Molecular Human Reproduction, Vol. 8, No. 5, 441-446,
May 2002
© 2002 European Society of Human Reproduction and Embryology
Uterine physiology |
Erythropoietin and erythropoietin receptor expression in human endometrium throughout the menstrual cycle
Department of Obstetrics and Gynecology, Tohoku University School of Medicine, 1–1, Seiryo-machi, Aoba-ku, Sendai, 980–8574, Japan
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Abstract |
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Erythropoietin (Epo) is an important regulator of erythropoiesis. Recent studies have demonstrated non-classical sites of Epo and Epo-receptor (Epo-R) expression, suggesting new physiological roles unrelated to erythropoiesis. Other studies have shown that the mouse uterus expresses Epo and its receptor, and produces Epo protein in an estrogen-dependent manner. We therefore hypothesized that Epo is one of the growth factors involved in cyclic endometrial changes. We determined Epo and Epo-R mRNA expression in isolated endometrial epithelial and stromal cells using RT–PCR. While both Epo and Epo-R were detected in all samples of isolated epithelial cells analysed throughout the menstrual cycle, neither one was detected in isolated stromal cells. In addition, using quantitative real-time RT–PCR with the TaqMan detection system, we showed that isolated epithelial cells had higher Epo mRNA levels in the secretory phase than in the proliferative phase. Immunohistochemical analyses revealed that Epo and Epo-R protein expression in glandular epithelial cells was increased during the mid-proliferative phase and was maintained during the late proliferative and the early, mid- and late secretory phases. These findings suggest that Epo may be involved in cyclic proliferation and differentiation of endometrial glandular epithelial cells, acting in an autocrine manner. In addition, we also hypothesize that ovarian steroids may stimulate Epo production in human endometrial glandular epithelial cells.
endometrium/erythropoietin/erythropoietin-receptor/immunohistochemistry/TaqMan RT–PCR
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Erythropoietin (Epo) plays a key role in the regulation of erythropoiesis. This
30 kDa factor (Tilbrook and Klinken, 1999
) stimulates proliferation of early erythroid precursors and differentiation of late erythroid precursors of the erythroid lineage by binding to the Epo receptor (Epo-R) (Krantz, 1991; Jelkmann, 1992). The kidney is a major site of Epo production in adults, and the kidney-derived Epo is responsible for the stimulation of erythropoiesis. In addition, studies have revealed non-classical sites of Epo and Epo-R expression, suggesting new tissue-specific physiological roles of Epo, unrelated to erythropoiesis (Masuda et al., 1994; Conrad et al., 1996). Other studies have demonstrated that the mouse uterus expresses Epo and Epo-R mRNA and produces Epo protein in an estrogen-dependent manner (Yasuda et al., 1998; Masuda et al., 1999). In the uterine endometrium, ovarian steroids modulate cyclic endometrial changes primarily by interacting with local growth factors to regulate growth, differentiation and function of the endometrium. Thus, we hypothesized that Epo is one of the growth factors involved in cyclic endometrial changes. This hypothesis was investigated by determining the presence of Epo and Epo-R mRNA and protein in human endometrium throughout the menstrual cycle of normal, fertile women by a real-time RT–PCR assay with the TaqMan detection system (Gibson et al., 1996; Heid et al., 1996; Matsuzaki et al., 2000, 2001) and by immunohistochemistry. |
Materials and methods |
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Experimental subjects
Endometrial tissue samples were obtained from 47 fertile premenopausal women (39–47 years old) undergoing a hysterectomy due to uterine leiomyoma or carcinoma in situ of the uterine cervix. Patients did not receive hormonal treatments, such as GnRH agonist or sex steroids, and did not use intrauterine contraception during a minimum 6 month period preceding surgery. All patients had regular menstrual cycles, confirmed by their menstrual history and measurements of serum 17ß-estradiol and progesterone levels. Each fresh tissue sample was fixed in 4% paraformaldehyde (pH 7.4) and embedded in paraffin for routine histopathological examination and immunohistochemical analysis. Tissue samples were classified according to time of sampling and histological dating by previously defined criteria (Noyes et al., 1950
) into six different groups: early proliferative (n = 8), mid-proliferative (n = 12), late proliferative (n = 5), early secretory (n = 8), mid-secretory (n = 6) and late secretory phases (n = 8). Out of 47 fresh samples, each of 14 samples (proliferative phase: n = 7, secretory phase: n = 7) was divided into two portions and one portion was immediately placed in an ice-cold 1:1 mixture of Dulbecco's Modified Eagle's medium and Ham's F-12 (DMEM/F12; Gibco BRL, Tokyo, Japan) for isolation of stromal and epithelial cells. All tissue samples were obtained with the full and informed consent of the patients and the research protocol was approved by the human research board of the ethical committee of Tohoku University School of Medicine.
Isolation of human endometrial stromal and epithelial cells
Tissue samples were minced into small pieces (1 mm3) and incubated at 37°C for 1 h (in a shaking water bath) in DMEM/F12 supplemented with 0.25% collagenase (Wako, Tokyo, Japan), 100 IU/ml penicillin (Sigma Chemical Co., St Louis, MO, USA), 100 µg/ml streptomycin (Sigma) and 2.5 µg/ml streptomycin B (Gibco). The resulting suspension consisted of single stromal cells and fragments of epithelial cells. These cells were then centrifuged at 400 g and pellets were resuspended in fresh DMEM/F12. The stromal and epithelial cells were isolated by differential sedimentation, as previously described (Sugawara et al., 1997a,b). In order to identify the isolated cells, immunohistochemical localization of cytokeratin and vimentin, which are markers for epithelial and stromal cells, was carried out.
RNA extraction
Prior to RT–PCR or TaqMan (Perkin-Elmer Applied Biosystems, Chiba, Japan) RT–PCR analyses, each sample of the frozen endometrial cells was homogenized and total RNA was extracted with ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer's instructions.
Oligodeoxynucleotide primers and TaqMan probes
Using Primer Express (version 1.0; Perkin-Elmer Applied Biosystems, Chiba, Japan) from the GenBank database, the oligodeoxynucleotide primer pair for RT–PCR and TaqMan RT–PCR and the probe for TaqMan RT–PCR of Epo were designed on the human Epo cDNA (Jacobs et al., 1985). The primer pair for RT–PCR of Epo-R was designed on the Epo-R cDNA (Jones et al., 1990). These primers and probes, as well as their locations on each cDNA, are shown in Table I.
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RT–PCR analysis
The cDNA was reverse transcribed at 42°C for 50 min in 19 µl of reaction solution containing 1 µg total RNA, 2.5 mmol/l MgCl2, 5 mmol/l dithiothreitol, 0.5 mmol/l dNTPs, random hexamers (50 ng/ml; Gibco) and 200 IU of Superscript II RT (reverse transcriptase; Gibco). As a negative control, 1 µl of RNA-free dietylpyrocarbonate-treated distilled water was subjected to the same RT reaction. PCR was performed in a 50 µl volume containing 2 µl of the RT reaction product, 5 µl 10xPCR buffer, 2.5 mmol/l MgCl2, 0.5 mmol/l dNTPs, 0.4 mmol/l of each of the sense and antisense primers for Epo or Epo-R, and 2.5 IU Taq DNA polymerase (Takara, Otsu, Japan). Amplification was performed in a Takara PCR Thermocycler (Takara) using a hot start. The same cycle profiles were used for both Epo and Epo-R: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 1 min at 94°C, 1 min at 60°C and 1.5 min at 72°C, with a final 10 min extension at 72°C. To test the integrity of the RT–PCR process, as an external positive control, expression of ß-actin mRNA was evaluated in each RT reaction product under the following amplification profile: 95°C for 1 min, followed by 35 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 2 min. The PCR products were subjected to electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. All RT–PCR procedures were performed at least twice in separate experiments to assess reproducibility.
Synthesis of recombinant RNA of Epo for generation of a standard curve
Recombinant RNA of Epo was synthesized for generation of a standard curve, as previously described (Matsuzaki et al., 2000, 2001). Plasmids, in which a 725 bp fragment within the Epo cDNA was ligated, were prepared as DNA templates for a standard curve in quantitative TaqMan PCR. Briefly, the Epo cDNA region was PCR amplified in a volume of 50 µl with the same primer sets and cycle profiles as used above for RT–PCR analysis. The PCR products were eluted from a 2% agarose gel using the QIAEX II Gel Extraction kit (Qiagen, Tokyo, Japan) and subcloned in plasmids (pGEM T-Easy vectors; Promega, Tokyo, Japan). E.coli DH5a was transformed with subcloned plasmids, and bacterial colonies containing the correct insert were cultured. After mini-preparation, plasmid DNA was linearized with SphI. Subsequently, recombinant RNA for Epo was generated by an in-vitro transcription reaction with T6 RNA polymerase, with the Dig labelling transcription kit (Boehringer Mannheim, Tokyo, Japan) following the manufacturer's instructions. Recombinant Epo RNA was quantified by spectrophotometry, aliquoted and stored at –80°C for future use in the TaqMan RT–PCR assay.
TaqMan RT–PCR
In a single-tube, single-enzyme system, RT and DNA polymerization take place without the addition of subsequent enzymes or buffers. The RT–PCR reaction was carried out in a 50 µl volume of reaction solution containing 5xTaqMan EZ buffer, 3 mmol/l Mn(OAc)2, 300 mmol/l dA/dC/dG/dUTP, 5.0 IU recombinant (r)Tth DNA polymerase, 400 nmol/l primers (forward and reverse), 100 nmol/l TaqMan probe, 0.5 IU AmpErase UNG and 1 µg total RNA. The TaqMan EZ RT–PCR Core Reagents kit for the RT–PCR reaction was purchased from Perkin-Elmer Applied Biosystems. TaqMan RT reaction conditions were 50°C for 2 min, 60°C for 30 min and 95°C for 10 min for 1 cycle. TaqMan PCR conditions were 95°C for 15 s and 60°C for 1 min for 40 cycles on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer). For each RT–PCR experiment, a standard curve assay was included with five RNA concentrations in duplicate (10-fold serially diluted recombinant RNA; 10–9 to 10–13 g) and a blank assay without the RNA template.
Determination of Epo mRNA levels
Epo mRNA levels were finally determined as the copy number per 1 µg of total RNA extracted from each tissue sample. Copy numbers for standard curves were calculated using the molecular weight of recombinant RNA of Epo. Each RT–PCR experiment included a standard curve assay with five RNA concentrations.
Immunohistochemical staining
Immunohistochemical staining was performed on endometrial paraffin sections using a goat polyclonal antibody against human Epo (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit polyclonal antibody against the human Epo-R (Santa Cruz Biotechnology). Immunohistochemistry for Epo and Epo-R was performed using a commercial streptavidin–biotin system (Histofine kit; Nicheirei, Tokyo, Japan). Specific immunolocalization of Epo and Epo-R has been reported previously in human placenta and fetal kidney; therefore, these tissues were chosen as positive controls (Conrad et al., 1996; Juul et al., 1998a,b; Fairchild Benyo and Conrad, 1999). Sections were deparaffinized and rehydrated in graded ethanols. After rinsing in distilled water, sections were digested with proteinase K (Dako A/S, Glostrup, Denmark) for 10 min at room temperature. Sections were then rinsed in distilled water and immersed in absolute methanol containing 0.3% (v/v) hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. Sections were blocked with 10% normal rabbit serum (for Epo) or normal goat serum (for Epo-R) in 0.01 mol/l phosphate-buffered saline (PBS, pH 7.2). Sections were then rinsed in PBS and incubated at 4°C overnight with the primary antibody, either anti-human Epo or anti-human Epo-R diluted 1:100 in PBS with 3% bovine serum albumin, followed by a rinse in PBS. Negative controls were performed by replacing the primary antibody with normal goat immunoglobulin (Ig)G (for Epo) or rabbit IgG (for Epo-R) diluted at the same concentration as the primary antibody. After rinsing with PBS, sections were incubated with alkaline phosphatase coupled anti-goat IgG (Histofine Kit) for Epo or alkaline phosphatase coupled anti-rabbit IgG (Histofine Kit) for Epo-R. Sections were subsequently washed with PBS, and incubated with 0.06 mmol/l 3,3'-diaminobenzidine and 2 mmol/l hydrogen peroxide in 0.05% Tris–HCl buffered at pH 7.6, then counterstained with Methyl Green and mounted.
Quantitation of Epo and Epo-R immunostained cells
To quantify immunopositivity, positively stained cells were counted using a semi-automatic computerized image analysis system. The computerized image analysis system consisted of a light microscope (Zeiss, Göttingen, Germany; 40x objective, 10x ocular) with a colour charge coupling device camera (Zeiss) connected to a Macintosh 9500/120 computer. For each section, the number of positive epithelial and stromal cells was counted, regardless of the staining intensity, in a total of 10 non-overlapping areas. The positivity index (PI, percentage of positive cells) for Epo or Epo-R was calculated for each sample.
Statistical analysis
Statistical analysis was performed with the StatView 4.5 program (Abacus Concepts Inc., Berkeley, CA, USA). The Kruskal–Wallis test or Mann–Whitney U-test was applied to compare results from different groups. Statistical significance was defined as P < 0.05.
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Standard curve
A strong linear relationship between the threshold cycle and the log of the starting RNA-copy number was always demonstrated (r2 > 0.99, Figure 1
). The 5-log dynamic range of the assay was revealed.
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RT–PCR and TaqMan RT–PCR analyses of Epo and Epo-R
As demonstrated in Figure 2
, we detected the expected 201 bp product for Epo mRNA and the 181 bp product for Epo-R mRNA in all isolated epithelial cells analysed at different phases of the menstrual cycle. Neither Epo nor Epo-R mRNA expression was detected in isolated stromal cells. TaqMan RT–PCR of isolated epithelial cells showed significantly higher Epo mRNA levels (P < 0.001) in the secretory than in the proliferative phase (Figure 3).
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Immunohistochemical analysis of Epo and Epo-R
The immunohistochemistry results are shown in Figures 4 and 5
and Table II. Homogeneous cytoplasmic immunoreactivities to Epo and Epo-R were detected in surface and glandular epithelial cells, while stromal cells stained more weakly and heterogeneously throughout the menstrual cycle. The PI of surface epithelial cells for both Epo and Epo-R was constantly high throughout the menstrual cycle and no cyclical change was observed. The PI of glandular epithelial cells for both Epo and Epo-R was significantly increased during the mid-proliferative phase compared with the early proliferative phase and was maintained during the late proliferative and the early, mid- and late secretory phases.
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The PI of stromal cells for both Epo and Epo-R was increased during the proliferative phase and decreased during the secretory phase.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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In this study, the presence of both mRNA and protein expression of Epo and Epo-R in endometrial epithelial cells indicates that Epo may be involved in cyclic proliferation and differentiation of endometrial epithelial cells, acting in an autocrine mechanism. Quantitative TaqMan RT–PCR analyses revealed an increased epithelial expression of Epo mRNA during the secretory phase. Furthermore, immunohistochemical analyses revealed that Epo protein expression in glandular epithelial cells was increased during the mid-proliferative phase, when serum estrogen levels are markedly elevated, and was maintained during late proliferative and early, mid- and late secretory phases. These findings suggest that ovarian steroids may stimulate Epo production in human endometrial glandular epithelial cells.
No detectable Epo and Epo-R mRNA was demonstrated in isolated stromal cells, whereas a low level of Epo and Epo-R protein expression was detected within the stroma. One possible explanation for the discrepancy is that Epo and Epo-R protein may be localized in endothelial cells and/or macrophages within the stroma. In addition, Epo is a soluble protein and Epo immunoreactivity within the stroma may reflect the ligand binding to Epo-R. Further studies should be required to clarify the localizations of Epo and Epo-R protein within the stroma.
Several growth factors are present in the human endometrium (Giudice, 1994). Among them, granulocyte colony-stimulating factor, colony-stimulating factor-1 and interleukin-6, which were formerly thought to be predominantly specific for the haematopoietic cells, are also considered to play central roles in cyclic proliferation and differentiation of endometrial cellular components (Giudice, 1994). The present study adds further evidence that haematopoietic growth factors could be important in uterine physiology. Further in-vivo and in-vitro investigations are required to clarify the functional roles of Epo in human endometrium.
Implantation represents the remarkable synchronization between development of the embryo and differentiation of the endometrium (Horne et al., 2000). The endometrium undergoes a series of changes leading to a period of uterine receptivity called the `window of implantation'. Outside of this time, the uterus is resistant to embryo attachment (Navot et al., 1991). After adhesion of the embryonic pole of the human blastocyst to the endometrial epithelial surface, trophoblastic differentiation develops as the trophoblast invades the stromal endometrium. Trophoblast cells of the human first-trimester placenta express both Epo and Epo-R mRNA, suggesting an autocrine role for Epo in the survival, proliferation or differentiation of placental trophoblast cells (Conrad et al., 1996; Fairchild Benyo and Conrad, 1999). Although there are no data regarding the expression of either Epo or Epo-R in human blastocysts, the present findings suggest that in endometrial epithelial cells, Epo could play a paracrine role in the processes of implantation. Further studies investigating the presence of Epo and Epo-R during human embryonic development are necessary to confirm this possibility. In addition, previous studies have demonstrated that the ampulla and isthmus regions of the oviduct produce Epo (Masuda et al., 2000), and that Epo stimulates epididymal sperm maturation and sperm-fertilizing activity (Yamamoto et al., 1997). These findings suggest that Epo may also play a role in fertilization.
As to other potential roles of Epo, important studies have demonstrated the angiogenic potential of Epo (Carlini et al., 1995; Yasuda et al., 1998; Masuda et al., 1999; Ribatti et al., 1999). Expression of the Epo-R has been demonstrated in endothelial cells both in vitro and in vivo (Anagnostou et al., 1994) and stimulation of endothelial cells by Epo may elicit an angiogenic response in vitro and in vivo (Carlini et al., 1995; Ribatti et al., 1999). In addition, previous studies have shown that Epo is involved in the estrogen-dependent cyclical angiogenesis occurring within the mouse uterus (Yasuda et al., 1998; Masuda et al., 1999). Although there are no data regarding the expression of Epo-R by human endometrial endothelial cells, it is possible that Epo may play some role as an angiogenic factor in the cycling human endometrium. Several growth factors, such as vascular endothelial growth factor, fibroblast growth factor (FGF) and epidermal growth factor, appear to be important for angiogenesis and blood vessel function in the cycling human endometrium (Möller et al., 2001). The angiogenic response of the chick embryo chorioallantoic membrane blood vessels to Epo is comparable with that elicited by the prototypic angiogenic cytokine-basic FGF (Ribatti et al., 1999). Thus, the relative strength of the action of Epo should be investigated in the cycling human endometrium.
In conclusion, the present study demonstrated mRNA and protein expression of Epo and Epo-R in human endometrium throughout the menstrual cycle. These findings provide further evidence that, in addition to erythropoiesis, Epo may have new tissue-specific physiological functions.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We are grateful to Ms Junko Fujiwara and Mr Satoshi Okamoto for their expert technical assistance. We are indebted to Dr F.K.Lin (Amgen Inc., Thousands Oaks, CA, USA) for the generous gift of plasmids, in which a 725 bp fragment of the Epo cDNA is ligated.
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Notes |
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1 Present address: Department of Gynecology, Polyclinique de l'Hotel-Dieu, 13, boulevard Charles-De-Gaulle, 63003 Clermont-Ferrand Cedex 1, France
2 To whom correspondence should be addressed. E-mail: sachikoma{at}aol.com
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Submitted on May 1, 2001; resubmitted on November 12, 2001; accepted on January 22, 2001.
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