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EMMPRIN) in human endometrium during menstrual cycle
40-45%). PRB is frequently expressed in both endothelial and smoothmuscle cells of myometrial blood vessels, irrespective of the phase of the cycle. Conclusions: Our findings demonstrate that in the cyclic rat uterus (1) PRB and PRA(⫹B) isoforms are coexpressed in the nuclei of endometrial cavum and glandular epithelial cells, as well as in stroma fibroblasts and myometrial smooth-muscle cells (2) Only PRB is expressed in endothelial and smooth muscle cells of myometrial vessels. (3) The immunoexpression of both PRA(⫹B) and PRB follows a cyclic pattern: an increase during proliferative phase to a periovulatory maximum and a continuous postovulatory reduction in all three endometrial target cell populations (glandular and cavum epithelial cells; stroma fibroblasts). The postovulatory reduction is retarded and only moderate in stroma fibroblasts. (4) PRA(⫹B) and PRB are expressed in a similar cyclic pattern.
P-482 Characteristic expression of extracellular matrix metalloproteinase inducer (CD147/EMMPRIN) in human endometrium during menstrual cycle. Tetsuaki Hara, Takashi Sato, Yutaka Noguchi, Koso Ohama, Michiko Hirata, Akira Ito. Dept of Obstetrics and Gynecology, Hiroshima Univ, Faculty of Medicine, Hiroshima, Japan; Dept of Biochemistry and Molecular Biology, Sch of Pharmacy, Tokyo Univ of Pharmacy and Life Science, Hachioji, Japan. Objective: In the process of menstruation and implantation, pericellular collagenolysis of endometrium is supposed to play a pivotal role. EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147 is a membrane-bound glycoprotein and participates in remodeling of extracellular matrices by augmenting matrix metalloproteinase (MMP) expression. In the present study, we identified EMMPRIN in the human endometrium and characterized the menstrual cycle-dependent expression of EMMPRIN in vivo. Design: Descriptive study of gene and protein expression and controlled in vitro experiment. Materials and Methods: Endometrial samples were collected from 14 pre-menopausal non-pregnant women. The localization of the protein was detected by immunohistochemistry using antibodies specific to EMMPRIN and matrix metalloproteinase-1 (MMP-1) in endometrium. To examine the change in messenger RNA levels of EMMPRIN during menstrual cycle, RT-PCR was performed. On the other hand, to determine the variation of EMMPRIN protein during menstrual cycle, Western blot analysis was performed. In order to examine whether the changes of EMMPRIN mRNA and protein expression are associated with estrodiol and/or progesterone treatment, we investigated the regulation of mRNA and protein expression of EMMPRIN using cultured epithelial and stromal cells obtained from endometrium with administration of estradiol (3.7x10-9 M) and/or progesterone (3.2x10-7 M) for 8 days. A one-way analysis of variance was used for statrical analysis, and the unpaired t-test was applied when multiple comparisons were performed. Results: At the proliferative phase of menstrual cycle, EMMPRIN was expressed in glandular epithelium of the basal layer of endometrium. In addition, at the superficial region of the functional layer, EMMPRIN was expressed in stroma, but not glandular epithelium. At the secretory phase, EMMPRIN was found in both stroma and glandular epithelium of the functional layer, and in glandular epithelium of the basal layer. Furthermore, EMMPRIN was found to co-localize with MMP-1 in glandular epithelium of endometrium in vivo. Western blot analysis using the functional layer showed that 35 and 47-kDa EMMPRIN were detected at the proliferative phase, while 35 and 51-kDa EMMPRIN were predominantly expressed at the secretory phase. In addition, the variant EMMPRIN molecules were found to differ in glycosylation. On the other hand, EMMPRIN was constitutively produced in primary cultured endometrial stromal and glandular epithelial cells. The production and glycosylation of EMMPRIN in the stromal cells were augmented by progesterone at the post-transcriptional and post-translational stages, respectively. Conclusion: : These results suggest for the first time that EMMPRIN is expressed in human endometrium during menstrual cycle and its expression and glycosylation are augmented by progesterone. EMMPRIN may be involved in menstruation and implantation through ECM remodeling at the interface between endometrial cells and ECM by utilizing EMMPRIN-boud MMP-1.
S280
Abstracts
Supported by: Bio-oriented Technology Research Advancement Institution (BRAIN).
P-483 The POU homeodomain protein oct-1 binds to a cis-regulatory element essential for the human GnRH upstream promoter activity in human placental trophoblasts. Benjamin C. Wong, Ke-wen Dong. Jones Institute for Reproductive Medicine, Dept of Ob/Gyn, Eastern Virginia Medical Sch, Norfolk, VA. Objective: This study aims at investigating the interaction between the placental tissue specific nuclear proteins and their binding elements in the human GnRH upstream promoter in human placental trophoblasts. Study Design: Following informed consent from patients, normal placentae were obtained from patients undergoing elective repeat cesarean sections or elective primary cesarean sections at term for fetal malpresentation. Minced placental tissue then underwent trypsin digestion followed by Percoll gradient centrifugation for isolation of trophoblasts. The trophoblasts were then plated and cultured in DMEM medium. After 48-72 hours of incubation, the medium was changed. To determine the role of the sequence (-994 to -723) that contains the four cis-regulatory elements in regulating the hGnRH upstream promoter activity in human placenta, transient transfection with the various GnRH-TK/luciferase reporter plasmid were performed in primary cultured human placental trophoblasts by the calcium-phosphate coprecipitation method. After 18 hours of incubation, the cells were rinsed with 1⫻ PBS and were incubated for another 24 hrs. The cells were then harvested for luciferase activity assay. All assays were repeated three times and results were expressed as mean ⫾ SEM. The data were evaluated by ANOVA. The electrophoretic mobility shift assay with nuclear proteins extracted from human placental trophoblasts and the tissue specific elements in the human GnRH upstream promoter was carried out to determine the interaction between the placental tissue specific nuclear proteins and their binding elements. Finally, the supershift assay with antibodies against transcriptional factors was performed to identify which DNA binding factors are involved in the mediation of human GnRH upstream promoter activity. Results: The results of transient transfection in human placental cells are very similar to the that in the JEG cell. Deletion of element 4 (E4, -987/-968) significantly decreased (four fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/940, or E3 in combination with E2, -919/-896) only slightly decreased the luciferase activity. In contrast, deletion of element 1 (E1, -876/-851) caused a two-fold loss of luciferase activity and elimination of E2 and E3 only lost less than two fold of the luciferase activity. Study performed with 5’ end deletion of this region confirmed these observations. Gel mobility shift showed that E1, E2 and E4 elements bound to the protein factors from the human placental trophoblasts. These mobility shifts could be competed with 500-fold excess of cold oligonucleotide indicating their specificity. These data suggest that the three elements are involved in tissue-specific expression of GnRH gene in human placental cells. Unlike in the JEG cells, the protein factors from human placental trophoblasts do not bind to the E3. Super shift assay using Oct-1 antibody showed that only E4 DNA-protein complex was supershifted by Oct-1 antibody, indicating that Oct-1 bound to E4. Conclusions: E1, E2 and E4 are required to confer tissue-specific expression of human GnRH gene in the human placental trophoblasts. However, the E4 element is the most important for the tissue-specific expression of human GnRH gene in the human placental trophoblasts. Oct-1 factor binds to the E4 element and may be involved in mediation of human GnRH upstream promoter activity.
P-484 Reproductive aging and ovarian secretory capacity—Is the monotropic FSH rise necessary to maintain adequate secretory function in older ovulatory women? Karl R. Hansen, Angela C. Thyer, Michael R. Soules, Nancy A. Klein. Univ of Washington, Seattle, WA. Objective: Monotropic FSH elevations and decreases in inhibin B in the early follicular phase of older reproductive aged women have been well characterized. However, secretory products of the dominant follicle (estradiol and inhibin A) in older ovulatory women remain at similar levels to
Vol. 80, Suppl. 3, September 2003
P-482 Characteristic expression of extracellular matrix metalloproteinase inducer (CD147/EMMPRIN) in human endometrium during menstrual cycle. Tetsuaki Hara, Takashi Sato, Yutaka Noguchi, Koso Ohama, Michiko Hirata, Akira Ito. Dept of Obstetrics and Gynecology, Hiroshima Univ, Faculty of Medicine, Hiroshima, Japan; Dept of Biochemistry and Molecular Biology, Sch of Pharmacy, Tokyo Univ of Pharmacy and Life Science, Hachioji, Japan. Objective: In the process of menstruation and implantation, pericellular collagenolysis of endometrium is supposed to play a pivotal role. EMMPRIN (extracellular matrix metalloproteinase inducer)/CD147 is a membrane-bound glycoprotein and participates in remodeling of extracellular matrices by augmenting matrix metalloproteinase (MMP) expression. In the present study, we identified EMMPRIN in the human endometrium and characterized the menstrual cycle-dependent expression of EMMPRIN in vivo. Design: Descriptive study of gene and protein expression and controlled in vitro experiment. Materials and Methods: Endometrial samples were collected from 14 pre-menopausal non-pregnant women. The localization of the protein was detected by immunohistochemistry using antibodies specific to EMMPRIN and matrix metalloproteinase-1 (MMP-1) in endometrium. To examine the change in messenger RNA levels of EMMPRIN during menstrual cycle, RT-PCR was performed. On the other hand, to determine the variation of EMMPRIN protein during menstrual cycle, Western blot analysis was performed. In order to examine whether the changes of EMMPRIN mRNA and protein expression are associated with estrodiol and/or progesterone treatment, we investigated the regulation of mRNA and protein expression of EMMPRIN using cultured epithelial and stromal cells obtained from endometrium with administration of estradiol (3.7x10-9 M) and/or progesterone (3.2x10-7 M) for 8 days. A one-way analysis of variance was used for statrical analysis, and the unpaired t-test was applied when multiple comparisons were performed. Results: At the proliferative phase of menstrual cycle, EMMPRIN was expressed in glandular epithelium of the basal layer of endometrium. In addition, at the superficial region of the functional layer, EMMPRIN was expressed in stroma, but not glandular epithelium. At the secretory phase, EMMPRIN was found in both stroma and glandular epithelium of the functional layer, and in glandular epithelium of the basal layer. Furthermore, EMMPRIN was found to co-localize with MMP-1 in glandular epithelium of endometrium in vivo. Western blot analysis using the functional layer showed that 35 and 47-kDa EMMPRIN were detected at the proliferative phase, while 35 and 51-kDa EMMPRIN were predominantly expressed at the secretory phase. In addition, the variant EMMPRIN molecules were found to differ in glycosylation. On the other hand, EMMPRIN was constitutively produced in primary cultured endometrial stromal and glandular epithelial cells. The production and glycosylation of EMMPRIN in the stromal cells were augmented by progesterone at the post-transcriptional and post-translational stages, respectively. Conclusion: : These results suggest for the first time that EMMPRIN is expressed in human endometrium during menstrual cycle and its expression and glycosylation are augmented by progesterone. EMMPRIN may be involved in menstruation and implantation through ECM remodeling at the interface between endometrial cells and ECM by utilizing EMMPRIN-boud MMP-1.
S280
Abstracts
Supported by: Bio-oriented Technology Research Advancement Institution (BRAIN).
P-483 The POU homeodomain protein oct-1 binds to a cis-regulatory element essential for the human GnRH upstream promoter activity in human placental trophoblasts. Benjamin C. Wong, Ke-wen Dong. Jones Institute for Reproductive Medicine, Dept of Ob/Gyn, Eastern Virginia Medical Sch, Norfolk, VA. Objective: This study aims at investigating the interaction between the placental tissue specific nuclear proteins and their binding elements in the human GnRH upstream promoter in human placental trophoblasts. Study Design: Following informed consent from patients, normal placentae were obtained from patients undergoing elective repeat cesarean sections or elective primary cesarean sections at term for fetal malpresentation. Minced placental tissue then underwent trypsin digestion followed by Percoll gradient centrifugation for isolation of trophoblasts. The trophoblasts were then plated and cultured in DMEM medium. After 48-72 hours of incubation, the medium was changed. To determine the role of the sequence (-994 to -723) that contains the four cis-regulatory elements in regulating the hGnRH upstream promoter activity in human placenta, transient transfection with the various GnRH-TK/luciferase reporter plasmid were performed in primary cultured human placental trophoblasts by the calcium-phosphate coprecipitation method. After 18 hours of incubation, the cells were rinsed with 1⫻ PBS and were incubated for another 24 hrs. The cells were then harvested for luciferase activity assay. All assays were repeated three times and results were expressed as mean ⫾ SEM. The data were evaluated by ANOVA. The electrophoretic mobility shift assay with nuclear proteins extracted from human placental trophoblasts and the tissue specific elements in the human GnRH upstream promoter was carried out to determine the interaction between the placental tissue specific nuclear proteins and their binding elements. Finally, the supershift assay with antibodies against transcriptional factors was performed to identify which DNA binding factors are involved in the mediation of human GnRH upstream promoter activity. Results: The results of transient transfection in human placental cells are very similar to the that in the JEG cell. Deletion of element 4 (E4, -987/-968) significantly decreased (four fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/940, or E3 in combination with E2, -919/-896) only slightly decreased the luciferase activity. In contrast, deletion of element 1 (E1, -876/-851) caused a two-fold loss of luciferase activity and elimination of E2 and E3 only lost less than two fold of the luciferase activity. Study performed with 5’ end deletion of this region confirmed these observations. Gel mobility shift showed that E1, E2 and E4 elements bound to the protein factors from the human placental trophoblasts. These mobility shifts could be competed with 500-fold excess of cold oligonucleotide indicating their specificity. These data suggest that the three elements are involved in tissue-specific expression of GnRH gene in human placental cells. Unlike in the JEG cells, the protein factors from human placental trophoblasts do not bind to the E3. Super shift assay using Oct-1 antibody showed that only E4 DNA-protein complex was supershifted by Oct-1 antibody, indicating that Oct-1 bound to E4. Conclusions: E1, E2 and E4 are required to confer tissue-specific expression of human GnRH gene in the human placental trophoblasts. However, the E4 element is the most important for the tissue-specific expression of human GnRH gene in the human placental trophoblasts. Oct-1 factor binds to the E4 element and may be involved in mediation of human GnRH upstream promoter activity.
P-484 Reproductive aging and ovarian secretory capacity—Is the monotropic FSH rise necessary to maintain adequate secretory function in older ovulatory women? Karl R. Hansen, Angela C. Thyer, Michael R. Soules, Nancy A. Klein. Univ of Washington, Seattle, WA. Objective: Monotropic FSH elevations and decreases in inhibin B in the early follicular phase of older reproductive aged women have been well characterized. However, secretory products of the dominant follicle (estradiol and inhibin A) in older ovulatory women remain at similar levels to
Vol. 80, Suppl. 3, September 2003