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.

Studies on the pathogenesis of choriocarcinoma by analysis of restriction fragment length polymorphisms.

The association of complete hydatidiform mole with choriocarcinoma has long been recognized, but it is unknown whether the pathogenesis of the two are identical. We investigated the pathogenesis of these trophoblastic tumors by analyzing restriction fragment length polymorphisms using a minisatellite DNA probe to choriocarcinoma, the complete mole, and normal trophoblasts as well as the parental cells. The polymorphic fragments of the complete mole were all transmitted from the paternal DNA, but some polymorphic fragments of the paternal DNA were not recognized in the complete mole. This confirms at a molecular level the androgenetic origin of the complete mole. In some cases of choriocarcinoma, the pattern of inheritance of restriction fragment length polymorphisms was the same as that in the complete mole, whereas in others all the polymorphic fragments in tumor tissues were identical to those in the host DNA. These results suggest that the pathogenesis of choriocarcinoma varies, being completely different from that of the complete hydatidiform mole in some cases.

A molecular basis for species differences in Thy-1 expression patterns.

Thy-1 is a membrane glycoprotein that displays species-specific differences in its pattern of expression. Although it is expressed on thymocytes and splenocytes in mice, it is only expressed on thymocytes in rats. Based on previous studies suggesting that the third intron of the mouse Thy-1 gene is required for its expression in thymocytes, in vivo footprinting analysis was performed on the third introns of both the mouse and rat Thy-1 genes, and led to the identification of homologous 36 bp "footprinted" regions. The mouse 36 bp region was found to be capable of specifically binding an Ets-1-like nuclear factor present in both mouse thymocytes and splenocytes. In contrast, the homologous 36 bp region of the rat which differs from the mouse 36 bp region by three nucleotides resulting in the loss of the Ets-1 binding site, is unable to bind a similar Ets-1-like factor present in rat thymocytes. Instead, this region of the rat third intron binds another nuclear factor which is present in rat thymocytes but not splenocytes. These observations suggest that the differential expression of the mouse and rat Thy-1 genes in thymocytes and splenocytes is the result of differential expression of nuclear factors that bind to this 36 bp region.

Steroid hormones induce macrophage colony-stimulating factor (MCSF) and MCSF receptor mRNAs in the human endometrium.

We investigated the biological effects of sex-steroid hormones, secreted from the corpus luteum and placenta, on the induction of mRNAs encoding macrophage colony-stimulating factor (MCSF) and c-fms proto-oncogene (MCSF receptor) in human endometrium. RNA was extracted from the placenta and endometrium of both pregnant and non-pregnant women, and Northern blot analysis was performed on poly(A)+ RNA using MCSF or c-fms proto-oncogene cDNA as the probe. Results showed: (1) that MCSF mRNA was expressed in the placenta and endometrium of the pregnant uterus, (2) that c-fms proto-oncogene mRNA was also expressed in the placenta and endometrium of the pregnant uterus, and (3) that exogenous sex-steroid hormones could induce the expression of MCSF and c-fms proto-oncogene mRNAs in the endometrium of non-pregnant women. These results indicate that sex-steroid hormones secreted by the corpus luteum and/or placenta influence endometrial and placental growth and differentiation via a mechanism of action involving local production of MCSF and its receptor.

Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-beta subunit: influence of mutation on hCG production.

The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the alpha and beta subunits. The beta subunit is specific for each hormone and is responsible for the biological specificity, but the beta subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different beta subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-beta subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the beta subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.

A new approach using DNA fingerprinting for the determination of androgenesis as a cause of hydatidiform mole.

A new method of DNA analysis has been used for the determination of androgenesis as a cause of complete hydatidiform mole. This method, using a minisatellite core probe, requires only a small amount of DNA and detects the restriction fragment length polymorphisms (RFLPs) due to allelic differences in the number of tandem repeats containing the core sequence. Southern blot hybridization showed an individual-specific DNA fingerprint, and each polymorphic band in molar tissues could be identified as being of paternal, but not maternal, origin. Some polymorphic bands of paternal DNA were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole. This method is sufficiently reliable and rapid that differential diagnosis could be made between complete hydatidiform mole, partial mole and hydropic change.

Structure and expression of the mouse oxytocin receptor gene.

To determine the structure of the mouse oxytocin receptor (OTR) gene, we have screened a mouse genomic library and confirmed cDNA sequence with rapid amplification of cDNA ends. Southern blot using human OTR cDNA probes indicated that the mouse genome has a single copy of the gene. The predicted amino acid sequence is 91% identical to human OTR. The gene contains 4 exons and 3 introns. Exons 1 and 2 contain the 5' untranslated region, with exons 3 and 4 encoding the amino acids of the receptor. Intron 3 interrupts the coding at the same location, after transmembrane domain 6, as in OTR genes of other species. The promoter region lacks an apparent TATA box but contains multiple putative interleukin-response elements and estrogen responsive elements. Expression of OTR gene in the uterus during pregnancy reached maximum at day 20 gestation. The information obtained from the mouse OTR gene facilitates further comparative and biochemical analysis for protein structure-function relationships and gene regulatory mechanisms.

Molecular characterization of a cloned human oxytocin receptor.

We describe here the binding and functional properties of a cloned human oxytocin receptor (OTR). We established a transient OTR expression system on COS-1 cells, which do not express vasopressin receptors. With the transfected cells and [3H]oxytocin, the dissociation constant (Kd) of OTR to oxytocin was 6.0 +/- 1.1 nmol/l; the binding properties of several oxytocin-related peptides were also examined. The functional properties of OTR were determined by an electrophysiological method, using a Xenopus laevis oocyte injected with in vitro transcribed OTR mRNA. These two methods showed that [Phe2,Orn8]vasotocin, a vasopressin agonist, was an OTR antagonist. A combination of these methods using cloned OTR cDNA is a novel and effective method for the investigation of oxytocin-related ligands.

Loss of H19 imprinting and up-regulation of H19 and SNRPN in a case with malignant mixed Müllerian tumor of the uterus.

In several human cancers, it has been recently reported that abnormally altered status of genomic imprinting is related to oncogenesis. In this study, we investigated the expression of three imprinted genes in a case with malignant mixed Müllerian tumor of the uterus (MMMT). In the tumor, expression of H19 showed marked upregulation (6.3-fold) with biallelic expression compared with that in the corresponding normal myometrium. The 5'-promoter region of H19 was hypomethylated in the tumor, whereas it was hemimethylated in the myometrium. Expression of the small nuclear ribonucleoprotein polypeptide N gene (SNRPN) was also upregulated by 1.9-fold. However, the insulin-like growth factor II gene (IGF2) was expressed at low levels in both myometrium and MMMT. The overexpression of H19 is caused by reactivation of the repressed allele of H19 due to demethylation of CpG islands within its 5'-promoter region. Whether upregulation of SNRPN is caused by its biallelic expression remains undetermined because restriction fragment length polymorphisms (RFLP) sites were not informative in SNRPN and IGF2. In conclusion, H19 and SNRPN may play significant roles in the tumorigenesis of MMMT and H19 may have tumor-promoting activity in addition to its known tumor-suppressing activity, probably depending on the tissue and the local milieu.

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.

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.

Bone reactions around hydroxyapatite-coated implants in ovariectomized rats.

OBJECTIVE: The purpose of this study was to investigate bone reaction after implantation in an estrogen-deficient state by examining the changes in bone reactions within tissue surrounding implants in ovariectomized rats. STUDY DESIGN: Ninety-six 12-week-old female Wistar rats were used in the study; they were divided into 2 groups, an ovariectomized group and a sham-operated group. Hydroxyapatite-coated implants were placed in the proximal metaphyses of the tibiae 21 days after surgery. The tibiae were examined histologically by undecalcified sections at various intervals from 7 to 168 days after surgery. RESULTS: In the cortical bone area of the ovariectomized rats, the procedure did not induce any apparent changes in bone volume around the implant or in bone contact with the implant in comparison with the sham-operated rats. In contrast, both bone volume around the implant and contact of the implant with new bone were significantly decreased in the cancellous bone area in the ovariectomized rats in comparison with the sham-operated rats. CONCLUSIONS: Ovariectomy did not seriously affect bone healing after the placement of implants in cortical bone areas, but it reduced the bone contact ratio and the bone in the cancellous bone area.

Bone reactions to titanium screw implants in ovariectomized animals.

OBJECTIVE: The purpose of this study was to investigate the reactions of bone tissue after the placement of implants into the tibiae of osteopenic model rats. STUDY DESIGN: Commercially pure titanium screw implants were placed in the bilateral proximal tibial metaphyses 168 days after ovariectomy had been performed on 12-week-old female Wistar rats. For control purposes, implants were similarly placed in sham-ovariectomy rats. The healing process was examined histologically by means of undecalcified sections at various intervals from 7 to 168 days after implantation. Through use of an automated imaging analytic system, changes in relative bone mass and implant-bone contact were histomorphometrically evaluated. RESULTS: In the cortical bone area, only a slight difference in bone contact was noted with the implant until 28 days after implantation. However, ovariectomy significantly affected bone contact at 56 days after implantation. The rate of bone contact in the cancellous bone area and the relative bone mass around the implant were significantly lower in the test group than in the control group. CONCLUSIONS: It is considered that a decrease in bone mass causes a reduction in the contact area between implant and bone and may also cause a reduction in the supporting ability of the implant because of thinning of the surrounding bone tissue.

Role of Thy-1 in T cell development.

Tolerance to self proteins is accomplished in part by elimination of autoreactive immature T cells as they develop in the thymus. Although many investigators have studied the cellular interactions that regulate this important process, the specific molecules involved in negative selection are still not well understood. Thy-1 is a glycosyl-phosphatidylinositol-linked protein that is expressed at high levels on immature thymocytes, and recent evidence suggests that it is involved in thymocyte apoptosis. Correspondingly, we have found that Abs to Thy-1 block Ag-dependent thymocyte deletion in an in vitro culture system. Thus, we investigated the role of Thy-1 in T cell development by using Thy-1 -deficient mice containing a TCR transgene specific for a class II MHC-restricted Ag. With this system, the role of Thy-1 in Ag-specific self-restriction and self-tolerance could be analyzed. Thy-1-null mice were found to undergo normal negative selection in three different models: the in vitro culture system, anti-CD3-induced thymocyte deletion in vivo, and Ag-induced thymocyte deletion in vivo. Self-restriction to MHC class II also appeared to occur normally in Thy-1-null mice. These results therefore suggest that Thy-1 is not essential for either self-restriction or self-tolerance to MHC class II-restricted Ags. This finding is discussed in light of recent data regarding the role of other glycosyl-phosphatidylinositol-linked proteins in thymocyte development.

Perinatal infection of human T-lymphotropic virus type I, the etiologic virus of adult T-cell leukemia/lymphoma. DNA amplification of specific human T-lymphotropic virus type I sequences.

A gene amplification technique, polymerase chain reaction, was used to detect human T-lymphotropic virus type I (HTLV-I), the etiologic agent of adult T-cell leukemia/lymphoma, in mononuclear cells in peripheral blood and breast milk of ten HTLV-I carrier gravida. The DNA in umbilical cord blood mononuclear cells of the neonates born to the HTLV-I carrier gravida were also amplified and examined for the possibility of HTLV-I infection via placenta during pregnancy. The HTLV-I sequences were detected both in the peripheral blood and milk of all ten carrier gravida by Southern blot analysis of amplified DNA. However, HTLV-I proviral DNA could not be detected in the cord blood of the carriers' neonates, indicating that transplacental infection of HTLV-I should be rare and that postpartum infection via breast milk is a likely major perinatal transmission route.

Vertical transmission of human T-cell leukemia virus type I (HTLV-I): detection of proviral DNA in HTLV-I carrier gravida.

The seroprevalence rate of human T-cell leukemia virus type I (HTLV-I) in pregnant women in the Osaka district was determined by enzyme-linked immunosorbent assay and Western blot analysis. Twenty-one (1.0%) of 2192 samples tested were positive for both assays and the seropositive parturients were found to be integrated with HTLV-I proviral DNA in their mononuclear cells by a DNA dot blot hybridization assay using HTLV-I DNA probe or by a selective DNA amplification technique using the polymerase chain reaction (PCR). On the other hand, proviral DNA was not detected in cord blood of the neonates born to the carrier mothers, indicating that transplacental infection of HTLV-I during pregnancy could be excluded. The results support the hypothesis that postpartum infection via breast milk plays a significant role among the possible perinatal transmission routes.

Differential diagnosis between complete mole and hydropic abortus by deoxyribonucleic acid fingerprints.

We used a new method of deoxyribonucleic acid fingerprint analysis to obtain the differential diagnosis between complete mole and hydropic abortus. This method with a deoxyribonucleic acid minisatellite probe requires only a small amount of tissue sample and peripheral blood, and presents individual specific restriction fragment length polymorphisms (deoxyribonucleic acid "fingerprints") by simultaneous detection of many hypervariable regions (minisatellite regions) widely dispersed in the human genome. Southern blot hybridization showed that in cases of complete mole, all polymorphic fragments were exclusively inherited from the father. Some of the polymorphic bands of paternal deoxyribonucleic acid were not observed in molar deoxyribonucleic acid. However, in the hydropic abortus, the polymorphic fragments could be traced back to its parent. These results indicate that deoxyribonucleic acid fingerprints could distinguish the abnormal fertilization of complete mole (androgenesis) from the normal fertilization of hydropic abortus by identifying the difference in genetic variations between complete mole and hydropic abortus at the deoxyribonucleic acid level.

Functional expression of human myometrial endothelin receptors in Xenopus laevis oocytes.

We demonstrate the existence of functional endothelin receptors in human uterine myometrium using the Xenopus oocyte expression system. Fifty nanograms of poly(A)+RNA from myometrium was injected into Xenopus laevis oocytes and incubated for 70-80 h. The membrane potential of the oocyte was clamped at -60 mV and membrane current was measured during and after endothelin stimulation. Endothelin-1 elicited a large inward membrane current in the oocytes injected with poly(A)+RNA; endothelin-2 elicited a small current; while endothelin-3 did not induce any membrane current. These results indicate the existence of messenger RNA encoding functional endothelin-1 receptors in human uterine myometrium.

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. METHODS: Conditioned medium from tumor cells and normal endometrial cells was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gel containing gelatin as a substrate. After electrophoresis, the gel was stained with Coomassie blue, and then the enzyme activity, expressed as the zone of dye clearing, was analyzed by densitometry. RESULTS: Densitometric analysis showed that all the endometrial cancers expressed a very high molecular weight enzyme activity (Mr 220,000), which was not detected in medium from normal endometrial cells. The analysis also showed that in endometrial cancer the activity of a Mr 92,000 enzyme was always superior to that of a Mr 64,000 enzyme, which was in contrast to the situation for normal endometrium. CONCLUSIONS: These results indicate that the expression of Mr 220,000 enzyme activity and the higher activity of the Mr 92,000 enzyme than the Mr 64,000 enzyme are involved in the malignant phenotype of native endometrial cancer.