Tenascin is highly expressed in endometriosis and its
expression is upregulated by estrogen
Orkun Tan, M.D.,a,bTurkan Ornek, M.D.,a,cYasemin Seval, M.Sc.,a,dLeyla Sati, B.Sc.,a,d and Aydin Arici, M.D.a
aDivision of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Sciences,
Yale University School of Medicine, New Haven, Connecticut; bDepartment of Obstetrics and Gynecology, Bridgeport
Hospital, Bridgeport, Connecticut;cDepartment of Obstetrics and Gynecology, Ufuk University School of Medicine, Ankara,
Turkey; anddDepartment of Histology and Embryology Faculty of Medicine, Akdeniz University, Antalya, Turkey
Objective: To investigate the localization of tenascin expression in the endometrium of women without endome-triosis and in endometriotic implants, and to determine the in vitro regulation of tenascin by E2in these tissues.
Design: Experimental laboratory study. Setting: University medical center.
Patient(s): Reproductive age women with or without endometriosis.
Intervention(s): Proliferative (n ¼ 14), and secretory (n ¼ 14) endometrium from women without endometriosis and endometriosis implants (n¼ 14) were used for immunohistochemical analysis. Endometrial and endometriotic stromal cells were grown in culture and treated with E2, the estrogen receptor antagonist ICI 182 780 (ICI) alone,
E2in combination with ICI, or vehicle (control) for 24 hours, and tenascin expression was analyzed by Western
blotting.
Main Outcome Measure(s): Expression levels of tenascin in normal endometrium and endometriotic implants and its regulation by E2.
Result(s): Tenascin immunostaining revealed an increasing intensity in the stromal cells, starting from normal se-cretory endometrium, then normal proliferative endometrium, and reaching the highest expression in endometri-otic implants. Estradiol induced a significant increase in tenascin protein levels in the endometriendometri-otic stromal cells in culture.
Conclusion(s): The modulation of tenascin as an extracellular matrix protein by E2in endometriotic stromal cells
may be one of the factors playing a role in the development of endometriosis. (Fertil Steril2008;89:1082–9.
2008 by American Society for Reproductive Medicine.) Key Words: Tenascin, endometriosis, endometrium, estradiol
Endometriosis is characterized by the presence of endome-trial stromal and glandular cells outside the uterine cavity, mainly in the pelvis. Endometriosis is diagnosed in 30% of women with infertility and in 10%–70% of women with pel-vic pain(1). The precise etiology and pathophysiology of the disease remains poorly understood. It is widely accepted that the disease arises as a result of retrograde menstruation and attachment of endometrial cells to the peritoneum (2). The extracellular matrix (ECM) has a fundamental role in the reg-ulation of diverse cellular processes such as cell adhesion, proliferation, differentiation, and invasion. Aberrant regula-tion of the adhesion of cells to the ECM is often associated with many diseases (3). Development of endometriosis is also likely to involve cell adhesion and local invasion into the underlying tissue. Because the ECM is involved in the regulation of these cellular events, it is possible that endome-triosis may be associated with aberrantly regulated cell-ECM interactions.
Tenascin is an ECM glycoprotein present throughout the body and it plays a role in cell differentiation, proliferation, and migration(4). It is also expressed in association with pro-cesses linked to embryogenesis, suggesting an involvement in the modulation of epithelial-mesenchymal interactions taking place during tissue development (5). In the human endometrium, tenascin expression changes physiologically throughout the menstrual cycle (6–8). Tenascin is found mostly as depositions in the stroma surrounding the endome-trial glands during the proliferative phase of the menstrual cycle(7). However, the role of tenascin in the endometrium and endometriosis is poorly understood.
Both epithelial and stromal cells in the endometrium un-dergo tissue-specific cyclic changes under the influence of sex steroid hormones. Earlier studies have shown that the proliferative effect of sex steroid hormones in the endome-trium is mediated by substances such as growth factors and ECM and that an epithelial-mesenchymal interaction plays a critical role in this mediation(9).
The effect of estrogen on tenascin expression in endome-trial and endometriotic stromal cells is still unknown. What role this effect may play in the pathogenesis of endometriosis
Received July 23, 2006; revised and accepted May 11, 2007.
Reprint requests: Aydin Arici, M.D., Section of Reproductive Endocrinol-ogy and Infertility, Department of Obstetrics, GynecolEndocrinol-ogy and Repro-ductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520-8063 (E-mail:aydin.arici@yale.edu).
has not been investigated. Tenascin may be one of the factors that regulate endometrial proliferation and function.
We hypothesized that estrogen may regulate tenascin pro-tein expression in endometriosis. Therefore, in the present study, we first investigated the localization of tenascin protein in normal endometrium and in endometriotic implants. We then investigated the in vitro regulation of tenascin protein expression by E2in these tissues.
MATERIALS AND METHODS Tissue Collection
Endometrial tissues were obtained from endometrial biop-sies and human uteri after hysterectomy conducted for be-nign diseases other than endometrial disease. The day of the menstrual cycle was established from the patient’s men-strual history and was verified by histologic examination of the endometrial histology using the criteria of Noyes et al. (10). The presence of endometriosis was confirmed by his-topathologic examination of the implants. Normal endome-trial samples were grouped according to menstrual cycle phases: proliferative (days 1–14 of the cycle) or secretory (days 15–28 of the cycle). Therefore, for immunohisto-chemical analysis, we evaluated 28 (14 proliferative, 14 secretory) samples of endometrium from women without endometriosis confirmed by laparoscopy and 14 ectopic en-dometrial samples (all of them were from proliferative phase according to the last menstrual date) from women with endometriosis confirmed by laparoscopy (6 ovarian endome-triomas, 8 peritoneal implants). For cell cultures, endometri-otic stromal cells were obtained from 11 women with endometriosis. Normal endometrial stromal cells were ob-tained from 10 women in proliferative phase and 9 women in secretory phase of the menstrual cycle. Endometrial stro-mal cells obtained from each patient were considered as sep-arate experiments. Each experimental set-up was repeated on at least three occasions using cells obtained from differ-ent patidiffer-ents.
Institutional Review Board approval was obtained for the study. Informed consent in writing was obtained from each patient before surgery. Consent forms and protocols were also approved by the Human Investigation Committee of Yale University. The mean age of patients without endometri-osis was 34.5 years (range 27–49 years). The mean age of pa-tients with endometriosis was 31.3 years (range 23–45 years). For the cell cultures, the tissues were placed in Hanks’ bal-anced salt solution (HBSS) and transported to the laboratory for endometrial stromal cell isolation and long-term culture. Cells obtained from each patient were considered as separate experiments.
Immunohistochemistry
Paraffin-embedded tissue samples were cut into 5 mm sec-tions and mounted on SuperFrost Plus slides (Erie Scientific Company, Portsmouth, NH). Slides were deparaffinized in xylene and rehydrated in a graded series of alcohol.
Endog-enous peroxidase activity was quenched by incubation in 3% H2O2 for 20 minutes and was followed by a rinse with phosphate-buffered saline (PBS). For antigen retrieval, slides were placed in 10 mmol/L citrate buffer (pH 6.0) and were microwaved twice for 5 minutes. Sections were then incubated with blocking horse serum (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature in a humidified chamber. Thereafter, sections were incubated overnight at 4C with primary antibody (500 mg/mL mouse monoclonal antihuman tenascin antibody; Sigma-Aldrich, St. Louis, MO), 1:2000 dilution, in 1% bovine serum albu-min in PBS (PBS-BSA). For negative control slides, normal mouse IgG (2.8 mg/mL; Vector) was used at the same con-centration instead of primary antibody for tenascin. The sections were washed in PBS, incubated with biotinylated horse antimouse IgG (1.5 mg/mL; Vector) at 1:250 dilution for 45 minutes at room temperature. After several PBS rinses, the antigen-antibody complex was detected by using an avidin-biotin-peroxidase kit (LabVision, Fremont, CA). Diaminobenzidine (3,3-diaminobenzidine tetrahydrochlor-ide dihydrate; LabVision) was used as the chromogen, and sections were counterstained with hematoxylin and mounted with Permount (Fisher Chemicals, Springfield, NJ) on glass slides and then evaluated under a light micro-scope.
Histologic score (HSCORE) evaluation was used as de-scribed elsewhere(11, 12). The intensity for tenascin immu-noreactivity in endometrial tissues was semiquantitatively evaluated as positively stained stromal cells using the follow-ing intensity categories: (no staining), 1þ (weak but de-tectable staining), 2þ (moderate or distinct staining), and 3þ (intense staining). For each tissue, an HSCORE value was derived by summing the percentages of cells that stain-ed at each intensity category and multiplying that value by the weighted intensity of the staining, using the formula SPi(iþ l), where i represents the intensity scores and Piis the corresponding percentage of the cells. In each slide, five different areas were evaluated under a microscope with60 objective magnification, and the percentage of cells for each intensity within these areas were determined by two in-vestigators blinded to the type and source of the tissues. The inter- and intraindividual coefficients of variations were 15% and 8%, respectively, for the HSCORE evaluation. The aver-age score of two was used.
Isolation and Culture of Human Endometrial Stromal Cells Endometrial stromal cells were separated and maintained in monolayer culture as described elsewhere(13). Briefly, endo-metrial tissue was minced with a sterile stainless surgical blade and digested by incubation of tissue minces in HBSS (Sigma-Aldrich) that contained HEPES (25 mmol), penicillin (200 U/mL), streptomycin (200 mg/mL), collagenase H (1 mg/mL, 15 U/mg; Roche, Mannheim, Germany), and deoxy-ribonuclease (0.1 mg/mL, 1500 U/mg; Roche) for 45–60 minutes at 37C with agitation every 5 minutes using a 20-mL syringe. The dispersed endometrial cells were separated
by filtration through a wire sieve (73-mm-diameter pore; Sigma-Aldrich). The endometrial glands (largely undis-persed) were retained by the sieve, whereas the dispersed stromal cells passed through the sieve into the filtrate.
The stromal cells were plated in Ham’s F-12/DMEM (1:1 v/v; Sigma-Aldrich) and fetal bovine serum (FBS; 10% v/v; Invitrogen Life Technologies, Gaithersburg, MD). Cells were plated in plastic flasks (75 cm2; Falcon, BD Biosciences, Franklin Lakes, NJ), maintained at 37C in a humidified at-mosphere (5% CO2in air), and allowed to replicate to conflu-ence. Thereafter, the stromal cells were passed by standard methods of trypsinization and plated on 6-well culture plates as appropriate for the experimental design and were allowed to replicate. When cells reached the desired confluence, they were incubated with serum-free, phenol red-free media for 24 hours before initiation of treatment for the experiment. Stro-mal cells were treated with E2(108mol/L; Sigma-Aldrich), estrogen receptor antagonist (ICI 182 780, 106mol/L; Toc-ris, Ballwin, MO), E2in combination with ICI (108mol/L and 106 mol/L, respectively) in ethanol, and vehicle only (control) for 24 hours and harvested for Western blot analysis.
Western Blot Analysis
Total protein from the cells was extracted using T-PER tissue protein extraction reagent (Pierce Biotechnology, Rock-ford, IL), supplemented with protease inhibitor cocktail (1 mmol/L Na3VO4, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 1 mol/L phenylmethylsulfonylfluoride; Calbiochem, San Diego, CA). The protein concentration was determined by Bradford assay (Pierce). Twenty micrograms of protein was loaded into each lane, separated electrophoretically by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 5% Tris-HCl Ready Gels (Bio-Rad Laboratories, Her-cules, CA), and electroblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in TBS-T buffer (0.05% Tween-20 in TBS) for 1 hour to reduce the nonspecific binding. Incubation with mouse monoclonal antihuman tenascin antibody (Sigma-Aldrich) diluted at 1:4000 was performed overnight at 4C and there-after washed with TBS-T for 20 minutes. The membrane was incubated for 1 hour with peroxidase-labeled antimouse IgG (Vector) and subsequently washed with TBS-T three times for 20 minutes. Immunodetection was developed with chemi-luminescent detecting reagents (NEN Life Science Products, Boston, MA), and subsequently the membrane was exposed to BioMax film (Kodak, Rochester, NY). After stripping the membrane with Western blot stripping buffer (Pierce), the membrane was washed with TBS-T and blocked with 5% nonfat dry milk in TBS-T for 1 hour. It was later incu-bated for 1 hour with goat antihuman b-actin (Abcam, Cam-bridge, MA) diluted at 1:1000 to confirm equal loading of proteins in each lane. Thereafter, the same protocol with te-nascin immunoblot was carried out to develop the b-actin bands. Tenascin expression was then normalized by dividing the arbitrary densitometry units for tenascin by those for
b-actin for each band. Similar experiments were conducted on at least three different occasions with cells prepared from three different endometrial tissues.
Statistical Analysis
Power calculation was performed based on previous studies that involved semiquantification of tenascin expression by im-munohistochemistry. A sample size of 14 in each group has 80% power to detect a difference between means of 22.07 with a significance level (alpha) of .05. For western blot analysis, a sample size of 8 in each group has 80% power to detect a difference between means of 0.22 with a significance level (alpha) of .05. The statistical power for each analysis was performed using GraphPad StatMate version 2.0 (GraphPad Software, San Diego, CA;http://www.graphpad. com). Levels of Western blot densitometries and HSCORE of immunohistochemistry were normally distributed as tested by Kolmogorov-Smirnov test. Therefore, differences in HSCORE values among the normal proliferative endome-trium, normal secretory endomeendome-trium, and ectopic endometri-osis were analyzed using one-way ANOVA test. Statistical significance was defined as P<.05. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific, San Rafael, CA). Each experiment was repeated at least three times using cells prepared from three different endometrial tissues.
RESULTS
In Vivo Expression of Tenascin in Normal Endometrium and in Endometriosis by Immunohistochemistry
In all samples evaluated, vascular endothelial cells showed immunohistochemical staining for tenascin throughout the cycle. In normal endometrium, tenascin immunoreactivity was extracellularly localized around the stromal cells during the proliferative phase (Fig. 1A). In secretory phase, strong staining for tenascin was confined to the ECM of a narrow layer of stromal cells immediately adjacent to the glandular epithelium and around the endothelium of tortuous blood vessels but was either absent or weakly positive in the rest of the stromal compartment (Fig. 1B). Overall, in the normal endometrium, tenascin immunoreactivity was observed strongly in the proliferative phase and its intensity decreased in the secretory phase, when its expression was observed mainly around the periglandular stromal area. Tenascin im-munoreactivity was observed in the vascular wall during both the proliferative and secretory phases of normal endometrium.
Endometriotic glandular tissue was morphologically het-erogeneous, and compared with the normal endometrium the glands appeared larger and were disorganized. All ectopic endometrial tissues were stained strongly for tenascin. Vascu-lar structures and periglanduVascu-lar areas were also stained strongly with tenascin, but no difference in staining inten-sity was observed in smooth muscle cells of arterioles, ve-nules, and capillaries of endometrium versus endometriosis.
Staining was localized around the periphery of stromal cells and in the intercellular spaces of all the tissues exam-ined. In contrast to the normal endometrium, tenascin staining was abundant throughout the stroma in ectopic endometrial tissues (Figs. 1C and1D). Mean staining intensi-ties in stromal cells were compared between normal endome-trium and endometriosis samples. Mean HSCORE values of the stromal tenascin immunostaining was higher in the endometriosis samples compared with normal endometrium (P<.05;Fig. 2).
Regulation of Tenascin Protein Expression in Normal Endometrial Stromal Cells in Culture by Estradiol
To investigate the regulation of tenascin protein expression by E2, normal endometrial stromal cells from both
prolifera-tive and secretory phases were separately incubated for 24 hours with E2 (108 mol/L), estrogen receptor antagonist (ICI; 106mol/L), E2in combination with ICI (108mol/L and 106 mol/L), and vehicle only (control), and tenascin protein levels were analyzed by Western blotting. Tenascin expression in cells from different phases of the cycle is shown inFigure 3. Untreated normal endometrial stromal cells ex-pressed tenascin protein. Estradiol exerted a more stimula-tory effect on tenascin expression in cells from the proliferative phase than in cells from the secretory phase. The ICI alone increased tenascin expression significantly higher than that in the control group (P<.05), but the levels remained lower than those of the E2-treated cells from the proliferative phase (Fig. 3B). Estradiol-induced tenascin ex-pression was significantly abrogated by cotreatment with ICI (Fig. 3B; P<.05).
FIGURE 1
Representative micrographs for tenascin immunohistochemistry in the proliferative and secretory phase of normal endometrium of women without endometriosis and in endometriotic implants. (A) Tenascin
immunoreactivity was observed strongly in the stroma during the proliferative phase of normal endometrium. (B) The intensity of tenascin staining was lower in the secretory phase of normal endometrium and was localized in the periglandular area. (C and D) Diffuse and strong immunostaining of tenascin was observed in endometriosis samples. (B and D, insets) Negative control slide in which normal mouse IgG was used instead of primary antibody. Original magnification: A and C100; B and D 60; insets 60.
Regulation of Tenascin Protein Expression in Endometriotic Stromal Cells in Culture by Estradiol To investigate the regulation of tenascin expression by estro-gen, endometriotic stromal cells were placed in serum-free medium, that did not include any macromolecular content, 24 hours before incubation with E2. Endometrial samples were not separated as proliferative or secretory, because ec-topic implants do not show a significant cycle changes. Te-nascin protein levels were analyzed by Western blotting. Untreated ectopic endometrial stromal cells expressed tenas-cin protein (Fig. 4). Tenascin expression was significantly up-regulated by E2 in ectopic stromal cells (P<.05). The ICI alone increased tenascin expression markedly higher than that in the control group but lower than that in the E2-treated cells. When ICI was combined with E2, they displayed a sig-nificantly lower stimulatory effect on tenascin expression compared with that observed with E2alone (P<.05). Estro-gen increased tenascin expression in endometriotic stromal cells significantly more than that observed in control group (P<.05).
DISCUSSION
The human endometrium undergoes vast architectural modi-fications during each menstrual cycle, and ECM proteins, which cause differentiation, adhesion, proliferation, and mi-gration of cells, play a key role in this process. Tenascin is
known to be involved in both the regulation of the endo-metrial menstrual cycle and in tissue breakdown during menstruation, as well as in the attachment and invasion of en-dometrial cells to the ECM(14). Earlier studies have shown that the proliferative effect of hormones on the endometrium is mediated by substances such as growth factors and the ECM, and it is thought that epithelial-mesenchymal interac-tions play a critical role in this mediation(15). It has been de-scribed that endometrial cells are stimulated to grow by estrogen in vivo(16), suggesting that a paracrine interaction between stromal and epithelial cells might play an important role in estrogen-induced growth of endometrial cells (17). Therefore, it is likely that the in vivo growth regulation of endometriotic cells is controlled by a complex interaction of ECM proteins and estrogen. The occurrence of endome-triosis is most prevalent in women of reproductive age and rare after menopause, and estrogen is known to be a factor in the progression of the disease (18). This idea is further supported by the observation that the suppression of estro-gen levels by GnRH agonists facilitates the regression of en-dometriotic lesions and that the return of normal estrogen levels after the discontinuation of the therapies induces a re-lapse of the lesions(18). However, despite estrogen’s clear role in the pathogenesis of endometriosis, the mechanism by which estrogen might mediate its effect is unknown. Thus, in the present study, we have investigated a novel role for estrogen in the regulation of tenascin expression and distribution in normal endometrium and endometriotic tissue.
Estrogen exerts multiple biologic effects on a diverse array of target tissues, and many estrogenic actions are mediated through the genomic pathway, which involves the binding of estrogen to its intracellular receptors. During binding with estrogen, the estrogen receptors undergo conformational changes that result in binding to the estrogen response ele-ment in the promoter region of target genes, leading to tran-scription and translation, which require hours to accomplish (19).
We have demonstrated that estrogen treatment up-regu-lates tenascin expression through classical estrogen receptors in endometriotic stromal cells in culture. Also, E2-induced te-nascin expression is abrogated by cotreatment with ICI. The ICI alone increased tenascin expression above that in the control group, but the levels remained lower than those in the E2-treated cells. This effect of ICI is actually not uncom-mon; ICI is a known estrogen receptor antagonist; however, contradictory reports on the ability of ICI to antagonize estro-gen have been published. For instance, it has been shown previously that ICI induced an increase in the phosphoryla-tion of p38 mitogen-activated protein kinase (MAPK) similar to the stimulatory effects of E2whereas the stimulatory ef-fects of E2on p38 MAPK phosphorylation was inhibited by cotreatment with ICI(20). Han et al.(21)also showed stim-ulatory effects of ICI. In their study, ICI effectively blocked the effect of E2on inducible isoform of nitric oxide synthase (iNOS) expression, indicating involvement of a classic
FIGURE 2
The distribution of endometrial stromal tenascin immunostaining intensity (HSCORE) in normal endometrial samples of women without
endometriosis and in endometriotic implants. A higher intensity in endometriotic implants was observed compared to normal endometrium samples. Bars represent mean SEM. *P< .05 between endometriotic implants and normal endometrium (both phases).
estrogen receptor pathway for mediating the effect of E2. They also stated that ICI also had effects of its own, stimulat-ing the expression of iNOS(21). Those results suggest that ICI in high enough concentrations acts more as an ER mod-ulator rather than as a pure ER antagonist in human endome-trial stromal cells(21).
We find E2-induced tenascin expression particularly inter-esting, as the bioavailability of tenascin is thought to be im-portant in epithelial-mesenchymal interactions, especially in certain processes involving tissue proliferation and migra-tion, suggesting that tenascin could also play a role in endo-metriosis (6). Consistent with our observations, Harrington et al.(14)have demonstrated that the distribution of tenascin
is widespread in the stroma of endometriotic tissue and ex-pressed at higher levels in proliferative than in secretory en-dometrium. Despite estrogen’s clear role in the pathogenesis of endometriosis, however, the mechanism by which estrogen might mediate its effect was not studied by Harrington et al., or any other researchers. We have shown here for the first time that, when treated with estrogen, ectopic endometrial stromal cells significantly up-regulated tenascin protein ex-pression levels to a greater extent than the cells from the pro-liferative phase of normal endometrium. The inhibition of the stimulatory effect of E2on tenascin expression by cotreat-ment with ICI implies the involvecotreat-ment of estrogen receptors in the achievement of this effect. Whether the stimulatory ef-fect of estrogen is due to an up-regulation of estrogen
FIGURE 3
Tenascin protein expression in normal endometrial stromal cells (proliferative and secretory phase). Cultured endometrial stromal cells from both phases were treated with 108mol/L E2, 106mol/L ICI alone, 106mol/L ICI 182 780 and 108mol/L E2, or vehicle only for 24 hours. C¼ Control; E ¼ estradiol; M ¼ molecular weight marker; ICI¼ ICI 182 780; EþICI ¼ E2plus ICI. (A) After the treatments, total protein was extracted and tenascin protein expression was analyzed by Western blot. A band around 220 kDa for tenascin was observed. b-Actin band provides an internal control. (B) Bars represent mean SEM; P< .05, control vs. E2, ICI, and EþICI. *Significantly different compared with control (P< .05). **Significantly different compared with E2(P< .05).
receptors in ectopic endometriotic cells or to the deregulation of tenascin expression or degradation needs to be determined in future studies.
The up-regulation of tenascin could have several roles in the pathogenesis of endometriosis. One role would be by contributing to the aberrant attachment or proliferation of endometriotic cells. Thorough understanding of the com-plex ECM protein and estrogen network is needed to under-stand normal endometrial physiology and to design specific treatments for endometriosis. We propose that the role of te-nascin in endometriosis should be further studied in future investigations.
Acknowledgments: The authors thank Anna M. Tan from the Section of Immunobiology and Dr. Umit Kayisli from the Department of Obstetrics and Gynecology, Yale University School of Medicine, for their technical assistance.
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Tenascin protein expression in ectopic endometrial stromal cells. Cultured ectopic endometrial stromal cells were incubated for 24 h with 108mol/L E2, 106mol/L ICI 182 780 alone, 106mol/L ICI and 108mol/L E2, or vehicle only. Abbreviations as inFigure 3. (A) After the treatments, total cellular protein was extracted and tenascin protein level was measured by Western blot analysis. A band around 220 kDa for tenascin was observed. b-Actin band provides an internal control. (B) Bars represent mean SEM; P< .05, control vs. E2, ICI, and EþICI. *Significantly different compared with control (P< .05). **Significantly different compared with E2(P< .05).
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