Objective
The purpose of this study was to investigate clomiphene citrate (CC)–induced modulation of uterine cell function in vivo.
Study Design
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.
Results
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.
Conclusion
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.
Clomiphene citrate (CC), a nonsteroid tissue-selective estrogen receptor (ER) modulator, is used commonly to treat various infertilities in women, most notably polycystic ovary syndrome (PCOS) and unexplained infertility. Several clinical and epidemiologic studies have indicated that successful ovulation occurs in 70–99% of women who are treated with CC, whereas ultimate pregnancy rates are only 27–40%. The total CC-treated pregnancy rates are approximately 10 times lower than “natural” rates. Furthermore, spontaneous abortion and miscarriage occur frequently in women who receive CC therapy. Although it has been proposed that the antiestrogenic effects of CC may lead to early pregnancy failure, the precise molecular events underlying CC-induced uterine defects remain incompletely understood.
For Editors’ Commentary, see Table of Contents
The mammalian uterus is a dynamic reproductive organ that undergoes cyclic changes in response to ovarian steroid hormones. The biologic effects of 17-β-estradiol (E2) and CC are mediated primarily by 2 nuclear receptors, ER-α and/or ER-β, which are encoded by 2 different genes and function as transcription factors. Although the distribution and relative levels of ER-α and ER-β expression are tissue specific and diverse, both ER-α and ER-β are expressed in the rodent uterus and human endometrium. It has been reported that inappropriate activation or inhibition of ER subtypes may cause or contribute to a variety of uterine diseases, such as endometriosis and endometrial cancer. The protective effects of E2 in uterine homeostasis are evident both from in vivo and in vitro studies: the presence of E2 inhibits uterine apoptosis in vivo; however, human endometrial cells undergo apoptosis when E2 is withdrawn. Previous studies from our laboratory and others have shown that CC enhances apoptotic processes in the ovaries, fallopian tubes, villi, and decidual tissues. Less is known, however, about the effects of CC on the apoptotic machinery in the uterus.
The maternal endometrium shows prominent steroid-dependent cyclic changes in structure and function in preparation for the process of implantation. Successful implantation requires precise coordination between the embryo and uterus under the influence of ovarian steroids. After fertilization, specific uterine cell types undergo differentiation and proliferation to provide a suitable environment for embryo implantation and development. Genomic endometrial responses to estrogen are essential for the regulation of the “implantation window.” The transformation of endometrial stromal cells into decidual cells has been recognized as a fundamental step during the process of implantation. Previously, it has been shown that in vivo treatment with CC delays and/or inhibits implantation in rodents, probably because of abnormalities that are seen in the reproductive tract (including the uterus) after CC treatment in both rats and humans. Furthermore, a significant decrease in the implantation rate has also been observed in rabbits that were treated with CC before and after ovulation. 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 in the uteri of rats that were treated with CC.
Materials and Methods
Animals
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.
Experimental design
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.
Experiment 1
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.
Experiment 2
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.
Experiment 3
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).
Antibodies
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.
In situ detection of DNA fragmentation
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.
Caspase activity assay
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 12,000 g 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.
Statistical analysis
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.
Results
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.
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