Toshikatsu Nobunaga

Clonal determination of uterine leiomyomas by analyzing differential inactivation of the X-chromosome-linked phosphoglycerokinase gene

To investigate the clonality of uterine leiomyomas, we developed a PCR-based method involving the differential inactivation of the X-chromosome-linked phosphoglycerokinase (PGK) gene. Small DNA samples of 22 leiomyomas from 9 Japanese patients, showing heterozygosity at the BstXI site of the PGK gene, were digested with the methylation-sensitive restriction enzyme HpaII. Only the inactive (methylated) PGK gene allele was selectively amplified by PCR followed by digestion with BstXI and electrophoresis. All leiomyoma samples consisted of a single type of inactive allele, even though alleles were detected that were specific to each nodule. The results indicated that all leiomyoma nodules were unicellular in origin but independently generated in the uterus.

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.

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.

Zygosity determination of multiple pregnancy by deoxyribonucleic acid fingerprints

We used a new method of deoxyribonucleic acid analysis to determine zygosity in multiple pregnancies. This method uses a minisatellite core probe, requires only a small amount of deoxyribonucleic acid, and detects the restriction fragment length polymorphisms that are a result of allelic differences in the number of tandem repeats that contain the core sequence. Southern blot hybridization showed an individual-specific deoxyribonucleic acid fingerprint and each polymorphic band in the sibling could be identified within one (but not both) of the parents. Identical deoxyribonucleic acid fingerprints among the siblings of multiple pregnancy indicate they must be monozygotic. This method is sufficiently reliable and rapid so the determination of zygosity in multiple pregnancy can be made the same day the fetal deoxyribonucleic acid is made available.

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.

BIPARENTAL ALLELES OF HLA-G ARE CO-DOMINANTLY EXPRESSED IN THE PLACENTA

SummaryHLA-G is the only major histocompatibility complex molecule expressed in the human placenta and thus has been considered to be necessary for maintenance of pregnancy. We investigated whether HLA-G expression is regulated in a parent-of-origin allele-specific manner. Of six first trimester and three third trimester placentas, three first trimester and two third trimester placentas showed heterozygosity at the PstI polymorphic site in the 3′-untranslated region. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed biallelic expression of HLA-G in all the informative cases, indicating that HLA-G is not imprinted during the gestational period, at least at the transcriptional level. As HLA-G has been postulated to be polymorphic not only at the DNA sequence level but also at the peptide level, co-dominant expression of the gene suggests that each parental allele is involved in the allogenic response during pregnancy.

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.