the functional PR isoform that is responsible for the regulation of uterine stromal differentiation during implantation,
it is interesting to note that CC is able not only to increase PRA expression when E2 is absent but is also able to block E2-induced PRA expression in rat uterine stromal cells, which suggests that CC converts from an estrogenic agonist to an estrogenic antagonist in the presence of E2. Previous studies from our laboratory and others have shown that p27 is a target gene of PR signaling in the uterus.
we propose that the antiestrogenic effects of CC on the regulation of p27 expression seem to be mediated by ER-α and do not require the presence of progesterone directly. There is no change in the expression of p53 after CC treatment in the uterus, which suggests that divergent regulatory pathways between p27 and p53 are triggered by CC treatment. Although both Hoxa10 and Hoxa11 are downstream targets of the action of estrogens in the mouse uterus,
we observed only the down-regulation of Hoxa11 in the CC-treated rat uterus. Because Hoxa11 is expressed only in the stromal cells, our results support the idea that the CC-induced expression of stromal signals may control the epithelial fate. Together, these data highlight the major role of the ER-α signaling pathway in the antiestrogenic effect of CC on the uterine stromal cells. Whether these molecules are independent of 1 another in CC-induced implantation defects will require further investigation.
In terms of the cell-type selection mechanism in the rat uterus in vivo, we have reported direct molecular mechanisms by which the antiestrogenic effects of CC induce stromal cell-specific apoptosis and regulate the expression of molecules that are important for the implantation process through the ER-α signaling pathway in the rat uterus. At least in part, the present study has resulted in a cellular and molecular mechanism-based explanation for the discrepancy between high ovulation rates and low successful pregnancy rates in women who are treated with CC.
Moving forward, it is noteworthy that there is also a need to evaluate such changes in women who undergo CC treatment. The use of endometrial biopsies in CC-treated women who do not become pregnant might help to explore our hypotheses that have been generated from this study.
We thank Prof Håkan Billig for his support with many aspects of this project and Profs Christina Bergh and Mats Brännström (Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Gothenburg, Sweden) for sharing their knowledge of clomiphene citrate.
Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene.
This study was supported by the Swedish Medical Research Council (Grant no. 5859 ), the Svenska Sällskapet för Medicinsk Forskning , Sahlgrenska Academy Research Council , Göteborgs Läkaresällskap, Fred G. and Emma E. Kanolds Stiftelse, Anna Cederbergs Stiftelse, Fredrik and Ingrid Thurings Stiftelse, and the Hjalmar Svenssons Research Foundation .
Cite this article as: Nutu M, Feng Y, Egecioglu E, et al. Stromal cell–specific apoptotic and antiestrogenic mechanisms may explain uterine defects in humans after clomiphene citrate therapy. Am J Obstet Gynecol 2010;203:65.e1-10.