Masahiko Takemura

Expression and localization of human oxytocin receptor mRNA and its protein in chorion and decidua during parturition.

Oxytocin (OT) is widely used to induce labor in the clinical setting. However, its physiological role in normal human parturition remains unclear. We demonstrated the enhanced expression of OT receptor (OTR) mRNA in chorio-decidual tissue, using the polymerase chain reaction after the reverse transcriptase reaction (RT-PCR) and Northern blot analysis. OTR gene expression in chorio-decidual tissue increased fivefold during the course of parturition. In situ hybridization of fetal membrane revealed the expression of OTR mRNA in maternally derived decidual cells. The OTR mRNA was also detected in fetally derived chorionic trophoblast cells. Immunohistochemistry, using a newly developed anti-OTR monoclonal antibody, demonstrated the distribution of OTR protein in fetal membrane. The distribution pattern of OTR protein and OTR mRNA was identical, indicating that the regulation of OTR expression occurs mainly at the transcriptional level. These results support the idea that the expression of decidual OTR regulates the initiation and amplification of labor. The implications of these findings with regard to the pathogenesis of preterm labor are also discussed.

Elevated Nitric Oxide Concentration in the Seminal Plasma of Infertile Males: Nitric Oxide Inhibits Sperm Motility

PROBLEM: To evaluate the “effect of nitric oxide in the seminal plasma on sperm motility. METHOD: Seminal plasma concentrations of NO2—, a stable end product of nitric oxide, of 108 males of infertile couples and 15 proven fertile donors were measured and compared with spermatogram parameters. Motile sperm was incubated with a nitric oxide‐generating drug, sodium nitroprusside, for 6 hr in the absence or presence of oxyhemoglobin, an inhibitor of nitric oxide.

Detection of oxidative stress in seminal plasma and fractionated sperm from subfertile male patients

OBJECTIVES Oxidative stress in the reproductive system is thought to affect the fertilizing ability of sperm. Since 8-hydroxydeoxyguanosine (8-OHdG) and lipid peroxides are widely used as markers to quantify oxidative stress, we compared 8-OHdG and lipid peroxide concentrations in seminal plasma and spermatozoa from subfertile and fertile men. STUDY DESIGN Semen obtained from 37 men of subfertile couples (21 men with normozoospermia and 16 with asthenozoospermia) and from eight fertile volunteers were examined. Seminal plasma and spermatozoa were fractionated by four-step discontinuous Percoll gradient centrifugation. 8-OHdG in seminal plasma was measured by ELISA, and lipid peroxides in seminal plasma and spermatozoa were determined using a thiobarbituric acid (TBA) assay. RESULTS The concentrations of 8-OHdG and lipid peroxides in the seminal plasma of the subfertile group were significantly higher than those of the fertile group. There were no significant differences in these values between patients with normozoospermia and asthenozoospermia. In all four fractions obtained by Percoll gradient fractionation, the lipid peroxide levels in spermatozoa recovered from subfertile males were significantly higher than those of fertile controls. CONCLUSIONS Seminal plasma and spermatozoa from subfertile males showed elevated levels of oxidative stress that were detectable in ejaculated semen specimens by ELISA or TBA assay. Even the spermatozoa fraction considered to be mature and normal showed elevated oxidative stress in the subfertile group. Our results confirm the importance of oxidative stress in male reproductive function, and could be applied for the selection of patients for antioxidant therapy.

NF‐κB Activation at Implantation Window of the Mouse Uterus

Problem:  Nuclear factor kappa B (NF‐κB) is one candidate transcriptional modulator, which might regulate many kinds of molecules that play sequential roles at implantation in the endometrium. However, temporal and spatial activation of NF‐κB at implantation window is unknown.

Triplet pregnancy involving complete hydatidiform mole andtwo fetuses: Genetic analysis by deoxyribonucleic acid fingerprint

A case of a triplet pregnancy involving a dizygous twin pregnancy and a complete hydatidiform mole after therapy with human menopausal gonadotropin and human chorionic gonadotropin is reported. Two female fetuses, two placentas in one mass with two amnions and two chorions, and a tumor mass with a grapelike appearance were spontaneously delivered at 19 weeks of gestation. The deoxyribonucleic acid fingerprints of the two placentas and tumor tissue were compared with those of the parents. The fingerprints of the placentas showed patterns different from each other; however, all their polymorphic fragments could be traced back to either the father or mother. All polymorphic fragments of the tumor tissue were inherited only from the father (androgenesis). These results indicated that this triplet pregnancy involved a dizygous twin pregnancy and a complete hydatidiform mole.

Estimation by an electrophysiological method of the expression of oxytocin receptor mRNA in human myometrium during pregnancy

In order to evaluate the changes in uterine oxytocin receptor-specific mRNA during pregnancy, receptor expression in Xenopus oocytes are examined electrophysiologically following microinjection of mRNA from human uterus. In voltage-clamped oocytes injected with term myometrial mRNA, oxytocin elicited an inward current response. The amplitude of the oxytocin-induced current increased with increasing dose of oxytocin, but no current was elicited following stimulation with vasopressin. The oxytocin-induced current was completely eliminated as a result of pretreatment with a specific oxytocin antagonist. 21 of 27 oocytes injected with term myometrial mRNA showed a large amplitude (77.0 +/- 16.1 nA) reaction to oxytocin. In comparison, only 3 of 13 oocytes injected with early gestational myometrial mRNA exhibited a small amplitude (4.6 +/- 1.4 nA) reaction to oxytocin. No oxytocin response was observed in oocytes injected with non-pregnant myometrial mRNA. These results indicate that the striking increment in oxytocin sensitivity in term uterus depends on the increase in mRNA encoding oxytocin receptors.

Type-IV collagenase and tissue inhibitor of metalloproteinase in ovarian cancer tissues

Objective: We examined the specific expression of gelatinase/type‐IV collagenase and tissue inhibitor of metalloproteinase (TIMP) in clinical ovarian cancer tissue. Methods: Molecular weight‐specific gelatinase/type‐IV collagenase activity was examined by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis in which substrate was included (zymography). The expression of TIMP mRNA was examined by Northern blot analysis. Results: Zymography revealed that in ovarian cancer the activity of a 92‐kDa gelatinase/type‐IV collagenase was always greater than that of a 64‐kDa gelatinase/type‐IV collagenase in contrast to the situation in the normal ovary. Northern blot analysis revealed no remarkable difference of TIMP mRNA expression between cancer and normal ovarian tissues. Conclusions: These results indicate that the higher activity of the 92‐kDa gelatinase/type‐IV collagenase enzyme, relative to that of the 64‐kDa enzyme, is involved in the malignant phenotype of ovarian cancer, while the inhibitor of these enzymes, TIMP, is distributed in a widespread fashion in the tissue, and its levels are not correlated with the malignancy.

Malignant cell‐specific gelatinase activity in human endometrial carcinoma

Background. The protease activity leading to degradation of the extracellular matrix was compared between human endometrial cancer and normal uterine endometrium.

Rapid detection of trisomy 21 by gene dosage analysis using quantitative real‐time polymerase chain reaction

Aim:  Rapid detection of fetal aneuploidy helps inform a mother’s choice about the course of her pregnancy. Obtaining results by fluorescent in situ hybridization (FISH) requires more than 24 h, and thus a more rapid method is needed.

The gene expressions of macrophage colony-stimulating factor (MCSF) and MCSF receptor in the human myometrium during pregnancy: regulation by sex steroid hormones.

We investigated the biological effect of sex-steroid hormones, secreted from the corpus luteum and placenta, on the induction of mRNA encoding macrophage colony-stimulating factor (MCSF) and c-fms proto-oncogene (MCSF receptor) in the human uterine myometrium. Poly(A)+RNA was extracted from the myometrium of pregnant and non-pregnant uterine myometrium and then Northern blot analysis was performed on poly(A)+RNA. The myometrium of non-pregnant women expressed neither mRNA of macrophage colony-stimulating factor (MCSF) nor any transcript related to the c-fms proto-oncogene. On the other hand the myometrium of pregnant women expressed MCSF mRNA (4.7 kb) and two kinds of transcript related to the c-fms proto-oncogene (3.9 and 1.3 kb). The mRNAs of both MCSF and c-fms proto-oncogene were induced in the uterine myometrium of non-pregnant women under pseudopregnant therapy of mestranol and norethindrone. These results indicate that sex steroid hormone secreted from the corpus luteum of pregnancy and/or placenta may be deeply involved in the hypertrophic change of uterus during pregnancy by inducing MCSF and MCSF receptor (c-fms proto-oncogene protein product) in the myometrium.