Oncostatin M is produced during pregnancy by decidual cells and stimulates the release of HCG.

Oncostatin M (OSM) is a member of the interleukin-6 superfamily and a multifunctional cytokine that effects the growth and differentiation of many different cell types. OSM concentrations in the sera of pregnant women were found to be significantly higher than those of non-pregnant women. Western blot analysis revealed that the OSM protein was present in the decidua and chorionic tissue in each trimester. Throughout pregnancy, the amount of the OSM protein in the decidua was larger than that in the chorionic tissue. Immunohistochemistry using an anti-OSM monoclonal antibody demonstrated that OSM was mainly localized in the decidual glands and stroma. OSM transcripts in the decidua and the chorionic tissue were detected during each trimester by reverse transcription-polymerase chain reaction (RT-PCR). The regulation of human chorionic gonadotrophin (HCG) release by the placenta in first trimester stimulated with recombinant OSM was also investigated. Stimulation of the placenta by OSM augmented HCG release in a time- and dose-dependent manner. HCG release induced by recombinant human OSM was completely blocked by antibodies against OSM and the signal transducer, gp130, but only partially inhibited by antibodies against the leukaemia inhibiting factor (LIF) receptor. These results suggest that OSM molecules produced by decidual glands and stromal cells during pregnancy have an important role in placental endocrine function.

Endometrial development was improved by transdermal estradiol in patients treated with clomiphene citrate.

The objective of this study was to examine the effect of transdermal estrogen therapy on the endometrial thickness and serum hormone levels in anovulatory patients treated with clomiphene citrate (CC). There was a significant difference in endometrial thickness between the CC + transdermal estrogen group and the CC only group from day -2 to day +2. Serum estradiol (E2) levels in the CC + transdermal estrogen group were significantly higher than those in the CC only group on day -2 and day 0. Our results support that addition of transdermal E2 to the treatment protocol of the women treated with CC elicited a favorable response of the endometrium.

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.

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.

Expression and immunolocalization of the oxytocin receptor in human lactating and non-lactating mammary glands.

The milk ejection reflex is mediated by the release of pituitary oxytocin and its interaction with specific receptors within the mammary gland. Although up-regulation of the oxytocin receptor during lactation has been shown for the rat mammary gland by ligand binding assay, investigation of the receptor expression in human breast at the molecular level has not yet been carried out in detail. Here we report the expression and immunolocalization of the oxytocin receptor in the human breast. It appears that the expression level of the receptor-specific mRNA is not significantly elevated during lactation and the protein remains at a relatively low level. However, this lack of increase may be only a dilution effect because of the high level of milk protein expression. Immunohistochemistry and immunoelectron microscopy using three anti-oxytocin receptor antibodies raised against different epitopes of the receptor indicated the presence of receptor immunoreactivity only to a very limited extent in the myoepithelial cells; more specific expression appeared to occur in the ductal/glandular epithelium in both the non-lactating as well as lactating breast. This finding was also confirmed in a New World monkey, the common marmoset (Callithrix jacchus). These results suggest that, at least for human and marmoset, in addition to--or even instead of--myoid cells, the ductal/glandular epithelium is also a target for oxytocin action, not only during lactation but also in the non-lactating breast. Thus, there may be other physiological effects of oxytocin besides direct myoid cell contraction in the breast.

Lipocalin-type prostaglandin D synthase in human male reproductive organs and seminal plasma.

Prostaglandin D synthase (PGDS) activity was detected in human seminal plasma (0.05-1.83 nmol/min per milligram protein). The enzyme was purified from human seminal plasma by immunoaffinity chromatography and found to be 27 kDa in size and N-glycosylated, similar to PGDS in the cerebrospinal fluid. The N-terminal amino acid sequence of 16 residues of the seminal enzyme, APEAQVSVQPNFQQDK, was identical to that of the cerebrospinal fluid PGDS. Although PGDS activity and the content determined by the immunoassay each highly varied in the seminal plasma, the concentration was significantly (p < 0.001) lower in the oligozoospermic group (2.47 +/- 0.51 microg/ml) than in the normozoospermic group (9.75 +/- 1.49 microg/ml). Prostaglandin (PG) D2 was detected in the seminal plasma (5.00 +/- 0.65 ng/ml) with a positive correlation to the PGDS concentration (p < 0.05). PGD2 was converted to the J series of PGs in the seminal plasma with a half-life of 6.5 h. Northern blot analysis revealed that mRNA for PGDS was expressed in the testis, prostate, and epididymis. Through immunohistochemistry, PGDS was localized in Leydig cells of the testis and in epithelial cells of the prostate and ductus epididymidis.

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.

Increased interleukin-1 and interleukin-1 receptor antagonist levels in cervical mucus in the ovulatory phase in comparison with the follicular phase.

Interleukin-1 (IL-1) receptor antagonist (IL-1ra) levels in the cervical mucus of women in the ovulatory phase are significantly higher than those in the follicular phase. IL-1 titers of women in the ovulatory phase are also significantly higher than those in the follicular phase. A positive correlation between IL-1ra and IL-1 levels in the cervical mucus was observed. Immunohistochemistry using an anti-IL-1ra monoclonal antibody revealed positive staining in the epithelial cells of the endocervix. These results suggest that IL-1ra from cervical epithelial cells protects the reproductive system from the toxicity of IL-1 produced in the endocervix.

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.

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.

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.

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. RESULTS: The NO2- concentration in the seminal plasma was 6.58 +/- 0.5.6 microM in 26 infertile males with leukocytospermia, 5.51 +/- 0.25 microM in 82 infertile males without leukocytospermia, and 3.91 +/- 0.16 microM in 15 controls. There was a significant correlation between the NO2- concentration and sperm motility. Sodium nitroprusside reduced the sperm motility in a dose- and time-dependent manner and its reduction was completely inhibited by the addition of oxyhemoglobin. CONCLUSION: These findings indicate that nitric oxide concentration in the seminal plasma of infertile males is elevated and that nitric oxide is an inhibitor of sperm motion.

Normal spatial learning despite regional inhibition of LTP in mice lacking Thy-1.

The process of learning involves stable changes in synaptic efficacy for which long-term potentiation (LTP) provides a widely adopted mammalian model. Synaptic modification induced by learning or LTP may involve the action of cell adhesion molecules. One such candidate is the ubiquitous neuronal glycoprotein Thy-1. In mice in which the gene encoding Thy-1 has been inactivated, we find a regionally selective impairment of LTP in vivo in the hippocampal formation: LTP is normal in area CA1 but strongly inhibited in the dentate gyrus. Spatial learning by Thy-1-deficient mice, as assessed in the watermaze, is unimpaired. Thus LTP in the cortical input to the dentate gyrus seems not to be required for spatial learning.

Maintenance of imprinting of the insulin-like growth factor II gene (IGF2) and the small nuclear ribonucleoprotein polypeptide N gene (SNRPN) in the human uterus and leiomyoma.

The insulin-like growth factor II gene (IGF2) is thought to be involved in the growth of uterine smooth muscle tumors. We studied the allele-specific expression of IGF2 in 20 patients with uterine leiomyomas by analyzing restriction fragment length polymorphisms (RFLP), because IGF2 is a maternally imprinted gene and only the paternal allele is exclusively expressed in human somatic tissue. We also studied the allelic expression of the small nuclear ribonucleoprotein polypeptide N gene (SNRPN), which is reportedly maternally imprinted in humans, and compared the imprinting status with that of IGF2. Nine patients (45%) were heterozygous at the ApaI site of IGF2, nine (45%) were heterozygous at the possible AccII polymorphic site of SNRPN, and three (15%) showed polymorphism in both genes. The genomic DNA of 15 patients showed heterozygosity in either or both of these genes, and the mRNA of these was expressed monoallelically in myometrial tissues and leiomyomas of these patients. These results demonstrated that IGF2 and SNRPN imprinting is completely maintained in human uteri and leiomyomas and that increased expression of IGF2 is not due to biallelic expression.

Plasma nitric oxide levels in pregnant patients with preeclampsia and essential hypertension.

Nitric oxide (NO) production may be an important causal factor in hypertensive disorders during pregnancy. The plasma concentrations of NO2-(+) NO3-, stable metabolites of NO, were measured in 70 nonpregnant women, 323 normotensive pregnant women, 23 pregnant patients with preeclampsia, and 7 pregnant patients with essential hypertension. The normotensive women had higher plasma concentrations (30.0 +/- 0.6 mumol/l) than nonpregnant women (18.3 +/- 1.0 mumol/l; p < 0.0001). The plasma concentrations in the patients with preeclampsia (45.6 +/- 2.3 mumol/l) were higher than in the normotensive women (30.3 +/- 1.0 mumol/l; p < 0.0001) and were correlated with the systolic blood pressure (r = 0.442; p < 0.05). However, pregnant patients with underlying essential hypertension had significantly lower plasma concentrations (19.1 +/- 3.0 mumol/l; p < 0.005). These findings suggest that NO contributes to maternal vasodilation, the maintenance of uterine quiescence, and the pathogenesis and clinical features of hypertensive disorders during pregnancy.

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.

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.

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.