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Localization of interferon regulatory factor-1 in human endometrium throughout the menstrual cycle
FERTILITY AND STERILITY威 VOL. 75, NO. 5, MAY 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Localization of interferon regulatory factor1 in human endometrium throughout the menstrual cycle Kotaro Kitaya, M.D.,a Jinsuke Yasuda, M.D., Ph.D.,a Shinji Fushiki, M.D., Ph.D.,b and Hideo Honjo, M.D., Ph.D.a Kyoto Prefectural University of Medicine, Kyoto, Japan
Objective: To determine the expression and localization of IRF-1 in human endometrium throughout the menstrual cycle. Design: A comparative study. Setting: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. Patients: Thirty-eight women aged 33 to 46 years, with regular menstrual cycles and nonpathological endometrium, undergoing hysterectomy. Intervention(s): Endometrial tissues were obtained from operative samples. Main Outcome Measure(s): Expression of IRF-1 mRNA throughout the menstrual cycle was investigated using semiquantitative reverse transcription–polymerase chain reaction. Localization of IRF-1 protein was determined using immunohistochemistry. Result(s): IRF-1 mRNA was expressed in the human endometrium at each phase of the menstrual cycle. The immunoreactivity for IRF-1 was observed in the extranuclear compartment of the surface and glandular epithelial cells, both during the proliferative and secretory phases, as well as in the gland secretion during the secretory phase. In contrast, stromal cells were nearly unstained. Conclusion(s): IRF-1 was localized in the human endometrium, implying that this nuclear protein plays some role other than as a transcription factor. (Fertil Steril威 2001;75:992– 6. ©2001 by American Society for Reproductive Medicine.) Key Words: IRF-1, human endometrium, gland secretion
Received August 3, 2000; revised and accepted, October 31, 2000. Reprint requests: Kotaro Kitaya, M.D., Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi, Hirokoji, Kamigyo-ku, Kyoto, 6028566, Japan (FAX: 81 75 212 1265; E-mail: [email protected]). a Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. b Pathology and Applied Neurobiology Research Institute. 0015-0282/01/$20.00 PII S0015-0282(01)01692-2
992
Interferon regulatory factor (IRF)-1 was originally identified as a transcription factor regulating the gene expression of interferon (IFN)-␣ and IFN- in response to phosphorylated signal transducer and activator of transcription (STAT)-1-␣ in various types of cells (1, 2). In addition, IRF-1 activates the gene of interleukin (IL)-15, a cytokine that plays a crucial role for the differentiation of natural killer (NK) cells (3, 4) in murine bone stromal cells (5, 6). Recently, we found that expression of IL-15 in the human endometrium increased during the secretory phase compared with during the proliferative phase (7, 8). Such cyclic fluctuation suggests that IRF-1 may be involved in the activation of the IL-15 gene also in the human endometrium. In the ovine endometrium, IRF-1 is not de-
tectable in a nonpregnant condition but appears transiently during pregnancy (9). On the contrary, in humans, IRF-1 is known to be present in the endometrium during the peri-implantation period (10). However, the relationship between its expression and the menstrual cycle has not been studied in detail. Accordingly, we herein studied the expression and localization of IRF-1 in the human endometrium during each phase of the menstrual cycle and discussed whether this nuclear protein may activate the IL-15 gene at the implantation site.
MATERIALS AND METHODS Samples Informed consent was obtained from each patient before sample collection. This study was approved by the Institutional Review
Board of Kyoto Prefectural University of Medicine.
Immunohistochemistry
Pieces of endometrial tissues were obtained from 38 women aged 33 to 46 years who had undergone hysterectomy for leiomyoma or carcinoma in situ of the uterine cervix. They had regular menstrual cycles ranging from 28 to 35 days. None of these samples displayed any pathological findings, such as polyps, inflammations, or tumors. Tissues were washed immediately in phosphate-buffered saline (PBS) to remove blood clots and fixed overnight in 4% paraformaldehyde (in phosphate buffer, pH 7.3) for immunohistochemistry and dated following the patients’ menstrual history and standard criteria (11). The remainder was homogenized in TRIzol Reagent (Gibco BRL, Gaithersburg, MD), and total RNA was extracted according to the manufacturer’s protocol.
Fixed samples were embedded in paraffin and cut into 4-m sections. After being deparaffinized in xylene and rehydrated in a graded series of ethanol, sections were subjected to microwave pretreatment for antigen unmasking in distilled water for 5 minutes. Subsequently, sections were immersed in 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase and then washed in PBS containing 10% fetal calf serum (JRH Biosciences, Lenexa, KS) for 10 minutes to suppress nonspecific antibody binding.
Reverse Transcription–Polymerase Chain Reaction Two micrograms of total RNA were converted to cDNA with 1 g of oligo dT primers by using a reverse-transcription kit (Gibco BRL) in a final volume of 20 L. One microliter of cDNA solution was amplified with 0.5 M of human IRF-1–specific primers: upper, 5⬘-ACC CTG GCT AGA GAT GCA GA-3⬘ (224 –243 base pairs [bp]); lower, 3⬘ GTG GAA GCA TCC GGT ACA CT-5⬘ (537–518 bp) in a final-volume 50-L solution containing Taq DNA polymerase (Gibco BRL). Each cycle consisted of 60 seconds at 94°C, 45 seconds at 63°C, and 90 seconds at 72°C. As an internal control, human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA, which was shown to be expressed constantly in the human endometrium (12), was simultaneously amplified with specific primers under the same polymerase chain reaction (PCR) conditions.
Sections were then incubated with rabbit anti-human IRF-1 polyclonal antibody C-20 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature in a moist chamber. After being washed in PBS three times, sections were covered with peroxidase-labeled goat antirabbit polyclonal antibody (DAKO, Kyoto, Japan) for 30 minutes. The primary antibody was omitted in the staining for negative control. After being washed, sections were developed with diaminobenzidine (DAKO) and observed under a light microscope with or without counterstaining with hematoxylin.
RESULTS Endometrial Dating Endometrial samples were classified as follows. There were three at the menstrual phase, six at the early proliferative phase (day 4 to 7), five at the mid-proliferative phase (day 8 to 11), four at the late proliferative phase (day 12 to 14), seven at the early secretory phase (day 15 to 18), eight at the mid-secretory phase (day 19 to 23), and five at the late secretory phase (day 24 onward).
Measurement of Relative IRF-1/G3PDH mRNA Ratio
Expression of IRF-1 mRNA in Human Endometrium
To determine the optimal condition for semiquantitation, six samples (three at the proliferative phase and three at the secretory phase) were subjected to PCR for 22 to 36 cycles. Ten microliters of PCR products were subsequently electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The PCR product was verified as a band on an ultraviolet (UV) transilluminator (Funakoshi, Tokyo, Japan).
PCR products consistent with IRF-1 mRNA (314 bp) were detected in all of the samples. The band intensity was weaker in the endometrium during the menstrual phase compared with in the other phases (Fig. 1). Densitometrical analysis revealed a higher relative IRF-1/G3PDH mRNA ratio of PCR products during the proliferative phase compared with the secretory phase and significantly varied throughout the menstrual cycle (P⬍.001; Fig. 2).
The images on the UV transilluminator were scanned into a computer, and the band intensity was measured by the densitometrical software NIH Image, version 1.61 (NIH, Bethesda, MD). The band intensity for PCR products increased exponentially up to 30 cycles and almost reached a plateau at more than 31 cycles. We therefore set the PCR condition at 28 cycles. Amplification was done in triplicate for each sample. The mean IRF-1/G3PDH mRNA ratio was calculated and used for statistical analysis. One-way analysis of variance (ANOVA) was used to evaluate significant difference among the phases. FERTILITY & STERILITY威
Localization of IRF-1 Protein in Human Endometrium Surface and glandular epithelial cells were constantly stained throughout the proliferative and secretory phases (Fig. 3). The immunoreactivity for IRF-1 was observed clearly in the cytoplasm and membrane in contrast to in the nuclei (Fig. 3B and D). Moreover, immunostaining was observed in the gland secretion of the secretory phase endometrium. This was prominent during the mid–secretory phase (Fig. 3D). In contrast, stromal cells were virtually unstained throughout the menstrual cycle. 993
FIGURE 1 Representative electrophoresis of RT-PCR products for IRF-1 mRNA derived from the human endometrium. M ⫽ menstrual phase; EP, MP, and LP ⫽ early, mid-, and late proliferative phases, respectively; ES, MS, and LS ⫽ early, mid-, and late secretory phases, respectively.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
DISCUSSION In the present study, we found that both the mRNA and protein of IRF-1 are expressed in the human endometrium throughout the menstrual cycle. Characteristic NK cells that display the phenotypes of CD3neg, CD16neg, and CD56bright have been found to infiltrate the human endometrium (13–15), although their functions remain unknown. Some investigations supported the hypothesis that these uterine NK cells are essential for successful pregnancy (16, 17), whereas others demonstrated an increase in the endometrium after the early pregnancy loss
FIGURE 2 Relative ratio of RT-PCR products for IRF-1 to G3PDH by semiquantitative analysis in the human endometrium at each menstrual phase. M ⫽ menstrual phase; EP, MP, and LP ⫽ early, mid-, and late proliferative phases, respectively; ES, MS, and LS ⫽ early, mid-, and late secretory phases, respectively.
(18, 19). Uterine NK cells are considered to be proliferating in situ because they express proliferation-associated nuclear marker Ki-67 (15, 20) and are numerously seen during the secretory phase, compared with the proliferative phase (21, 22). More recently, we found that the expression of IL-15 protein in the human endometrial stroma elevated during the secretory phase compared with the proliferative phase and corresponded with the fluctuation of uterine NK cells (8). This finding indicates that progesterone is a potent stimulator of the IL-15 gene in the human endometrial stromal cells, but the progesterone-responsive element is not found in the promoter region of the human IL-15 gene. Therefore, the regulatory mechanisms involved in the transcription of IL-15 gene remained unclear. The nuclear protein IRF-1 can bind a wide range of genes (2, 23) in various types of cells. The mouse lacking the IRF-1 gene has severe deficiency of NK cells (24). IRF-1 has been reported to activate the IL-15 gene in the murine-bone stromal cells (5, 6). Accordingly, we presumed that IRF-1 might induce the expression of IL-15 also in the endometrium, where NK cells are proliferating in situ. IRF-1 is classified into two isoforms: an active-form that is capable of inducing various genes, and an inactive-form lacking this ability (25). The active-form IRF-1 is translated from wild-type mRNA, whereas the inactive form is synthesized from exon 2– deleted or exon-2- and -3– deleted mRNA. To detect the presence of wild-type mRNA for IRF-1, we set the upper primer in the sequence of the exon 2 region of the IRF-1 gene and found the active-form mRNA in all of the endometrial samples examined.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
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IRF-1 localization in human endometrium
Using immunohistochemistry, we investigated the localization of IRF-1 in the human endometrium during the each phase of the menstrual cycle. Recently, Jabbour et al. reported that IRF-1 protein was detectable in the epithelial Vol. 75, No. 5, May 2001
FIGURE 3 Localization of IRF-1 protein in the human endometrium of the proliferative phase (A and B) and secretory phase (C and D). Arrows point to the immunostaining. The immunoreactivity for IRF-1 is observed in the extranuclear compartment of the glandular and surface epithelial cells (A–D) and in the gland secretion (C and D). Scale bars indicate 100 m.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
cells of the human endometrium during the peri-implantation period, whereas IRF-1 was not evident in the stroma (10). We also confirmed the immunoreactivity for IRF-1 in the surface and glandular epithelial cells throughout the menstrual cycle. The role of IRF-1 in implantation or placentation remains unresolved. There is no significant difference in abortion rates between the IRF-1⫺/⫺ and IRF-1⫹/⫹ mouse. On the contrary, the intraperitoneal administration of IFN-␥ drastically increases the abortion rates in the IRF-1⫹/⫹ mouse but not in the IRF-1⫺/⫺ mouse (26). This may be explained by the activated IRF-1 in response to the IFN-␥ signaling pathway inducing the production of some fetotoxic substance. However, our observation has disclosed that IRF-1 was mainly localized in the cytoplasm and membrane of epithelial cells. The immunoreactivity on or close to the cell membrane is intriguing, although immunoelectron microscopical studies are required to elucidate the localization at the ultrastructural level. It is interesting that immunostaining FERTILITY & STERILITY威
for IRF-1 was also observed in the gland secretion during the secretory phase, suggesting possible roles beyond a nuclear transcription factor at the fetal–maternal interface. It may be worthwhile to compare the expression pattern of IRF-1 in the endometrium in fertile women with that of patients with implantation failure or habitual abortion. In conclusion, we demonstrate that IRF-1 is expressed in the human endometrium throughout the menstrual cycle. The immunoreactivity for IRF-1 is found in the extranuclear compartment of the epithelial cells and gland secretion. In contrast, virtually no staining was found in the endometrial stroma, where NK cells were proliferating in situ. IRF-1 may thus be involved in phenomena other than intranuclear gene transcription in the human endometrium. References 1. Miyamoto M, Fujita T, Kimura Y, Maruyama M, Harada H, Sudo Y, et al. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell 1988; 54:903–13. 2. Fujita T, Kimura Y, Miyamoto M, Barsoumian EL, Taniguchi T.
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Induction of endogenous IFN-alpha and IFN-beta genes by a regulatory transcription factor, IRF-1. Nature 1989;337:270 –2. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 1994;264:965– 8. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al. Interleukin (IL)-15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 1994;180:1395– 403. Ogasawara K, Hida S, Azimi N, Tagaya Y, Sato T, Yokochi-Fukuda T, et al. Requirement for IRF-1 in the microenvironment supporting development of natural killer cells. Nature 1998;391:700 –3. Ohteki T, Yoshida H, Matsuyama T, Duncan GS, Mak TW, Ohashi PS. The transcription factor interferon regulatory factor 1 (IRF-1) is important during the maturation of natural killer 1.1⫹ T cell receptor-alpha/ beta⫹ (NK1⫹ T) cells, natural killer cells, and intestinal intraepithelial T cells. J Exp Med 1998;187:967–72. Okada S, Okada H, Sanezumi M, Nakajima T, Yasuda K, Kanzaki H. Expression of interleukin-15 in human endometrium and decidua. Mol Hum Reprod 2000;6:75– 80. Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H. IL-15 expression at human endometrium and decidua. Biol Reprod 2000;63: 683–7. Spencer TE, Ott TL, Bazer FW. Expression of interferon regulatory factors one and two in the ovine endometrium: effects of pregnancy and ovine interferon tau. Biol Reprod 1998;58:1154 – 62. Jabbour HN, Critchley HO, Yu-Lee LY, Boddy SC. Localization of interferon regulatory factor-1 (IRF-1) in nonpregnant human endometrium: expression of IRF-1 is up-regulated by prolactin during the secretory phase of the menstrual cycle. J Clin Endocrinol Metab 1999; 84:4260 –5. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;35:751– 4. Dou Q, Williams RS, Chegini N. Expression of integrin messenger ribonucleic acid in human endometrium: a quantitative reverse transcription polymerase chain reaction study. Fertil Steril 1999;71:347–53. Starkey PM, Sargent IL, Redman CWG. Cell populations in human early pregnancy decidua: characterization and isolation of large granular lymphocytes by flow cytometry. Immunology 1988;65:129 –34. Nishikawa K, Saito S, Morii T, Hamada K, Ako H, Narita N, et al. Accumulation of CD16⫺CD56⫹ natural killer cells with high affinity
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interleukin 2 receptors in human early pregnancy decidua. Int Immunol 1991;3:743–50. King A, Balendran N, Wooding P, Carter NP, Loke YW. CD3⫺ leukocytes present in the human uterus during early placentation: phenotypic and morphologic characterization of the CD56⫹⫹ population. Develop Immunol 1991;1:169 –90. Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome. J Immunol 1996;156:4027–34. Fukui A, Fujii S, Yamaguchi E, Kimura H, Sato S, Saito Y. Natural killer cell subpopulations and cytotoxicity for infertile patients undergoing in vitro fertilization. Am J Reprod Immunol 1999;41:413–22. Clifford K, Flanagan AM, Regan L. Endometrial CD56⫹ natural killer cells in women with recurrent miscarriage: a histomorphometric study. Hum Reprod 1999;14:2727–30. Fukui K, Yoshimoto I, Matsubara K, Hori R, Ochi H, Ito M. Leukocyte function-associated antigen-1 expression on decidual natural killer cells in patients with early pregnancy loss. Mol Hum Reprod 1999;5: 1083– 8. Kammerer U, Marzusch K, Krober S, Ruck P, Handgretinger R, Dietl J. A subset of CD56⫹ large granular lymphocytes in first-trimester human decidua are proliferating cells. Fertil Steril 1999;71:74 –9. Starkey PM, Clover LM, Rees MCP. Variation during the menstrual cycle of immune cell populations in human endometrium. Eur J Obstet Gynecol Reprod Biol 1991;39:203–7. Lachapelle MH, Miron P, Hemmings R, Baron D, Roy DC. Flowcytometric characterization of hematopoietic cells in non-pregnant human endometrium. Am J Reprod Immunol 1996;35:5–13. Taniguchi T, Tanaka N, Taki S. Regulation of the interferon system, immune response and oncogenesis by the transcription factor interferon regulatory factor-1. Eur Cytokine Netw 1998;9(suppl 3):43– 8. Duncan GS, Mittrucker HW, Kagi D, Matsuyama T, Mak TW. The transcription factor interferon regulatory factor-1 is essential for natural killer cell function in vivo. J Exp Med 1996;184:2043– 8. Harada H, Kondo T, Ogawa S, Tamura T, Kitagawa M, Tanaka N, et al. Accelerated exon skipping of IRF-1 mRNA in human myelodysplasia/leukemia: a possible mechanism of tumor suppressor inactivation. Oncogene 1994;9:3313–20. Clark DA, Chaouat G, Arck PC, Mittruecker HW, Levy GA. Cytokinedependent abortion in CBA ⫻ DBA/2 mice is mediated by the procoagulant fgl2 prothrombinase. J Immunol 1998;160:545–9.
Vol. 75, No. 5, May 2001
Localization of interferon regulatory factor1 in human endometrium throughout the menstrual cycle Kotaro Kitaya, M.D.,a Jinsuke Yasuda, M.D., Ph.D.,a Shinji Fushiki, M.D., Ph.D.,b and Hideo Honjo, M.D., Ph.D.a Kyoto Prefectural University of Medicine, Kyoto, Japan
Objective: To determine the expression and localization of IRF-1 in human endometrium throughout the menstrual cycle. Design: A comparative study. Setting: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. Patients: Thirty-eight women aged 33 to 46 years, with regular menstrual cycles and nonpathological endometrium, undergoing hysterectomy. Intervention(s): Endometrial tissues were obtained from operative samples. Main Outcome Measure(s): Expression of IRF-1 mRNA throughout the menstrual cycle was investigated using semiquantitative reverse transcription–polymerase chain reaction. Localization of IRF-1 protein was determined using immunohistochemistry. Result(s): IRF-1 mRNA was expressed in the human endometrium at each phase of the menstrual cycle. The immunoreactivity for IRF-1 was observed in the extranuclear compartment of the surface and glandular epithelial cells, both during the proliferative and secretory phases, as well as in the gland secretion during the secretory phase. In contrast, stromal cells were nearly unstained. Conclusion(s): IRF-1 was localized in the human endometrium, implying that this nuclear protein plays some role other than as a transcription factor. (Fertil Steril威 2001;75:992– 6. ©2001 by American Society for Reproductive Medicine.) Key Words: IRF-1, human endometrium, gland secretion
Received August 3, 2000; revised and accepted, October 31, 2000. Reprint requests: Kotaro Kitaya, M.D., Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi, Hirokoji, Kamigyo-ku, Kyoto, 6028566, Japan (FAX: 81 75 212 1265; E-mail: [email protected]). a Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine. b Pathology and Applied Neurobiology Research Institute. 0015-0282/01/$20.00 PII S0015-0282(01)01692-2
992
Interferon regulatory factor (IRF)-1 was originally identified as a transcription factor regulating the gene expression of interferon (IFN)-␣ and IFN- in response to phosphorylated signal transducer and activator of transcription (STAT)-1-␣ in various types of cells (1, 2). In addition, IRF-1 activates the gene of interleukin (IL)-15, a cytokine that plays a crucial role for the differentiation of natural killer (NK) cells (3, 4) in murine bone stromal cells (5, 6). Recently, we found that expression of IL-15 in the human endometrium increased during the secretory phase compared with during the proliferative phase (7, 8). Such cyclic fluctuation suggests that IRF-1 may be involved in the activation of the IL-15 gene also in the human endometrium. In the ovine endometrium, IRF-1 is not de-
tectable in a nonpregnant condition but appears transiently during pregnancy (9). On the contrary, in humans, IRF-1 is known to be present in the endometrium during the peri-implantation period (10). However, the relationship between its expression and the menstrual cycle has not been studied in detail. Accordingly, we herein studied the expression and localization of IRF-1 in the human endometrium during each phase of the menstrual cycle and discussed whether this nuclear protein may activate the IL-15 gene at the implantation site.
MATERIALS AND METHODS Samples Informed consent was obtained from each patient before sample collection. This study was approved by the Institutional Review
Board of Kyoto Prefectural University of Medicine.
Immunohistochemistry
Pieces of endometrial tissues were obtained from 38 women aged 33 to 46 years who had undergone hysterectomy for leiomyoma or carcinoma in situ of the uterine cervix. They had regular menstrual cycles ranging from 28 to 35 days. None of these samples displayed any pathological findings, such as polyps, inflammations, or tumors. Tissues were washed immediately in phosphate-buffered saline (PBS) to remove blood clots and fixed overnight in 4% paraformaldehyde (in phosphate buffer, pH 7.3) for immunohistochemistry and dated following the patients’ menstrual history and standard criteria (11). The remainder was homogenized in TRIzol Reagent (Gibco BRL, Gaithersburg, MD), and total RNA was extracted according to the manufacturer’s protocol.
Fixed samples were embedded in paraffin and cut into 4-m sections. After being deparaffinized in xylene and rehydrated in a graded series of ethanol, sections were subjected to microwave pretreatment for antigen unmasking in distilled water for 5 minutes. Subsequently, sections were immersed in 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase and then washed in PBS containing 10% fetal calf serum (JRH Biosciences, Lenexa, KS) for 10 minutes to suppress nonspecific antibody binding.
Reverse Transcription–Polymerase Chain Reaction Two micrograms of total RNA were converted to cDNA with 1 g of oligo dT primers by using a reverse-transcription kit (Gibco BRL) in a final volume of 20 L. One microliter of cDNA solution was amplified with 0.5 M of human IRF-1–specific primers: upper, 5⬘-ACC CTG GCT AGA GAT GCA GA-3⬘ (224 –243 base pairs [bp]); lower, 3⬘ GTG GAA GCA TCC GGT ACA CT-5⬘ (537–518 bp) in a final-volume 50-L solution containing Taq DNA polymerase (Gibco BRL). Each cycle consisted of 60 seconds at 94°C, 45 seconds at 63°C, and 90 seconds at 72°C. As an internal control, human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA, which was shown to be expressed constantly in the human endometrium (12), was simultaneously amplified with specific primers under the same polymerase chain reaction (PCR) conditions.
Sections were then incubated with rabbit anti-human IRF-1 polyclonal antibody C-20 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature in a moist chamber. After being washed in PBS three times, sections were covered with peroxidase-labeled goat antirabbit polyclonal antibody (DAKO, Kyoto, Japan) for 30 minutes. The primary antibody was omitted in the staining for negative control. After being washed, sections were developed with diaminobenzidine (DAKO) and observed under a light microscope with or without counterstaining with hematoxylin.
RESULTS Endometrial Dating Endometrial samples were classified as follows. There were three at the menstrual phase, six at the early proliferative phase (day 4 to 7), five at the mid-proliferative phase (day 8 to 11), four at the late proliferative phase (day 12 to 14), seven at the early secretory phase (day 15 to 18), eight at the mid-secretory phase (day 19 to 23), and five at the late secretory phase (day 24 onward).
Measurement of Relative IRF-1/G3PDH mRNA Ratio
Expression of IRF-1 mRNA in Human Endometrium
To determine the optimal condition for semiquantitation, six samples (three at the proliferative phase and three at the secretory phase) were subjected to PCR for 22 to 36 cycles. Ten microliters of PCR products were subsequently electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. The PCR product was verified as a band on an ultraviolet (UV) transilluminator (Funakoshi, Tokyo, Japan).
PCR products consistent with IRF-1 mRNA (314 bp) were detected in all of the samples. The band intensity was weaker in the endometrium during the menstrual phase compared with in the other phases (Fig. 1). Densitometrical analysis revealed a higher relative IRF-1/G3PDH mRNA ratio of PCR products during the proliferative phase compared with the secretory phase and significantly varied throughout the menstrual cycle (P⬍.001; Fig. 2).
The images on the UV transilluminator were scanned into a computer, and the band intensity was measured by the densitometrical software NIH Image, version 1.61 (NIH, Bethesda, MD). The band intensity for PCR products increased exponentially up to 30 cycles and almost reached a plateau at more than 31 cycles. We therefore set the PCR condition at 28 cycles. Amplification was done in triplicate for each sample. The mean IRF-1/G3PDH mRNA ratio was calculated and used for statistical analysis. One-way analysis of variance (ANOVA) was used to evaluate significant difference among the phases. FERTILITY & STERILITY威
Localization of IRF-1 Protein in Human Endometrium Surface and glandular epithelial cells were constantly stained throughout the proliferative and secretory phases (Fig. 3). The immunoreactivity for IRF-1 was observed clearly in the cytoplasm and membrane in contrast to in the nuclei (Fig. 3B and D). Moreover, immunostaining was observed in the gland secretion of the secretory phase endometrium. This was prominent during the mid–secretory phase (Fig. 3D). In contrast, stromal cells were virtually unstained throughout the menstrual cycle. 993
FIGURE 1 Representative electrophoresis of RT-PCR products for IRF-1 mRNA derived from the human endometrium. M ⫽ menstrual phase; EP, MP, and LP ⫽ early, mid-, and late proliferative phases, respectively; ES, MS, and LS ⫽ early, mid-, and late secretory phases, respectively.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
DISCUSSION In the present study, we found that both the mRNA and protein of IRF-1 are expressed in the human endometrium throughout the menstrual cycle. Characteristic NK cells that display the phenotypes of CD3neg, CD16neg, and CD56bright have been found to infiltrate the human endometrium (13–15), although their functions remain unknown. Some investigations supported the hypothesis that these uterine NK cells are essential for successful pregnancy (16, 17), whereas others demonstrated an increase in the endometrium after the early pregnancy loss
FIGURE 2 Relative ratio of RT-PCR products for IRF-1 to G3PDH by semiquantitative analysis in the human endometrium at each menstrual phase. M ⫽ menstrual phase; EP, MP, and LP ⫽ early, mid-, and late proliferative phases, respectively; ES, MS, and LS ⫽ early, mid-, and late secretory phases, respectively.
(18, 19). Uterine NK cells are considered to be proliferating in situ because they express proliferation-associated nuclear marker Ki-67 (15, 20) and are numerously seen during the secretory phase, compared with the proliferative phase (21, 22). More recently, we found that the expression of IL-15 protein in the human endometrial stroma elevated during the secretory phase compared with the proliferative phase and corresponded with the fluctuation of uterine NK cells (8). This finding indicates that progesterone is a potent stimulator of the IL-15 gene in the human endometrial stromal cells, but the progesterone-responsive element is not found in the promoter region of the human IL-15 gene. Therefore, the regulatory mechanisms involved in the transcription of IL-15 gene remained unclear. The nuclear protein IRF-1 can bind a wide range of genes (2, 23) in various types of cells. The mouse lacking the IRF-1 gene has severe deficiency of NK cells (24). IRF-1 has been reported to activate the IL-15 gene in the murine-bone stromal cells (5, 6). Accordingly, we presumed that IRF-1 might induce the expression of IL-15 also in the endometrium, where NK cells are proliferating in situ. IRF-1 is classified into two isoforms: an active-form that is capable of inducing various genes, and an inactive-form lacking this ability (25). The active-form IRF-1 is translated from wild-type mRNA, whereas the inactive form is synthesized from exon 2– deleted or exon-2- and -3– deleted mRNA. To detect the presence of wild-type mRNA for IRF-1, we set the upper primer in the sequence of the exon 2 region of the IRF-1 gene and found the active-form mRNA in all of the endometrial samples examined.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
994
Kitaya et al.
IRF-1 localization in human endometrium
Using immunohistochemistry, we investigated the localization of IRF-1 in the human endometrium during the each phase of the menstrual cycle. Recently, Jabbour et al. reported that IRF-1 protein was detectable in the epithelial Vol. 75, No. 5, May 2001
FIGURE 3 Localization of IRF-1 protein in the human endometrium of the proliferative phase (A and B) and secretory phase (C and D). Arrows point to the immunostaining. The immunoreactivity for IRF-1 is observed in the extranuclear compartment of the glandular and surface epithelial cells (A–D) and in the gland secretion (C and D). Scale bars indicate 100 m.
Kitaya. IRF-1 localization in human endometrium. Fertil Steril 2001.
cells of the human endometrium during the peri-implantation period, whereas IRF-1 was not evident in the stroma (10). We also confirmed the immunoreactivity for IRF-1 in the surface and glandular epithelial cells throughout the menstrual cycle. The role of IRF-1 in implantation or placentation remains unresolved. There is no significant difference in abortion rates between the IRF-1⫺/⫺ and IRF-1⫹/⫹ mouse. On the contrary, the intraperitoneal administration of IFN-␥ drastically increases the abortion rates in the IRF-1⫹/⫹ mouse but not in the IRF-1⫺/⫺ mouse (26). This may be explained by the activated IRF-1 in response to the IFN-␥ signaling pathway inducing the production of some fetotoxic substance. However, our observation has disclosed that IRF-1 was mainly localized in the cytoplasm and membrane of epithelial cells. The immunoreactivity on or close to the cell membrane is intriguing, although immunoelectron microscopical studies are required to elucidate the localization at the ultrastructural level. It is interesting that immunostaining FERTILITY & STERILITY威
for IRF-1 was also observed in the gland secretion during the secretory phase, suggesting possible roles beyond a nuclear transcription factor at the fetal–maternal interface. It may be worthwhile to compare the expression pattern of IRF-1 in the endometrium in fertile women with that of patients with implantation failure or habitual abortion. In conclusion, we demonstrate that IRF-1 is expressed in the human endometrium throughout the menstrual cycle. The immunoreactivity for IRF-1 is found in the extranuclear compartment of the epithelial cells and gland secretion. In contrast, virtually no staining was found in the endometrial stroma, where NK cells were proliferating in situ. IRF-1 may thus be involved in phenomena other than intranuclear gene transcription in the human endometrium. References 1. Miyamoto M, Fujita T, Kimura Y, Maruyama M, Harada H, Sudo Y, et al. Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell 1988; 54:903–13. 2. Fujita T, Kimura Y, Miyamoto M, Barsoumian EL, Taniguchi T.
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Vol. 75, No. 5, May 2001