Mol. Hum. Reprod. Advance Access originally published online on January 18, 2006
Molecular Human Reproduction 2006 12(1):7-10; doi:10.1093/molehr/gah254
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Oct-4 expression in human endometrium
1Department of Gynecological Endocrinology and Reproductive Medicine, 2Department of Clinical Pathology and 3Department of Gynecology and Obstetrics, Medical University of Vienna, Waehringer Guertel, Vienna, Austria
4 To whom correspondence should be addressed at: Department of Gynecological Endocrinology and Reproductive Medicine, Medical University of Vienna, Waehringer Guertel 18-20, 5Q, Vienna A-1090, Austria. E-mail: andrea.kolbus{at}meduniwien.ac.at
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Abstract |
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The transcription factor Oct-4 is crucial for the maintenance of cell pluripotency and is known to be expressed in embryonic stem cells, germ cells and whole embryos at various stages of development. Oct-4 regulates cell fate in a dose-dependent manner and plays a key role in germ-cell tumours. In the past, several stem-cell markers have been detected, and their role in the pathogenesis of diseases has been discussed frequently. Thus, we investigated the expression of Oct-4 comparing its occurrence in endometrium of healthy and diseased women using immunohistochemistry (IHC) and RT–PCR. IHC demonstrated Oct-4 expression in 25 of 60 sections (42%), respectively in 11 out of 25 patients (44%). Oct-4 mRNA was detected by RT–PCR in all tested samples (9 of 9) of endometrium, although the levels of expression varied. To our knowledge, this is the first study demonstrating Oct-4 expression in human endometrium.
Key words: endometrium/Oct-4/stem cells/transcription factor
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Introduction |
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The transcription factor Oct-4 (also known as Oct-3 and POU5f1) is a member of the Pit-Oct-Unc (POU) transcription factor family (Scholer et al., 1990a
). Recent data indicate that Oct-4 is expressed in embryonic stem (ES) cells, germ cells and embryonal carcinoma cells, in whole embryos at different stages of development and germ-cell tumours (Scholer et al., 1990b; Nichols et al., 1998; Niwa et al., 2000; Pesce and Scholer, 2000; Gidekel et al., 2003). In co-operation with other factors (e.g. FOXD3, SOX2, STAT3), Oct-4 regulates transcription by either activating or repressing the respective target genes. The POU transcription factors are DNA-binding proteins that regulate genes containing an octamer motif important for tissue- and cell-specific transcription within their enhancer or promoter regions by binding to this consensus motif, ATGCAAAT (Clerc et al., 1988; Scheidereit et al., 1998; Pesce and Scholer, 2001). The Oct-4 gene itself contains two structural elements: the proximal enhancer (PE) and the distal enhancer (DE) that are important for cell type-specific expression of the transcription factor, and the four conserved domains CR1-CR4, which may be important for Oct-4 basal expression (Yeom et al., 1996).
Expression of Oct-4 is restricted to pluripotent cells, and down-regulation of Oct-4 is associated with loss of pluripotentiality of these cells. An aberrant expression of Oct-4 in mouse clones leads to abnormalities at various embryonic stages and thus underlines the importance of the transcription factor during development (Boiani et al., 2002).
It is uncertain whether there is a connection between the appearance of cells expressing Oct-4 and any kind of pathologies, and whether stem cells do play a role in oncogenesis. In the human breast cancer cell line MCF-7, at least four POU gene products including Oct-4 are expressed (Jin et al., 1999). Furthermore, Monk and Holding (2001) described a re-expression of embryonic genes including Oct-4 in cancer cells. In fact, stem cells and cancer cells do have common characteristics. Both stem and cancer cells are immortal, undifferentiated and invasive. Adult stem cells, which maintain the tissue in which they reside, are rare cells and have been identified so far in diverse tissues, including human bone marrow, breast, prostate, brain and liver. Recently, clonogenic epithelial and stromal cells were described indicating the presence of putative stem cells in human endometrium (Chan et al., 2004). Based on these findings, we investigated whether Oct-4 expression can be detected in human endometrium and whether there is a possible correlation between Oct-4 expression and specific phases of the menstrual cycle.
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Materials and methods |
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Immunohistochemistry
We used 60 formalin-fixed paraffin-embedded tissue samples of endometria from 25 patients, aged 29–51 years (mean age 43 years), who underwent a hysterectomy. The main indications being uterine fibroids (n = 16) and endometriosis (n = 4). Surgery was performed irrespective of the day of patient’s menstrual cycle (Table I). Formalin-fixed paraffin-embedded tissue from heart, myometrium and embryonic carcinoma of testis were used as negative or positive controls.
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For detection of Oct-4 in these sections, we used immunohistochemistry (IHC). Negative control slides (embryonic carcinoma of testis) in the absence of primary antibody but in the presence of mouse IgG were included for each staining (Figure 1B). After drying the sections in the oven at a temperature of approximately 60°C for at least 15 min, deparaffinization was performed with xylene two times for 10 min, followed by rehydration through an ethanol series. The sections were washed with phosphate-buffered saline (PBS) for 5 min. Endogenous peroxidase activity was blocked for 10 min with a 0.3% H202 solution in methanol. The sections were washed again with PBS for 5 min. After hydration, tissue sections were boiled in a 0.01 molar sodium-citrate retrieval solution (pH = 6) diluted with PBS using the microwave oven at 500 W three times for 5 min. Deionized water was added in between to replace the evaporated liquid. Next, slides were cooled down to room temperature for 30 min and washed with PBS several times. For the last washing step, we utilized deionized water for 5 min.
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The slides were incubated with primary antibodies recognizing Oct-4 [Oct-4 (C-10): sc-5279 (Santa Cruz Biotechnology, CA, USA); or anti-Oct-4 MAB4305 (Chemicon, Temecula, CA, USA)] or Vimentin (M7020, Dako, USA) and Cytokeratin (MAB3412, Chemicon) diluted 1:200 with a 0.1% bovine serum albumin (BSA)/PBS solution over night at 4°C. The sections were then warmed up for half an hour at room temperature and washed with PBS for 5 min.
The reagents from ChemMateTM Detection Kit, were used as follows: sections were covered with the secondary antibody (biotinylated secondary antibody, ready-to-use) and incubated at room temperature for 30 min. Afterwards, they were washed with PBS for 5 min. Subsequently, the slides were covered with streptavidin peroxidase and incubated for 30 min at room temperature and washed again with PBS for 5 min. Finally, a mixed solution of diaminobenzidine and HRP substrate buffer was used to cover the sections. This solution was stored in the refrigerator for 15 min before use. Then the slides were covered with the mixed solution to develop the colour and washed with deionized water. After counterstaining with hematoxylin, the sections were washed again and mounted with glycerin gelatine. The diaminobenzidine substrate solution gives a brown colour at the site of the target antigen recognized by the primary antibody. Nuclei are stained blue by the haematoxylin counterstain.
RT–PCR
Nine randomly chosen endometrium samples were used in this study from women with a mean age of 48 years. These tissues were obtained after hysterectomy. For RNA extraction, frozen tissue samples were triturated and total RNA was extracted using the TRI REAGENT—method by MRC (Molecular Research Centre, Inc., Ohio). RNA concentration was determined by measuring the optical density at 260 nm. About 1µg RNA was reversely transcribed into first strand complementary DNA (cDNA) using Superscript (Invitrogen Ltd., Paisley, UK). The resulting cDNA was amplified by PCR using primers specific for Oct-4 for 30 cycles (Monk and Holding, 2001). The following primers were used for RT–PCR reactions: forward 5'-GAC AAC AAT GAA AAT CTT CAG GAG A-3' and Oct-4 reverse 5'-TTC TGG CGC CGG TTA CAG AAC CA-3'. The PCR was started with a denaturating step at 94°C for 5 min and the amplification of the 218 bp product was performed for 20 cycles at 94°C for 30 s, 61°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 10 min. As control for genomic DNA, we used extracted RNA only (no cDNA). As a positive control for the expression of Oct-4, we used RNA from embryonic carcinoma of testis. The human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was amplified in parallel reactions as a housekeeping reference gene serving as an internal control for the quantity and quality of the cDNA. Primers for GAPDH: forward 5'-TCT GGT AAA GTG GAT ATT GTT G-3', reverse 5'-GAT GGT GAT GGG ATT TCC-3'. The expected amplified product for GAPDH was 156 base pairs. Amplified probes were separated on 1.5% agarose gels in the presence of ethidium bromide and visualized by using the eagle eye system (Stratagene, Amsterdam, The Netherlands).
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Results |
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Oct-4 protein found in endometrial cells
Endometrial samples from a cohort of 25 patients who had undergone hysterectomy for different reasons as summarized in Table I were analysed. As a positive control for the specificity of the immunohistochemical staining, embryonic carcinoma cells of testis origin were used, known to express Oct-4 (Figure 1A); only sections with markedly brown stained cells, showing a reliable and clear cell structure, respectively, were scored positive for Oct-4 expression (Figure 1C and D). To confirm the obtained results, we used a second antibody recognizing Oct-4. Importantly, with both antibodies Oct-4-expressing cells could be detected in endometrial tissue but not in heart or myometrium (Figure 1C, D, G and H). Several single cells expressing Oct-4 were regularly found in the stroma of the endometrium, which was the most frequent location for Oct-4+ cells (Figure 1C and D). As stromal and epithelial markers, we detected Vimentin and Cytokeratin expression in serial sections, respectively. Eleven out of 25 patients analysed (44%) showed endometrial cells expressing Oct-4 (summarized in Table I).
When we searched for a link between the expression of Oct-4 and patient characteristics, e.g. age, phase of the menstrual cycle or the different pathologies patients suffered from, we could not establish a correlation between patient’s age or gynaecologic disorders and Oct-4 expression, yet most of the positive sections were found in patients hysterectomized in the first half of their menstrual cycle (Table I).
Oct-4 mRNA expression in human endometrium
The observed Oct-4 protein levels can be ascribed to ongoing Oct-4 mRNA expression in the endometrium. Using RT–PCR, Oct-4 mRNA expression was detected in nine out of nine endometrial samples. Although the mRNA quantities varied, expression of Oct-4 could be detected in all samples analysed (Figure 2). By including control reactions with input RNA instead of cDNA obtained after reverse transcription of RNA, we feel very confident that the Oct-4 PCR product of the endometrial samples is correct and not due to a genomic DNA contamination. To validate that the amplified PCR product is specific for Oct-4, we isolated and sequenced the amplified product. The comparison of the obtained sequence by nucleotide–nucleotide Blast (blastn with NCBI) confirmed that the amplified product was a 218 bp fragment (nt 432–649) of the published sequence: Accession number NM_203289
[GenBank]
.2. As a positive control, we used RNA from embryonic carcinoma tissue (Figure 2). Thus, both Oct-4 mRNA and protein expression could be detected in human endometrial cells. In contrast to protein expression, which was found in only about 44% of the patients studied, Oct-4 mRNA was seen in all patients analysed, a difference, which may be explained by the far more sensitive method of RNA detection.
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Discussion |
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It has been reported that the expression of Oct-4 is restricted to oogenesis and preimplantation development and becomes more restricted to the inner cell mass (ICM) at the blastocyst stage. Later, the expression is found throughout the early epiblast, and then in the developing germ cells (for review, see Rossant, 2001
). Additionally, aberrant expression of Oct-4 in mouse clones leads to abnormalities at various embryonic stages (Boiani et al., 2002) and is also involved in germ-cell tumours (Gidekel et al., 2003). So far, expression of Oct-4 has not been investigated in human endometrium. Our work represents the first study investigating Oct-4 expression in human endometrium. Consequently, our scientific aim was at first to investigate whether Oct-4 is expressed at all in human endometrium of healthy women and secondly to determine whether Oct-4-expressing putative endometrial stem/progenitor cells may be implicated in proliferative and invasive pathologies. To the best of our knowledge, this is the first time cells expressing Oct-4 were detected in the endometrium mainly in stromal cells. The quantity of Oct-4 expression plays an essential role in the maintenance of stem-cell totipotency and confers oncogenicity (Niwa et al., 2000; Niwa, 2001; Gidekel et al., 2003). Although we found Oct-4 mRNA expression in all samples tested, most likely due to the sensitive method of RT–PCR analysis, the levels of expression varied and quantification of immunohistochemically detected Oct-4 protein levels is extremely difficult. Accordingly, Hansis et al. could also detect Oct-4 mRNA in differentiated trophectoderm, although these cells are known to lose Oct-4 expression (Hansis et al., 2000). It is not known whether the fate of the cells expressing Oct-4 in the endometrium is to remain undifferentiated and totipotent, to develop into other functional cells and tissues or under certain circumstances undergo malignant transformation.
Although we could not correlate the amount of Oct-4-expressing cells to a disease state, we nevertheless, made the interesting observation that most of the Oct-4-expressing cells could be seen in endometrium at the beginning or the middle of the menstrual cycle. The female reproductive tract contains the full complement of immune cells, which are responsible for innate and specific immunity. The number and the activity of the immune cells vary throughout the phases of the menstrual cycle, believed to be controlled by changes in the level of female sex hormones (Wira and Kaushic, 1996). Unfortunately, we could not find a significant correlation between specific phases of the menstrual cycle and Oct-4-expressing cells.
Recently, evidence for the existence of stem cells in the uterine endometrium was provided by detecting expression of the stem-cell markers c-kit, CD34, bcl-2 and Ki67 (Cho et al., 2004). Interestingly, the presumptive stem cells expressing these markers seem to be located mainly in the stroma of the endometrium, which is in accordance to our findings. Lately, clonogenic epithelial and stromal cells with high proliferative potential indicating the presence of putative stem cells in human endometrium have been identified (Chan et al., 2004; Gargett, 2004). Because endometrial stromal cells are known to be able to differentiate into other mesenchymal tissues, such as muscle, bone and cartilage (Dizerega et al., 1980; Andrews et al., 1997; Cano et al., 2000; Alison et al., 2002), all these data, taken together, strongly suggest the existence of endometrial stromal stem cells.
Interestingly, Taylor et al. provided new evidence of endometrial regeneration by bone marrow stem cells in endometrial tissue. They could show that cells of extrauterine origin could repopulate the endometrium (Taylor, 2004). These findings demonstrate that bone marrow stem cells have the ability to transdifferentiate into endometrial cells. The Oct-4-expressing cells that we found in the endometrium may originate from haematopoietic stem cells that eventually could differentiate into endometrial or malignant cells.
Whether or not the presence of these Oct-4-expressing stromal cells is required for physiological maintenance of endometrium functions, or even responsible for pathological conditions remains to be investigated in the future. Importantly, preliminary analyses by our group indicate that in all IHC investigations concerning the endometrial carcinoma, Oct-4 positive cells could be detected and the amount of Oct-4-expressing cells was also enhanced within the carcinoma compared to the normal, non-pathologic endometrial tissue (data not shown). Starzinski-Powitz et al. proposed the presence of adult stem cells in peritoneal endometriotic lesions due to the detection of the markers E-cadherin and cytokeratin and hypothesized that stem cells may contribute to the persistence of endometriosis by detaching and forming secondary lesions (Starzinski-Powitz et al., 2003). Recent data also demonstrated that human adult stem cells maintain expression of Oct-4, consistent with the stem-cell hypothesis of carcinogenesis (Tai et al., 2005).
In conclusion, we provide first evidence for cells expressing the stem-cell marker Oct-4 in the human endometrium. In conjunction with previous reports on the existence of stromal endometrial cells positive for stem-cell markers such as E-cadherin and cytokeratin (Starzinski-Powitz et al., 2003), c-kit, CD34, bcl-2 and Ki67 (Cho et al., 2004) as well as the clonogenic appearance and high proliferative potential (Chan et al., 2004; Gargett, 2004), our data strongly indicate the existence of endometrial stromal stem cells. In the future, more research is inevitable to clarify the link between this stem-cell population and their possible role in the pathways of various diseases.
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Acknowledgements |
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The authors want to thank Katja Hiesboeck-Philipp, Alexander Dangl and Ladislaus Szabo for excellent technical assistance, Josefine Stani for help with the IHC photographs and Dr Marina Schorpp-Kistner for critical reading of the manuscript.
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Submitted on September 7, 2005; accepted on November 29, 2005.
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