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Hiroshi Fujiwara,
Yoshihiro Nishioka,
Hisanori Matsumoto,
Koh Suginami,
Akihito Horie,
Hirohiko Tani,
Noriomi Matsumura,
Tsukasa Baba,
Yukiyasu Sato,
Yoshihiko Araki,
Ikuo Konishi
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ABSTRACT: The Eph-ephrin system is a unique system that can induce multiple cellular responses such as cell migration, regulation of angiogenesis, and axonal guidance. Previously, the Eph-ephrin system was reported to regulate human extravillous trophoblast invasion. In this study, we examined the possible involvement of the Eph-ephrin system in the invasion of malignant gestational trophoblastic diseases using a human choriocarcinoma-derived cell line, JEG-3.
The mRNA expression of class A Ephs and ephrins on JEG-3 cells was examined by reverse transcription-polymerase chain reaction. The effects of recombinant human Eph A1 (r-Eph A1) and r-ephrin A4 on the proliferation and invasion of JEG-3 cells were investigated by cell proliferation and Matrigel invasion assays. The alterations of integrin expression on JEG-3 cells in the presence of r-Eph A1 and r-ephrin A4 were investigated by flow cytometry. The induction of phosphorylation of focal adhesion kinase in JEG-3 cells by r-ephrin A4 was examined by Western blot analysis.
By reverse transcription-polymerase chain reaction, mRNAs of Eph A1, A2, and A4 and ephrin A1, A4, and A5 were detected on JEG-3 cells. In Matrigel invasion assay, both r-Eph A1 and r-ephrin A4 promoted the invasion of JEG-3 cells without affecting cell proliferation. During 24-hour culture with r-Eph A1 and r-ephrin A4, the increase in integrin α 5 expression on JEG-3 cells was observed by flow cytometry. Western blotting analysis showed that r-ephrin A4 induced dephosphorylation of focal adhesion kinase in JEG-3 cells.
These findings suggest that Eph-ephrin interaction plays some role in the regulation of choriocarcinoma invasion in cooperation with integrins.
International Journal of Gynecological Cancer 03/2013; 23(3):576-82. · 1.65 Impact Factor
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ABSTRACT: In primate placenta, extravillous trophoblast (EVT) invades maternal tissue in temporally- and spatially-regulated fashions. We previously identified a novel placenta-specific cell-surface aminopeptidase, laeverin/aminopeptidase Q, which is expressed on EVT-lineage cells in the fetal membrane. Laeverin possesses a peptide-binding site that is evolutionally unique to primates, suggesting possible involvement of laeverin in a primate-specific phenomenon during placentation. Thus, this study was designed to elucidate the molecular characteristics and physiological roles of laeverin in human EVT.
Placental tissues of various developmental stages were subjected to immunostaining and western blotting. Effects of siRNA and a soluble form of recombinant laeverin on EVT cells isolated from primary villous explant cultures were examined using Matrigel invasion assays and cell proliferation assays.
Laeverin was specifically immunolocalized to HLA-G-positive EVT in placentas from early and term pregnancy. In primary villous explant cultures, laeverin expression was induced on the cell surface of the outgrowing EVT. In western blotting, laeverin protein was detected as two distinct bands at 130 and 160 kDa along with a broad band ranging from 200 to 270 kDa. De-glycosylation treatment showed that these native laeverin isotypes are N-linked glycoproteins sharing a common 115-kDa core protein. In invasion assays, the reduction of laeverin expression by siRNA suppressed migration of the isolated EVT, while the soluble form of recombinant laeverin enhanced its migration.
Laeverin is a specific cell-surface marker for human EVT and plays a regulatory role in EVT migration.
Human Reproduction 03/2012; 27(5):1267-76. · 4.47 Impact Factor
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Journal of Obstetrics and Gynaecology Research 06/2011; · 0.94 Impact Factor
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Journal of Obstetrics and Gynaecology Research 06/2011; 37(7):966 - 966. · 0.94 Impact Factor
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ABSTRACT: Amniotic fluid embolisms (AFE) are one of the most fatal complications of pregnancy. We describe a case of AFE that occurred 2 h after vaginal delivery at 41 weeks of gestation. The diagnosis of AFE was made by symptoms of dyspnea, coagulopathy, and severe hypotension. ZnCP-1, the characteristic component of meconium, was elevated in the serum. Cardiac compressions after repeated cardiac arrests were required during the initial 2 h of resuscitation. Primary resuscitation was performed with airway management and aggressive fluid management, including infusion of 33 units of red cell concentrates and 57 units of fresh frozen plasma. The patient recovered without any aftereffects. This case report warrants that AFE should be considered when coagulopathy and dyspnea are observed during the postpartum period.
Journal of Obstetrics and Gynaecology Research 04/2011; 37(8):1122-5. · 0.94 Impact Factor
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ABSTRACT: Management of second- and third-trimester fetal death in the presence of placenta previa is a dilemma for obstetricians. We herein describe a case of fetal death occurring at 23 weeks' gestation in the presence of placenta previa. Three weeks of expectant management failed to reduce uteroplacental blood perfusion evaluated with pulsatility index of the uterine artery. Labor was then induced with gemeprost vaginal pessary following overnight laminaria pretreatment. Vaginal delivery was achieved with total blood loss of 1900 ml. Homologous blood transfusion was obviated owing to autologous blood that had been stored during the waiting period.
The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians 03/2011; 25(2):196-9. · 1.36 Impact Factor
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ABSTRACT: During implantation, the human embryo invades endometrial stromal tissues, reducing the intercellular connections among epithelial cell layers. Since Eph-ephrin interaction can induce repulsive forces to control cell position and movement, we examined the possible involvement of this system in intercellular dissociation among endometrial epithelial cells.
The expression of Eph A receptor on human endometrial epithelial cells and endometrial carcinoma-derived Ishikawa cells was examined by RT-PCR, immunohistochemistry and western blotting. The effects of recombinant ephrin A1 on Eph A2 phosphorylation in Ishikawa cells were also examined by western blotting. A permeability assay was performed to determine the effects of ephrin A1 on cell-to-cell adhesion.
Eph A1, A2 and A4 mRNAs were detected in human endometrial epithelial cells and Ishikawa cells, and ephrin A1 was present in human blastocysts. Immunohistochemical staining showed that Eph A1, A2 and A4 receptors were expressed on the cell surface region of luminal and glandular epithelial cells in human endometrium in both the proliferative and secretory phase. The presence of Eph A2 protein in the human endometrium was confirmed by western blot analysis. Recombinant ephrin A1 was bound to Ishikawa cells and induced phosphorylation of Eph A2 expressed in Ishikawa cells. In addition, stimulation by ephrin A1 for 20 min increased the permeability of monolayer Ishikawa cells versus control cultures (P < 0.01), without affecting cell viability.
This study demonstrated that the Eph-ephrin A system can promote intercellular dissociation in Ishikawa cells suggesting an important role in the initial step of embryo implantation by opening the endometrial epithelial cell barrier.
Human Reproduction 02/2011; 26(2):299-306. · 4.47 Impact Factor
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ABSTRACT: Recently, the Eph-ephrinA system was proposed to contribute to the initial interaction between the maternal endometrial epithelium and embryonic trophectoderm. Since the Eph-ephrin interaction can induce adhesive and/or repulsive forces into the cells, we examined the possible role of this system in functional changes in endometrial epithelial cells using endometrial carcinoma-derived Ishikawa cells.
The expressions of EphA1, A2 and A4 on Ishikawa cells were examined by RT-PCR and western blotting analyses. The effects of recombinant ephrinA1 on Ishikawa cells were also examined by western blot analysis and cell attachment and aggregation assays.
EphA1, A2 and A4 were expressed on Ishikawa cells. Recombinant ephrinA1 bound to the surfaces of Ishikawa cells and induced phosphorylation of EphA2 and A4. In bovine serum albumin-blocked nitrocellulose-coated dishes, Ishikawa cells remained floating and aggregated with each other. Under these conditions, immobilized ephrinA1 promoted Ishikawa cell attachment with increased tyrosine phosphorylation in focal adhesion kinase. In addition, immobilized ephrinA1 reversibly inhibited Ishikawa cell aggregation. Gene-reduction of EphA1, A2 and A4 by siRNAs attenuated the inhibitory effects of ephrinA1 on cell aggregation, confirming that ephrinA1 affects Ishikawa cell functions through Eph-ephrinA interaction.
This study demonstrated that the Eph-ephrinA system can promote cell attachment along with intercellular dissociation in Ishikawa cells. These findings suggest that this system can induce functional changes in endometrial epithelial cells.
Human Reproduction 02/2011; 26(5):1163-70. · 4.47 Impact Factor
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ABSTRACT: We here describe a case of congenital leukemia that ended in intrauterine fetal demise at 30 weeks of gestation. Acute enlargement of the fetal trunk, elevated pulsatility index of the umbilical artery with concomitant decline of pulsatility index of the middle cerebral artery, pleural effusion, and polyhydramnios preceded the fetal death. Diagnosis of congenital myeloid leukemia was suggested by microscopic examination of the placental tissue, revealing immature myeloid precursors filling the lumina of fetal vessels in the umbilical cord and chorionic villi. Extensive vascular involvement of the placenta by leukemic cells was considered to be a primary cause of the fetal death.
Fetal Diagnosis and Therapy 02/2011; 29(4):325-30. · 1.05 Impact Factor
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ABSTRACT: Cases of retained products of conception (RPOC) with marked vascularity present a clinical challenge because simple dilation and curettage (D&C) can lead to life-threatening hemorrhage. We describe here two cases of hypervascular RPOC that were successfully managed with two different approaches. Case 1: A 26-year-old woman with history of 3 D&Cs was transported to the emergency room for heavy vaginal bleeding 45 days after a spontaneous abortion. Diagnosis of RPOC with aneurysm-like structure was considered and uterine artery embolization was performed. Four days after the uterine artery embolization, reduction of the vascularity of RPOC was confirmed on color Doppler ultrasonography and D&C was successfully carried out. Case 2: A 37-year-old woman with history of one cesarean section became pregnant after the regular menses. She underwent D&C for missed abortion at 8 weeks' gestation. Seven days after the D&C, sonographically heterogenous mass emerged in the vicinity of the previous cesarean scar. Thereafter, the mass gradually grew larger and diagnosis of hypervascular placental polyp was considered. As the amount of vaginal bleeding was small, expectant management was instituted. Sixty-one days after the first D&C, reduction of the vascularity of RPOC was confirmed on color Doppler ultrasonography and D&C was successfully completed.
Journal of Obstetrics and Gynaecology Research 01/2011; 37(5):458-64. · 0.94 Impact Factor
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ABSTRACT: Middle cerebral artery-peak systolic velocity (MCA-PSV) has been reported to predict fetal anemia with similar accuracy as amniotic ΔOD450 assay. Alloimmunized dizygotic twin pregnancy allows us to compare anemic and non-anemic twins in the same intrauterine environment. We herein present a case of Rh (E)-incompatible dizygotic twin pregnancy, where MCA-PSV could precisely detect the anemia in one of the twins. A 36-year-old woman, whose previous child required exchange transfusion due to hemolytic anemia of newborn (HFDN), conceived twins after in vitro fertilization-embryo transfer. At 24 weeks' gestation, MCA-PSV of twin A and twin B were 23.9 cm/s (0.8 multiples of median; MoM) and 30.7 cm/s (1.0 MoM), respectively. At 31 weeks' gestation, MCA-PSV values of both twins were sharply elevated to nearly 1.4 MoM. Thereafter, MCA-PSV of twin A fell to 1.0 MoM, whereas MCA-PSV of twin B exceeded 1.5 MoM at 34 weeks' gestation. Development of fetal anemia was suspected and emergency cesarean section was performed. Twin B showed moderate anemia with positive direct Coombs' test and was diagnosed as HFDN due to anti-E alloimmunization. Twin B required phototherapy and red cell transfusion, but exchange transfusion was safely obviated.
Journal of Obstetrics and Gynaecology Research 10/2010; 36(6):1236-9. · 0.94 Impact Factor
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ABSTRACT: We describe a case of congenital meconium peritonitis with progressive fetal ascites and polyhydramnios. Fetal ascites could be only partially reduced on paracentesis at 29 weeks' gestation, and it subsequently increased. Urinary trypsin inhibitor (UTI), a physiological anti-inflammatory substance, was administered into the fetal abdominal cavity at a second paracentesis performed at 35 weeks' gestation. There was a significant amount of fetal ascites remaining 1 day after the second paracentesis, but this completely resolved within 5 days. A healthy infant was delivered vaginally and no surgical intervention was required. The case suggests that UTI can reduce meconium-induced chemical peritonitis and thereby facilitate intrauterine remission of fetal ascites.
Ultrasound in Obstetrics and Gynecology 09/2010; 37(3):366-8. · 3.01 Impact Factor
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ABSTRACT: Anterior sacral meningocele is an extremely rare condition and there has been only one previous report of a prenatal diagnosis. We report the case of a 36-year-old primigravida who was referred following detection of a huge fetal pelvic cyst on routine ultrasound examination at 19 + 4 weeks' gestation. Neither fetal ultrasound nor magnetic resonance imaging (MRI) at 20 + 5 weeks' gestation could detect communication between the cyst and the spinal cord. Because extension of the pear-shaped cyst through the pelvic diaphragm down to the perineum was reminiscent of dilated vagina and uterine cervix, a tentative diagnosis of hydrometrocolpos secondary to imperforate hymen was considered. On follow-up MRI at 33 + 5 weeks' gestation, a narrow stalk connecting the pelvic cyst and the spinal canal through the anterior sacral foramen was clearly delineated, allowing us to reach the prenatal diagnosis of anterior sacral meningocele.
Ultrasound in Obstetrics and Gynecology 09/2010; 37(4):493-6. · 3.01 Impact Factor
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ABSTRACT: In the human placenta, embryo-derived trophoblasts aggressively invade maternal spiral arteries and transform the arteries to low-resistance large-caliber vessels. This process, which ensures adequate placental perfusion, is called maternal vascular remodeling. Histological examination showed deposition of maternal platelets in the trophoblast aggregates formed in the spiral arteries. Several lines of evidence suggest that these platelets are activated. Soluble factors released from the activated platelets, as a whole, enhanced invasive capacity of isolated trophoblasts in vitro. These findings suggest the importance of nonhemostatic platelet function in maternal vascular remodeling. In contrast, gene knockout studies suggest that maternal platelet defects are compatible with successful pregnancy in mice. Moreover, pregnant women with severe platelet defects usually accomplish an uneventful pregnancy. Thus, promotion of endovascular trophoblast infiltration by maternal platelets might not be the only mechanism that regulates maternal vascular remodeling. The maternal vascular remodeling is an essential component of human reproduction and should be secured by several complementary mechanisms. Future studies should aim to elucidate other mechanisms that could regulate endovascular trophoblast infiltration.
Medical Molecular Morphology 09/2010; 43(3):129-33. · 1.39 Impact Factor
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ABSTRACT: Attachment to endometrial epithelium is an essential process for human embryos. Although it is widely accepted that this process is largely regulated by the endocrine system, the precise molecular mechanism(s) remains unclear. Recent evidence suggests that immune cells actively contribute to the establishment of embryo implantation. In accordance with this, we found that peripheral blood immune cells positively affect the differentiation of maternal endometrium to facilitate embryo implantation during early pregnancy. From these findings, we propose a novel concept that circulating immune cells are important regulators of embryo implantation. Lately, implantation failure in patients treated with in vitro fertilization and embryo transfer has received increasing attention. Based on our hypothesis, we have successfully developed a new therapy for implantation failure using autologous peripheral blood immune cells. These findings suggest that supportive mechanisms via the immune system facilitate embryo implantation and will be useful in the field of assisted reproductive technology.
Journal of Mammalian Ova Research 12/2009;
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ABSTRACT: Human laeverin/aminopeptidase Q (LVRN/APQ) is a novel member of the M1 family of zinc aminopeptidases and is specifically expressed on the cell surface of human extravillous trophoblasts. Multiple sequence alignment of human M1 aminopeptidase revealed that the first Gly residue within the conserved exopeptidase motif of the M1 family, GXMEN motif, is uniquely substituted for His in human LVRN/APQ. In this study, we evaluated the roles of nonconserved His(379), comprising the exopeptidase motif in the enzymatic properties of human LVRN/APQ. We revealed that the substitution of His(379) with Gly caused significant changes in substrate specificity both toward fluorogenic substrates and natural peptide hormones. In addition, the susceptibilities of bestatin, a sensitive inhibitor for human LVRN/APQ, and natural inhibitory peptides were decreased in the H379G mutant. A molecular model suggested a conformational difference between wild-type and H379G human LVRN/APQs. These results indicate that His(379) of the enzyme plays essential roles in its distinctive enzymatic properties and contributes to maintaining the appropriate structure of the catalytic cavity of the enzyme. Our data may bring new insight into the biological significance of the unique exopeptidase motif of LVRN/APQ obtained during the evolution of primates.
Journal of Biological Chemistry 10/2009; 284(50):34692-702. · 4.77 Impact Factor
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ABSTRACT: Mammalian mothers undergoing embryo implantation must specifically recognize the developing embryo in a species-restricted manner. We previously observed that immune cells derived from early pregnant mice could promote endometrial differentiation and embryo implantation in blastocyst-transferred pseudopregnant mice. Although the precise mechanism remains unknown, it is suggested that the maternal immune system undergoes functional changes after recognizing developing embryos from the very early stages of pregnancy. Since it is physically impossible for immune cells to directly interact with the developing embryo while it is surrounded by the zona pellucida (ZP), it is speculated that the embryo produces certain embryo- and species-specific soluble factor(s) in the oviduct before hatching. As a candidate for this factor, we have paid attention to the ZP that is normally protected from immunological attack during oogenesis in the ovarian follicle. ZP-specific glycoproteins are known to play important roles in the species- and oocyte-specific binding of sperm, and the ZP can also be considered an abundant store of oocyte- and species-specific glycoproteins. In contrast to unfertilized oocytes, developing embryos may degrade the ZP starting just after fertilization and proceeding until hatching using enzymes that are released from cortical granules or produced by the developing embryo. Accordingly, the developing embryo might provide ZP-degradation products including oligosaccharide chains to the immune system from the very early stages. Taken together, we propose here a novel hypothesis that these ZP-derivatives can act as an intrinsic signal from the developing embryo for maternal recognition by the immune system.
Journal of Reproductive Immunology 07/2009; 81(1):1-8. · 2.97 Impact Factor
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Hiroshi Fujiwara
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ABSTRACT: In early pregnancy, human chorionic gonadotrophin (HCG) stimulates the corpus luteum (CL) of pregnancy to produce progesterone, which in turn maintains human embryo implantation in the uterus. In addition to this embryo-maternal cross-talk via the endocrine systems through blood circulation, accumulating evidence suggests that circulating blood cells also play an important role in embryo implantation. Peripheral blood mononuclear cells (PBMC) derived from pregnant women increased the progesterone production by luteal cells and promoted the invasion of embryos in vitro. Recombinant-HCG increased chemokine production by PBMC through lectin-glycan interaction and enhanced the effects of PBMC on embryo invasion. Later, it was shown that not only PBMC, but also circulating platelets were possible sources of these chemokines that promote extravillous trophoblast invasion to reconstruct maternal endometrial artery. Circulating platelets were also proposed to induce neovascularization during CL formation. Furthermore, intrauterine administration of autologous PBMC effectively improved live birth, pregnancy and implantation rates in patients with repeated (four or more) implantation failures during in vitro fertilization therapy. These findings suggest that circulating blood cells positively contribute to maternal tissue remodeling and embryo-maternal cross-talk around the implantation period in cooperation with the endocrine system.
Molecular Human Reproduction 05/2009; 15(6):335-43. · 3.85 Impact Factor
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ABSTRACT: To investigate the physiological characteristics of the corpus luteum (CL) of pregnancy, we raised a mAb, human corpus luteum (HCL)-4, against human luteal cells obtained from CL of pregnancy. The affinity-purified antigen from human CL of pregnancy or placenta using HCL-4 was a 61 kDa protein. The partial amino acid sequence of the antigenic protein was identical to that of human monoamine oxidase A (MAOA, EC1.4.3.4). MAOA has been shown to catabolize catecholamines that were reported to regulate luteal function in CL and vasoconstriction in various organs. Immunohistochemistry using HCL-4 mAb showed that MAOA was intensely expressed on large luteal cells and moderately expressed on small luteal cells in the CL of pregnancy. In the CL of menstrual cycle, MAOA was weakly detected on large luteal cells but not detected at all on small luteal cells. Western blotting analysis confirmed the high expression of MAOA in CL of pregnancy. Northern blot analysis also showed the expression of MAOA mRNA in human CL, and showed that its expression was higher in CL of pregnancy than in CL of menstrual cycle. The increased expression of MAOA in the CL of pregnancy suggests the contribution of MAOA to the function of the CL of pregnancy.
Reproduction 06/2008; 136(3):367-75. · 2.58 Impact Factor
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ABSTRACT: Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/APQ contains the HEXXHX(18)E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/APQ is a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo-maternal interface.
Journal of Biological Chemistry 08/2007; 282(28):20088-96. · 4.77 Impact Factor
