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ObjectivePulmonary oedema is recognised as a severe side effect of ritodrine hydrochloride. Recently, the number of twin pregnancies has been increasing. Few studies have reported the association between total dose of ritodrine hydrochloride prior to delivery and pulmonary oedema in twin pregnancy. We aimed to examine this association and determine the optimal cut-off threshold of total ritodrine hydrochloride dose to predict the incidence of pulmonary oedema in twin pregnancy based on obstetric records.DesignRetrospective cohort study.SettingYamanashi Prefectural Central Hospital, Japan.ParticipantsTwo hundred and twenty-six women with twin pregnancy who delivered at Yamanashi Prefectural Central Hospital between September 2009 and November 2016.MethodsThe obstetric records of the participants were analysed. We defined 1 unit of ritodrine hydrochloride as 72 mg per 24 hours continuous transfusion at 50 µg/min to calculate the dose of ritodrine used for tocolysis.Outcome measuresMultivariable logistic regression analysis was performed to examine the association between total dose of ritodrine hydrochloride used for threatened preterm labour and pulmonary oedema, while controlling for potential confounding factors. Then, a receiver–operating characteristic curve was used to determine the optimal cut-off of total ritodrine dose to predict pulmonary oedema incidence.ResultsMean maternal age was 32 (range, 18–46) years; 143 participants were nulliparous (63.3%), 109 had (48.2%) term deliveries and 194 (85.8%) had caesarean deliveries. The overall incidence of pulmonary oedema was 13.7% (31/226). Multivariable analysis showed that the total dose of ritodrine was significantly associated with pulmonary oedema (adjusted OR 1.02; 95% CI 1.004 to 1.03). The best cut-off point to predict the incidence of pulmonary oedema was 26 units (1872 mg) (sensitivity, 61.3%; specificity, 87.8%).ConclusionOur results suggest that consideration of the total dose of ritodrine hydrochloride is helpful in the management of patients with threatened preterm labour in twin pregnancy.

We analyzed the gene expression of sperm flagellum-movement associated proteins, namely, adenylate kinase domain containing protein (RGD1303144) and outer dense fiber protein 1. These gene expressions were up-regulated in a maturing rat testis, and RGD1303144 gene was expressed dominantly in spermatocytes. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin administration reduced sperm motility, these gene expressions were not affected.

Mitochondria play a central role to provide ATP for fertilization and preimplantation embryo development in the ooplasm. The mitochondrial dysfunction of oocyte has been proposed as one of the causes of high levels of developmental retardation and arrest that occur in preimplantation embryos generated using Assisted Reproductive Technology. Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been applied to infertility due to dysfunctional ooplasm, with resulting pregnancies and births. However, neither the efficacy nor safety of this procedure has been appropriately investigated. In order to improve embryogenesis, we observed the mitochondrial distribution in ooplasma under the several conditions using mitochondrial GFP-transgenic mice (mtGFP-tg mice) in which the mitochondria are visualized by GFP. In this report, we will present our research about the mitochondrial distribution in ooplasm during early embryogenesis and the fate of injected donor mitochondria after CT using mtGFP-tg mice. The mitochondria in ooplasm from the germinal vesicle stage to the morula stage were accumulated in the perinuclear region. The mitochondria of the mtGFP-tg mouse oocyte transferred into the wild type mouse embryo could be observed until the blastocysts stage, suggesting that the mtGFP-tg mice oocyte is very useful for visual observation of the mitochondrial distribution in the oocyte, and that the aberrant early developmental competences due to the oocyte mitochondrial dysfunction may be overcome by transferring the "normal" mitochondria.

The present study used immunohistochemical staining and in situ hybridization (ISH) to examine whether progression of cervical intraepithelial neoplasia, grade 2 (CIN 2) can be predicted by p16INK4a immunoexpression and high-risk human papilloma virus (HPV) ISH signal types. We studied 52 cases histologically diagnosed with CIN 2: dysplasia regressed in 28 cases; 13 cases progressed to CIN 3; and CIN 2 persisted in 11 cases. Expression of p16INK4a and high-risk HPV signal both related to grade of CIN. Stronger p16INK4a immunoexpression and a higher frequency of expression of a punctate nuclear signal were observed in CIN 2 lesions before progression compared with those before regression. CIN 2 cases in which moderate to strong immunoexpression of p16INK4a and a punctate signal were observed simultaneously progressed to CIN 3 in 10 (91%) of 11 cases. CIN 2 cases with moderate to strong immunoexpression of p16INK4a and a high-risk HPV punctate signal should be treated because of the great risk of progression.

Many temporarily functioning proteins are generated during the replacement of nucleoproteins in the nuclei of late spermatids and seem to be degraded in the nucleus. This study was designed to clarify the involvement of the ubiquitin-proteasome degradation system in the nucleus of rat developing spermatids. Thus, we studied the nuclear distribution of polyubiquitinated proteins (pUP) and proteasome in spermiogenic cells and sperm using postembedding immunoelectron microscopy. We divided the nuclear area of late spermatids into two regions: (1) a dense area composed of condensed chromatin and (2) a nuclear pocket in the neck region. The latter was located in the caudal nuclear region and was surrounded by redundant nuclear envelope. We demonstrated the presence of pUP in the dense area and nuclear pocket, proteasome in the nuclear pocket, and clear spots in the dense area of rat spermatids. Using quantitative analysis of immunogold labeling, we found that fluctuation of pUP and proteasome levels in late spermatogenesis was mostly synchronized with disappearance of histones and transitional proteins reported previously. In the nuclei of human sperm, pUP was detected in the dense area, whereas proteasome was in the nuclear vacuoles and clear spots. These results strongly suggest that pUP occur in the dense nuclear area of developing spermatids and that the ubiquitin-proteasome system is more actively operational in the nuclear pocket than dense area. Thus, the nuclear pocket might be the degradation site for temporarily functioning proteins generating during condensation of chromatin in late spermatids.

We report a rare case of extranodal NK/T-cell lymphoma, nasal type of the uterine cervix that showed cytologic features mimicking cervical cancer. A 65-year-old woman presented with vaginal bleeding. Gynecological examination revealed a bulky tumor of the cervix. A conventional Papanicolaou-stained cervical smear showed hypercellularity consisting of numerous variably sized cohesive clusters that mimicked epithelial tumors, with a necrotic and inflammatory background. A small number of individually scattered cells were also identified. These scattered cells showed pleomorphic, often cleaved, or horseshoe-shaped nuclei and pale cytoplasm. Biopsy specimens revealed a diffuse growth of atypical cells with an angiocentric pattern. Extensive necrosis and infiltration of inflammatory cells were present. There were numerous mitotic figures. The tumor cells were positive for CD45RO, CD3ε, CD56, granzyme B, TIA-1, CD7, and Epstein-Barr virus (EBV)-encoded small RNA (EBER) by in situ hybridization, and negative for cytokeratin, chromogranin A, synaptophysin, CD4, CD5, CD8, CD20, and CD30. Based on these findings, this tumor was diagnosed as extranodal NK/T-cell lymphoma, nasal type of the uterine cervix.

Human papillomavirus (HPV)-negative adenocarcinoma (AC) is a minor subset of endocervical cancer, but its pathogenesis has yet to be elucidated. This study investigated the clinicopathologic features of HPV-negative endocervical AC (n=14) in comparison with HPV-positive endocervical AC (n=30), and further studied aberrations of cell-cycle regulators. Expression patterns of cyclin-dependent kinase inhibitors (p16, p14, p27, and p21) and p53 were evaluated immunohistochemically, and nuclear high-risk HPV DNA signals were detected by in situ hybridization and polymerase chain reaction. Immunoexpression of p16, p14, p27, p21, and p53 were observed in 90%, 67%, 77%, 40%, and 20% of HPV-positive ACs, and in 0%, 0%, 29%, 14%, and 57% of HPV-negative ACs, respectively. A higher frequency of lymph node metastasis and worse prognosis were significantly associated with HPV-negative AC. Our findings suggest that alteration of cyclin-dependent kinase inhibitors and p53 status may contribute to carcinogenesis and the clinical behavior of HPV-negative AC of the uterine cervix.

There is no consensus opinion regarding a possible relation between low 50-g glucose challenge test (GCT) results and small-for-gestational-age infants (SGA). This study aimed to clarify the relation between low 50-g GCT results and SGA, after adjusting for potential confounding factors, using a large clinical dataset.Our retrospective cohort study evaluated the obstetric records of women who delivered at Kofu Municipal Hospital (Japan) between January 2011 and December 2013. The exclusion criteria were gestational diabetes mellitus, diabetes in pregnancy, and GCT results of ≥140 mg/dL. Based on these criteria, we included 1603 women who had undergone a GCT between 24 and 28 weeks of gestation, and divided the subjects into a low-GCT group (≤90 mg/dL) and a non-low-GCT group (91-139 mg/dL). The χ(2) -test and multivariate logistic regression analysis were used to investigate the association between low GCT results and SGA.The mean subject age was 30.4 ± 5.4 years, with 45.1% nulliparity (n = 723) and 1538 (96.0%) term deliveries. The incidence of SGA was 10.7% (172/1603), and 17.9% (287/1603) of the women had low GCT results. Low GCT results were significantly associated with an increased risk of SGA (odds ratio, 1.66; 95% confidence interval, 1.14-2.42), after controlling for maternal age, pre-pregnancy maternal weight, maternal weight gain during pregnancy, and pregnancy-induced hypertension.It appears that there is a significant association between low GCT results and SGA. However, further studies are needed to explore the detailed mechanisms of this association.

We investigated the localization of several markers for lysosomes and aggresomes in the chromatoid bodies (CBs) by immunoelectron microscopy. We found so-called aggresomal markers such as Hsp70 and ubiquitin in the core of the CBs and vimentin and proteasome subunit around the CBs. Ubiquitin-conjugating enzyme (E2) was also found in the CBs. In tubulovesicular structures surrounding the CBs, lysosomal markers were detected but an endoplasmic reticulum retention signal (KDEL) was not. Moreover, proteins located in each subcellular compartment, including the cytosol, mitochondria, and nucleus, were detected in the CBs. Signals for cytochrome oxidase I (COXI) coded on mitochondrial DNA were also found in the CBs. Quantitative analysis of labeling density showed that all proteins examined were concentrated in the CBs to some extent. These results show that the CBs have some aggresomal features, suggesting that they are not a synthetic site as proposed previously but a degradation site where unnecessary DNA, RNA, and proteins are digested.

Background, Aims and Methods Clomiphene citrate (CC) has been widely used for induction of ovulation; however, despite the high rate of ovulation, the pregnancy rate is only 30%. The anti-estrogenic effect of CC on the endometrium is one explanation for this finding. It is well known that repeated administration of CC enhances its anti-estrogenic effect. To overcome this adverse affect, a number of techniques have been used. One technique is the early administration of CC, in an attempt to decrease the anti-estrogenic effects of CC on the endometrium. The aim of the present study was to retrospectively evaluate if repeated administration of CC at varying times may affect the endometrium during preovulatory period; pregnancy rates were compiled for patients with unexplained infertility undergoing intrauterine insemination (IUI). The patients were divided into four groups based on the number of repeat administrations and the time of CC administration.

In the summer of 2002, standard guidelines for the application of assisted reproductive technology were reported by a research group of the Ministry of Health, Labor and Welfare. The present study aimed to examine the relationship between the number of cycles of artificial insemination and the cumulative pregnancy rates according to the cause of infertility. Patients who experienced their first cycle of artificial insemination during the period of January 1999-December 2002 were included in the study and were divided into a male factor infertility group and an idiopathic infertility group. Cumulative pregnancy rates resulting from artificial insemination with the husband’s semen were calculated by the life-table approach. During the study period, 139 couples entered the assisted reproduction program and underwent 581 cycles. Significant differences were observed in cumulative pregnancy rates between the two groups. It is recommended that couples with male factor infertility and who fail to conceive within six or seven cycles of intrauterine insemination, consider a modification of treatment strategy such asin vitro fertilization, because cumulative pregnancy rates of this group were reached at a plateau within six or seven cycles. In contrast, patients with idiopathic infertility, the cumulative pregnancy rates appeared to increase constantly with each subsequent cycle. It is important to consider modifications of treatment strategy in the light of the cause of infertility.

We studied temporal changes in the subcellular localization and levels of a moonlighting protein, phospholipid hydroperoxide glutathione peroxidase (PHGPx), in spermatogenic cells and mature sperm of the rat by immunofluorescence and immunoelectron microscopy. The PHGPx signals were detected in chromatoid bodies, clear nucleoplasm, mitochondria-associated material, mitochondrial aggregates, granulated bodies, and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes as previously reported. Within mitochondria, PHGPx moved from the matrix to the outermost membrane region in step 19 spermatid, suggesting that this spatiotemporal change is synchronized with the functional change of PHGPx in mitochondria. In the nucleus, PHGPx was associated with electron-lucent spots and with the nuclear envelope, and PHGPx in the latter region increased after step 16. In early pachytene spermatids, PHGPx signals were noted in the nuclear material exhibiting a very similar density to chromatoid bodies and in the intermitochondrial cement, supporting the previous proposal that chromatoid bodies originate from the nucleus and intermitochondrial cement. The presence of PHGPx in such various compartments suggested versatile roles for this protein in spermatogenesis. Quantitative immunoelectron microscopic analysis also revealed dynamic changes in the labeling density of PHGPx in different subcellular compartments as follows: 1). Total cellular PHGPx rapidly increased after step 5 and reached a maximum at step 18; 2). mitochondrial labeling density increased after step 1 and achieved a maximum in steps 15-17; 3). nuclear labeling density suddenly increased in steps 12-14 to a maximum; 4). in cytoplasmic matrix, the density remained low in all steps; and 5). the labeling density in chromatoid bodies gradually decreased from pachytene spermatocytes to spermatids at step 18. These spatiotemporal changes in the level of PHGPx during the differentiation of spermatogenic cells to sperm infer that PHGPx plays a diverse and important biological role in spermatogenesis.

The open reading frames of human sex steroid hormone receptors (hSSHRs) are composed of eight exons. In addition, the presence of various exons - including 5'-untranslated exons, alternative coding exons and novel 'intronic' exons - has been demonstrated in the genes encoding hSSHRs. The isoform/variant hSSHR mRNAs generated from thes e exons can be tentatively classified into seven types. In type 1, different mRNAs are generated with the use of alternative transcription start sites. In type 2, one or more exons are skipped. In type 3, one or more exons are duplicated. In type 4, distinct mRNAs containing different 5'-untranslated exon(s) are synthesized. In type 5, distinct mRNAs possessing different coding exon(s) are generated. In type 6, mRNA is synthesized by intronic exons and coding exons 4/5-8. In type 7, mRNA with insertion of intronic exon(s) is generated. Here, we review the isoform/variant hSSHR mRNAs and the structure of the genes encoding them.

In order to analyze the structures of the 5'-untranslated region of estrogen receptor alpha (ER alpha) mRNA in human uterine endometrium (Em), total RNA from Em was analyzed by 5'-rapid amplification of the cDNA ends method with antisense primer located on exon 1 of human ER alpha gene. Three isoforms of 5'-RACE clones were obtained: ER alpha mRNAs containing exon (A) (the upstream region of exon 1), exon C, and exons F-E2 (we adopted the nomenclature of 5'-untranslated exons of the Gannon group). The results imply that the major isoforms of ER alpha mRNA expressed in Em are these three isoforms. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis was carried out on Em, ovary (Ov) and liver (Li) mRNAs to detect the novel isoforms of ER alpha mRNA in these tissues, using sense primers located on exons (A), B, C, F, and E1, and antisense primer located on exon 1. As a result, in addition to the previously reported ER alpha mRNA isoforms containing exons (A), B, C, F-E2 and E1-E2 on exon 1, we identified two novel isoform mRNAs in which exons F and E1 were directly spliced onto exon 1. Differential distributions of these isoforms of ER alpha mRNAs in Em, Ov and Li were demonstrated by RT-PCR-Southern blot analysis. These results, together with the previous reports by others, indicate that there are at least ten isoforms of ER alpha mRNA containing different 5'-untranslated regions, exons (A), B, C, D, T1-T2, T1, F-E2, F, E1-E2 and E1, expressed in human, and that these are involved in tissue specific expression of the gene.

The multiple untranslated first exons and promoters system has been reported to be involved in the tissue-specific expression of the estrogen receptor alpha (ERalpha) in humans and rats. However, a few reports are available concerning tissue-specific regulation of the expression of the estrogen receptor beta (ERbeta) gene. To investigate the mechanism regulating the expression of the rat ERbeta gene, we analyzed the structure of the 5'-untranslated region (UTR) of the rat testicular ERbeta mRNA using 5'-rapid amplification of the cDNA ends (5'-RACE) method. Sequence analysis revealed the presence of two isoforms of the ERbeta mRNA containing distinct 5'-UTRs. Although the 5'-UTR of one isoform of the messages was identical to the 5'-UTR of the previously reported ERbeta cDNA, the other isoform had a novel sequence in its 5'-UTR. Genomic analysis revealed that the 5'-UTRs of these two mRNA isoforms originated from two distinct untranslated first exons, the previously identified exon termed "exon 0N," and the novel exon we termed "exon 0H," both of which were spliced onto exon 1. We termed these isoforms of the messages containing the exon 0N and exon 0H, the ERbeta mRNA (0N-1) and ERbeta mRNA (0H-1), respectively. Furthermore, the distributions of these mRNA isoforms in several rat tissues were analyzed using the reverse transcription-polymerase chain reaction (RT-PCR) method. The distributions of the two mRNA isoforms differed; the ERbeta mRNA (0N-1) was widely distributed in the tissues examined, while expression of the ERbeta mRNA (0H-1) was restricted to a few tissues such as the anterior pituitary, amygdala, and some peripheral tissues. In conclusion, our findings indicate that the tissue-specific expression of the rat ERbeta gene is regulated, at least in part, by the multiple untranslated first exons system which consists of exon 0N and exon 0H.

HER-2 /neu is a 185-kDa glycoprotein and a transmembrane receptor with tyrosine kinase activity. Its overexpression is observed in 25–30% of primary breast carcinomas and is associated with a poor clinical prognosis. Recently, the U.S. Food and Drug Administration and the Japanese Ministry of Health, Welfare, and Labor approved the use of trastuzumab (Herceptin, Genentech, South San Francisco, CA) for the treatment of patients with metastatic breast carcinomas overexpressing HER-2 /neu. Results of clinical trials with Herceptin suggest that it may prolong the survival of patients with advanced metastatic breast carcinoma. Relatively little is known concerning the relationship between HER-2 /neu status and ovarian clear cell carcinoma. If HER-2 /neu overexpression status were demonstrable in ovarian clear cell carcinoma and a clinical correlation between overexpression and prognosis could be established, a rationale for clinical use of Herceptin for this tumor could be established. Our aim was to evaluate HER-2 /neu status in ovarian clear cell carcinomas. Fifteen ovarian clear cell carcinoma cases were immunostained for HER-2 /neu using HercepTest (DAKO, Glostrup, Denmark). Overexpression of HER-2 /neu was detected in only one case. Unlike in breast carcinoma, HER-2 /neu overexpression appeared to be uncommon in ovarian clear cell carcinomas. Herceptin may thus target only a small proportion of ovarian clear cell carcinomas and be of limited clinical value for treatment of this carcinoma.

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.

Background and Aims Nuclear proteins in mature mammalian spermatozoa nuclei are oxidized to form numerous disulfide bonds. Reduced glutathione (GSH) in the oocyte has been linked to spermatozoan nuclear decondensation after fertilization. In this study, we analyzed whether GSH reduced protamines in sperm nuclei in vitro, and examined the zona-binding ability of treated nuclei.

In eukaryotic cells, mitochondria are the major site of ATP production, which is achieved through the electron-transport chain and oxidative phosphorylation, according to the energy demand. Mitochondria contain their own genome (mitochondrial DNA, mtDNA) on which a limited number of genes are encoded. In the human sperm, mitochondria helically wrap the midpiece of the tail and supply the energy for the driving force of motility While various mutations in mtDNA in somatic cells are found to be associated with a wide spectrum of diseases, it is also reported that the abnormal mtDNA causes astenozoospermia and male infertility. At fertilization, the paternal mitochondria and mtDNA are rapidly degraded early in embryogenesis, thus, only maternal mtDNA is transmitted to the descendant. We briefly review here the basic characteristics of mtDNA and its maternal transmission during fertilization, as well as male infertility.

In order to clone the novel isoform of the cDNA for the human estrogen receptor-alpha (ERalpha), the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human ERalpha cDNA. As a result, a novel isoform of the ERalpha cDNA (termed the ERalpha isoform S cDNA), which consists of a previously unidentified 5'-sequence and the exons 4-8 of the ERalpha gene, has been cloned. The structure of the ERalpha isoform S cDNA is essentially similar to that of the progesterone receptor (PR) isoform S cDNA that was identified in our recent report. Analysis of the genomic DNA revealed that the 5'-sequence of the ERalpha isoform S mRNA originated from a novel exon (termed the exon S). Moreover, the reverse transcription-polymerase chain reaction (RT-PCR) was carried out using the primers specific to the ERalpha isoform S mRNA on the total RNA from the human spermatozoon (Sp), liver (Li), uterine endometrium (Em) and myometrium (Mm). The ERalpha isoform S mRNA was detected in the uterine Em and Sp. Moreover, the molecular size of the ERalpha isoform S encoded by the ERalpha isoform S mRNA, which was analyzed by the transfection of the expression vector with ERalpha isoform S cDNA into the 293T cell, was approximately 39kDa. It was indicated that the one of the ATGs in the exon S could be used as the translation initiation codon. This is the first report on the ERalpha mRNA isoform that is not caused by exon-skipping or alternative utilization of the untranslated 5'-exons.

A renal angiomyolipoma (AML) is a rare benign tumour of kidney origin. Pregnancy is known to be associated with an increased risk of tumour rupture causing hypovolaemic shock, which is usually managed surgically or through an embolisation procedure. However, having surgery during pregnancy predisposes the mother to a preterm delivery, and the unknown influences of radiation exposure to the fetus make the management of such cases very challenging. A 30-year-old pregnant woman had a sudden onset of gross haematuria at the 20th week of her pregnancy. The MRI showed a 10 cm mass suggestive of AML in the left kidney, with evidence of an intrarenal haematoma. To avoid an iatrogenic preterm delivery and unnecessary fetal exposure to radiation, conservative management was conducted until 34 weeks of gestation, when she came to our hospital reporting of flank pain. An endovascular treatment was performed immediately after an emergency caesarean delivery.

Fetal cell microchimerism is defined as the persistence of pluripotent fetal cells in the maternal body long after delivery. The exact process by which fetal cells cross the placental barrier and enter maternal circulation is still being investigated. We reported that fetal cells persist only in the maternal bone marrow and may give rise to subpopulations with the ability to differentiate into the tissue-specific mature cells within injured maternal organs. Moreover, most of the fetal cells enter the maternal circulation during the early stages of pregnancy. These results indicate that the fetal cells with a multilineage potential, which were detected in a variety of maternal organs during pregnancy did not pass through the placental barrier; rather, they were derived from the fetal cells that entered maternal circulation early after implantation, and sustained their population long after delivery.

Three different melanocyte-specific mRNAs are studied as potential markers for circulating melanoma cells in the serum of mice inoculated subcutaneously with B16F10 melanoma cells. These three mRNAs encode tyrosinase, tyrosinase related protein-2 (TRP-2) and Pmel17, proteins that are essential for the synthesis of melanin and are expressed specifically in melanocytes. We used reverse-transcription polymerase chain reaction (RT-PCR) to detect these three different melanocyte-specific mRNAs in the sera of B16F10 bearing mice. Since melanocytes would not normally be present in the blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT-PCR detection of all three mRNAs was highly sensitive and specific. Our in vitro studies show that as few as 10 melanoma cells can be detected in 125 microl blood and that in vivo, melanoma cells can be detected in blood samples from B16F10 melanoma bearing mice. Of these three mRNAs, Pmel17 mRNA is the most sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA and TRP-2 mRNA. Moreover, this mouse model might be useful for basic research of malignant melanoma patients with haematogenous metastasis.

Two different melanocyte-specific mRNAs are studied as markers for circulating melanoma cells in vitro using the human melanoma cell line G361 and in vivo using blood samples from Japanese melanoma patients at different clinical stages. These mRNAs encode tyrosinase, the most essential enzyme for melanin synthesis, and gp100, a melanosomal matrix glycoprotein recognized by monoclonal antibody HMB-45. We used reverse-transcription polymerase chain reaction (RT-PCR) to detect tyrosinase mRNA and gp100 mRNA in peripheral blood. Since melanocytes would not normally be present in peripheral blood, the detection of those transcripts should indicate the presence of circulating melanoma cells. RT-PCR detection of these two mRNAs was highly sensitive and specific. Our in vitro study showed that as few as 10 melanoma cells in 0.125 ml normal blood could be detected. In in vivo study, 130 blood samples from 55 melanoma patients gave positive and variably sensitive results, whereas no samples from healthy controls or patients with other cancers gave positive results. Tyrosinase mRNA was not detected in any of the melanoma patients. gp100 mRNA was detected in 12 of 55 melanoma patients, in none of five stage I patients (0%), in four of 26 stage II patients (15.4%), in one of six stage III patients (16.7%) and in seven of 18 stage IV patients (38.9%). Thus gp100 mRNA is a more sensitive marker for detecting circulating melanoma cells compared with tyrosinase mRNA.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenium (Se)-dependent glutathione peroxidase. It is reported that the relative PHGPx mRNA levels are much higher in the testis than in the other tissues. We have analyzed the existence and structure of the PHGPx mRNA in rat sperm and the changes in the level of the PHGPx mRNA after feeding with Se-deficient diets. We used 8-wk-old male Wistar strain rats given Se-adequate feed (control group, n = 5) and Se-deficient diets with marginal levels of Se (0.03 ppm or less) (Se-deficient group, n = 5) for 4 wk. The existence and level of the PHGPx mRNA in the cauda epididymal sperm, testis, and liver from the Se-adequate rats were analyzed by the reverse transcription-polymerase chain reaction and the Southern blotting method. As a result, the existence of the PHGPx mRNA was demonstrated in the cauda epididymal sperm as well as in the testis and liver. Moreover, the subtype of the PHGPx mRNA in the rat sperm was the mitochondrial-type mRNA, which included a region corresponding to the mitochondrial transfer leader sequence. These results imply that the intracellular localization of PHGPx may be regulated by the transcription level. On the other hand, there was no significant difference between the control group and the Se-deficient group in the Se level of the cauda epididymal sperm and the level of the PHGPx mRNA. In conclusion, it has been demonstrated that the PHGPx mRNA exists in rat sperm for the first time. The analysis of the PHGPx mRNA in the sperm would be a useful tool for investigating the disfunction caused by the disorder of the level or structure of the PHGPx in the sperm.

Objective: To evaluate the propriety of oocyte retrieval (OR) from a small leading follicle during a fixed-schedule in vitro fertilization (IVF) program. Methods: OR was fixed only to take place on Wednesdays. Gonadotropin-releasing hormone agonist treatment was initiated on the first day of the cycle, human menopausal gonadotrophin was given for 7 days starting on the next Monday, and human chorionic gonadotrophin (hCG) was given on the Tuesday before the OR. Patients were divided into 2 groups according to the follicular size observed before the day of hCG administration: Group 1 (141 cycles), with follicles > 16 mm in mean diameter; and Group 2 (38 cycles) with follicles of 10 to 16 mm in mean diameter. Results: Fertilization rates and the incidence of mature oocytes were higher in Group 1 than in Group 2. However, the mean number of oocytes recovered and pregnancy rates were similar in the 2 groups. Conclusion: It is worthwhile to retrieve the oocyte from a small leading follicle in a fixed-schedule IVF program.

Presence of alternatively spliced-estrogen receptor (ER) mRNA variants has been revealed in the breast cancer tissues. The ER variants transcribed from these mRNA variants were supposed to cause changes in the estrogen responsiveness of breast cancer. Although uterine endometrial cancer also has an estrogen-dependent profile, these ER mRNA variants have not yet been reported in the tumor. In the present study, we attempted to detect the exon 7 deletion- (del.7-) and exon 5 deletion (del.5) ER mRNA variants in normal human uterine endometrium (hEM) and uterine endometrial cancer tissue (hEC) by the use of reverse transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) with the PCR primers: hE4 (forward), hE6 (reverse), and hE8 (reverse), which were located in exons 4, 6, and 8, respectively. Two major products were generated from RNAs of both hEM and hEC with primers hE4 and hE8. The nucleotide sequence of the longer product was identical to exon 4-8 of human ER cDNA, whereas that of the shorter one completely deleted exon 7. Moreover, when the RT-PCR was done with the primers hE4 and hE6, the shorter product lacking exon 5 was detected with the longer one having the same sequence as exon 4-6 of human ER cDNA. Since the RT-PCR-SB with primers hE4 and hE8 produced a very low or undetectable level of the signals corresponding to del.5 ER mRNA variant, the level of del.7 ER mRNA variant seemed to be higher than that of del.5 ER mRNA variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Microchimeric fetal cells are present in the maternal body over a long period and thought to have the ability to colonize multiple tissues and organs. They are found in a wide range of maternal tissues affected with variety of diseases, thus, there is a possibility that they may contribute tissue repair and regeneration. To assess their possibility of use in regenerative medicine, we investigated whether fetal cells regenerate infracted maternal organs. We crossbred wild female mice with male transgenic mice, expressing enhanced green fluorescent protein (EGFP), and total hysterectomies were performed at the day 6 of pregnancies. On day 60 after the operations, the mice were injected with streptozotocin (STZ) to induce multiple organs injuries. We also created the ischemic organ injury model ; myocardial infarction model and cerebral infarction model. Detection and quantification of fetal cells were then attempted in a variety of maternal organs via a fluorescent microscope and quantitative PCR amplification of the gfp transgene. Fetal cells were detected only in maternal bone marrow before maternal organs injuries were induced, however, they were detected not only in bone marrow but also in the maternal injured organs. Histological analysis showed that differentiated fetal cells were observed and their morphological appearance was indistinguishable from their maternal counterparts. These results indicate that fetal cells sustained their population in the maternal bone marrow, may have contributed to the maternal tissue regeneration.

The successful generation of cloned animals and the establishment of embryonic stem (ES) cell lines from somatic cells suggest that these techniques may be used in human regenerative medicine. However, the fact that oocytes must be donated by women undergoing infertility treatment remains a fundamental ethical objection, as they might be concerned about the potential exploitation of their genome. Here, we investigated the reprogramming potential of enucleated and cryopreserved oocytes for the development of full-term cloned mice. BDF1 strain mouse oocytes were cryopreserved at metaphase II, before and after enucleation. After thawing, cumulus cell nuclei were microinjected to generate clones. Although the rate of development of cloned embryos to the blastocyst stage using the treated oocytes was lower than that obtained using fresh oocytes, three live pups were delivered after embryo transfer into pseudopregnant females (0.4% of the oocytes used). Thus, although cryopreservation reduces the potential of oocytes, these cells retain the ability to support the full-term development of cloned embryos. In addition, the removal of DNA from human oocytes may alleviate the ethical and psychological problems for women who are undergoing infertility treatment and are considering oocyte donation for research or therapeutic purposes.

In an attempt to examine regional synthesis of the progesterone receptor (PR) in the brain, the distribution of mRNA encoding PR was investigated in the female adult rat di- and telencephalon by in situ hybridization using T7 RNA polymerase transcripts of a 320 base pair rat PR cDNA clone. The rat PR cDNA had been partially cloned and sequenced by using the reverse transcription-polymerase chain reaction (RT-PCR) method. The primer set corresponds to a part of the progesterone binding domain of human PR cDNA. Large numbers of strong labeling were observed in the arcuate nucleus, medial preoptic nucleus, and ventrolateral part of the ventromedial nucleus which are relative to sexual behavior. Moderate labeling was found in layers II and IV of the isocortex, in the pyramidal layer of the CA1 and CA3 fields of the hippocampal formation, in the cortical nucleus of the amygdala, in the nucleus of the diagonal band, and in the anterior periventricular nucleus. Weak labeling was found in many other regions. These results were largely in agreement with the distribution of PR previously reported by ligand binding assay and autoradiographic studies. This present in situ hybridization study may provide a useful tool for the analysis of the regional regulation of PR synthesis in the rat brain.

Reverse transcription-polymerase chain reaction (RT-PCR)-Southern blotting was employed for biochemical detection and measurement of the estrogen receptor messenger ribonucleic acid (ERmRNA) in various parts of the Wistar strain female rat brain. Total RNA extracted from the tissues was subjected to RT-PCR using the primer set which flanked the part(287bp) of the estrogen binding domain-corresponding region of the rat ERcDNA. It was confirmed that the RT-PCR product corresponded to the part of the ERcDNA by direct nucleotide sequencing of the product. Moreover, the level of ERmRNA could be determined by Southern blotting which was highly sensitive and produced quantitative results. The RT-PCR product of about 290 bp, corresponding in length to the distance between two primers, was generated from RNA of all the tissues examined. The levels of the product were as follows; anterior hypophysis greater than hypothalamus and preoptic area, amygdala much greater than cerebral cortex, cerebellum. These results indicate that ERmRNA is widely distributed in the whole brain, implying some physiological action of estrogen on target and 'non-target' brain regions.

In order to examine sex steroid hormone receptor expression in the central and peripheral tissues relative to reproduction, we have analysed estrogen (ER) and progestin receptors (PR) and their mRNAs in rat brain, and human uterine and other tissues, using immunocytochemical assay (ICA), reverse transcrip-tion-polymerase chain reaction (RT-PCR) and in situ hybridization technique (ISH) Summary and conclusion are as follows;1) ICA studies confirmed nuclear localization of ER or PR in human uterus and ovarian endometriosis. Good correlation exists between the ER-ICA and ligand binding assay (LH20 assay) or ER-enzyme immunoassay (EIA), indicating the usefulness of the ER-ICA for the semiquantitative measurement in humanuterine tissues. The ER score of ovarian endometriotic tissue was much less than that of the normal endometrium, suggesting much lower hormonal responsiveness in the pathologic tissue.2) ERmRNA analysis:(1) Northern blot analysis using rat ERcRNA probe demonstrated 6.6 kb ERmRNA in rat brain (the hypothalamus-preoptic area, HPOA), anterior hypophysis (AP), uterus, tube, ovary, and testis. Additional subspecies of 4.2, 2.6 and 2.0 kb were identified in the testis.(2) A very sensitive and specific RT-PCR assay for ERmRNA was developed to analyse the receptor ex-pression in more detail. The RT-PCR product 287 bp was generated from tissue RNA using the primer set derived from the rat ERcDNA sequence and its authenticity was confirmed by direct sequencing. Quan-tification of the RT-PCR assay was made possible by measurement of the hybridization signals in a Bio-Im-age Analysing System, BAS-2000. A standard curve was obtained from graded dilutions of the uterine RT-PCR products.The levels of ERmRNA were as follows; AP>HPOA, amygdala (AMY) >cerebral cortex (CC), cerebellum (Ce). The existence of a small amount of ERmRNA in the “non-target” CC and Ce implied adirect action of estrogen on these brain regions.3) PRmRNA analysis:In analogous experiments as ERmRNA, a very sensitive and specific RT-PCR assay for PRmRNA was also developed to detect and quantify PRcDNA in the tissues. Since rat PRcDNA has been not cloned and sequenced, rat PRmRNA was synthesized with the primer set derived from the human PRcDNA sequence. The RT-PCR product of 320 bp was generated from tissue RNA, and the authenticity of the rat uterine product was confirmed by direct sequencing.The levels of PRmRNA in adult rat brain were as follows; AP, HPOA, AMY, CC>Ce, with smaller differential distribution than that of ERmRNA, and roughly paralled the levels of PR with exception of the CC where the PR level is low. The expression mechanism of PR in the CC may be different from that in the other brain regions.The early postnatal development of PRmRNA was found. The increment of the cerebral PRmRNA might be associated with the early drastic increase of PR in the neonatal rat brain cortex.4) ER-and PRmRNAs analysis in human uterus:(1) Primary structure of the uterine ERmRNA, amplified by the RT-PCR and determined by direct sequencing of the product, showed that ERmRNA expressed in the normal human endometrium has Gly-400 (GGG) and Thr-594 (ACA).(2) Northern blot analysis of ER-and PRmRNAs using riboprobes demonstrated greater message levels in the endometrium than the myometrium, respectively. The results were in agreement with estrogendependent induction of PR.5) In situ hybridization studies.We have detected ER- and PRmRNAs and mapped out the distribution of the meassages in the intact female rat brain using rat ER- and PRcRNA synthetized. The distribution of both mRNAs was similar, with higher density in the arcuate nucleus and the ventrolateral part of the ventromedial nucleus of hypothalamus, indicating a possible coexpression in the same neurons.

It has been demonstrated that several messenger RNA (mRNA) isoforms have been transcribed from the alpha-fetoprotein (AFP) gene. In rats, it was reported that the novel exon, termed the exon V, exists between the exons 7 and 8, and the novel mRNA isoform (termed AFP-V mRNA) is synthesized using the exon V. In this study, a reverse transcription-polymerase chain reaction was performed and quantitative analysis was done on the AFP mRNA to identify the exon V and the AFP-V mRNA in humans. As a result, 2 novel exons, the exons VA and VB, were identified. Furthermore, 3 novel AFP mRNAs, the AFP-V1, -V2, and -V3 mRNA, were demonstrated to be expressed through alternative splicing. Expression of the AFP-V2 mRNA isoform and the wild-type mRNA was differentially regulated, implying that the AFP-V mRNA isoforms could be used in diagnosis and classification of hepatocellular carcinoma and ovarian carcinoma.

The present study evaluated the clinical impact of preoperative screening for gastric mucin in cervical discharge, using a latex agglutination test with HIK1083, a monoclonal antibody against gastric mucin. HIK1083-labeled latex agglutination tests were performed preoperatively using cervical secretions from 44 patients (group 1) with profuse watery vaginal discharge, multiple cervical cysts on vaginal ultrasonography, or yellowish mucin on a Papanicolaou smear and from 31 patients (group 2) with none of these clinical signs. The results were positive in 26 patients in group 1 and in no patients in group 2. Glandular lesions with a gastric phenotype were identified histologically in all 26 cases positive for the HIK1083 latex test, such as minimal deviation adenocarcinoma, lobular endocervical glandular hyperplasia (LEGH), and pyloric gland metaplasia, but not in negative cases. In 2 cases of LEGH, adenocarcinoma in situ was identified. Screening of gastric mucin in cervical discharge may facilitate preoperative detection of some early cervical adenocarcinoma.

Selection of good quality oocytes is important for improvement of assisted reproductive technology. Here, we studied the relationship of the mitochondrial distribution in metaphase II stage (MII) oocytes with fertility, since mitochondria in ooplasm are essential for energy production required for fertilization and embryo development. To observe mitochondria non-invasively, we used oocytes from a transgenic mouse, in which enhanced green fluorescent protein is targeted to the mitochondrial matrix and thus fluorescence is observed exclusively in the mitochondria. Control oocytes with mitochondria distributed around the nucleus showed normal embryo developmental competence, whereas oocytes with abnormal diffuse and fragmented mitochondria showed a significantly lower rate of embryo development after activation by intracytoplasmic sperm injection or strontium, which is a very effective agent for activation of mouse oocytes. Also, we showed that the reduced developmental competence of oocytes with diffuse and fragmented mitochondria caused by vitrification and thawing is similar to that of oocytes with abnormal mitochondrial foci obtained naturally. These findings suggest that abnormal mitochondrial distribution in oocytes at MII is a cause of developmental retardation and therefore normal mitochondrial distribution could be used as a criterion for selection of good oocytes.

In rare cases, patients with heterozygous hemophilia show bleeding tendency due to a low factor VIII activity of

Recently, we have isolated the untranslated first exon ‘exon 0N’ of the rat estrogen receptor (ER) gene from the liver by the use of the 5′-rapid amplification of cDNA end (5′-RACE) method. To investigate the existence of other untranslated first exon(s), we further analyzed the 5t'-unsaturated region (UTR) of ER mRNA in the rat liver in this study. Total RNA from the livers of 8-week-old male Wistar rats was subjected to 5′-RACE with the antisense primers located in exon 1 of the rat ER gene. The inserts of four clones (clones 3, 4, 7 and 8) were sequenced. The nucleotide sequences of the clones revealed the existence of a previously unidentified untranslated first exon (we termed it ‘exon 0S’) which was spliced onto exon 1 of the rat ER mRNA. The distribution of ER mRNA containing ‘exonn 0S’ (ER mRNA (0S-1)) in several brain regions and various peripheral tissues of 8-week-old male and female Wistar rats was further analyzed by the use of the reverse transcription-polymerase chain reaction. ER mRNA (0S-1) was found to be widely distributed in the rat brain and peripheral tissues. The distribution of the message was different from that of ER mRNA containing exon 0 (the first reported 5′-UTR form of rat ER mRNA) or of ER mRNA with exon 0N which was reported in our recent report. These results indicate that (1) ‘exon 0S’ is a novel untranslated first exon of the rat ER gene, (2) rat ER mRNAs possess at least three forms of 5′-UTRs which are exon 0, exon 0N, and exon 0S, (3) the tissue specific expression of ER is regulated, at least in part, by the usage of differential promoters in the rat.

Alpha-fetoprotein (AFP) is a major fetal serum protein, the biologic role of which has not been not fully elucidated. Recently, existence of a novel AFP mRNA isoform (del.1 AFP mRNA isoform), which is transcribed from the intron A (the intron between exons 1 and 2), has been reported in murine yolk sac and fetal liver. In the present study, we intended to identify the human homologue of the murine AFP mRNA isoform in the yolk sac tumor.To investigate the existence of the mRNA isoform (which we termed the "AFP-C mRNA isoform"), reverse transcription-polymerase chain reaction (RT-PCR) was used. Moreover, the expression analysis of the AFP-C cDNA isoform using the AFP-negative human cell line was carried out.RT-PCR revealed the existence of the AFP-C mRNA isoform in the yolk sac tumor and human hepatocellular carcinoma cells. The expression analysis clarified that the molecular size of the AFP-C was approximately 65 kd, and that the protein was not secreted, in contrast to the traditional AFP.From these results, the existence of the AFP-C mRNA isoform has been demonstrated for the first time in humans. The AFP-C located in cytoplasm possibly plays physiologic/pathogenic roles distinct from those of the traditional AFP in the yolk sac tumor and hepatocellular carcinoma.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4–5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6–8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9–11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.

Cytoplasmic transfer (CT) from a donor to a recipient oocyte has been used clinically to facilitate human pregnancies. Data reported here describe the first characterization of CT coincident with intracytoplasmic sperm injection in the mouse system. Sibling oocytes were used to transfer 2, 4, or 6 pl of ooplasm to a recipient egg along with a sperm head using piezo-actuated injection. Survival and fertilization after CT were comparable to controls at 2 pl and 4 pl, but survival was significantly reduced with 6 pl volumes. Development to the blastocyst stage was also inversely related to CT volume, with some decline beginning with the 4 pl CT group. However, some blastocysts did develop in all of the groups. The results are in contrast with human eggs, which tolerate larger CT volumes. Results indicate that the mouse system can be used to characterize the transfer of exogenous materials concomitant with sperm injection, provided that the CT volume is not excessive.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. The majority of patients are sporadic cases, while 5–10% of the patients have a family history of ALS (fALS). Mutations in the gene that encodes cytoplasmic Cu/Zn superoxide dismutase (SOD1) have been identified in about 25% of fALS cases. Although the precise pathogenesis of ALS is still unknown, experimental studies including animal models suggest that fALS is caused by the toxic gain-of-function of the SOD1 mutant. We have analyzed not only SOD1 gene mutation by genomic sequencing, but also SOD1 mutant protein by liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS). We analyzed 33 fALS patients and found 10 mutations in SOD1 gene, in which two were novel: Asp101His substitution in exon 4 and Gly141Glu substitution in exon 5. Here, we present their mass spectrometric protein analyses and clinical features.

Case reportA 29 year old woman attended at our clinic complainingof secondary amenorrhoea. She was found to have anandrogenic adult granulosa cell tumour without androgenicmanifestations. Signs of virilisation (increased facial orabdominal hair, hirsutism or clitoromegaly) were absent.Concentrations of luteinising hormone, follicle stimulatinghormone, prolactin, dehydroepiandesterone sulphate, dehy-droepiandesterone and cortisol were all within theirreference ranges. The patient was initially treated withclomiphene citrate to induce menstruation; however, whenshe did not respond, she was treated with monophasic oralcontraceptive pills. The amenorrhoea persisted for the nextthree months; a pelvic ultrasound scan revealed a rightadnexal mass measuring 7.5 6.4 4.8 cm, with cysticand solid components. Her plasma testosterone waselevated to 192 ng/dL (normal range: 10–85 ng/dL);however, her plasma oestradiol was within normal limitsat 41 pg/mL (normal range: 12–49 pg/mL). Her activatedpartial thromboplastin time (APTT) was 76.1 s (normalrange: 29.7–47.4 s). Her prothrombin time, clotting factorsVIII, IX, XI, XII, fibrinogen, lupus anticoagulant, glycopro-tein 1 complex anti-cardiolipin h2 and tests of fibrinolysiswere normal. Plasma factor VIII procoagulant activitiesand plasma factor IX procoagulant activities were normal.The mixing test using 25%, 50%, and 75% normal pooledplasma did not completely correct the elevated APTT(56, 45.5 and 37 s). The possibility of a mild coagulationfactor inhibitor was considered; however, she did notexhibit thrombotic or haemorrhagic phenomena.The woman underwent a wedge resection of the leftovary and right salpingo-oophorectomy. The right ovariantumour measured 7.0 cm in its greatest diameter. No othergross evidence of disease was noted. Microscopic exam-ination of the right ovarian tumour revealed the typicalfeatures of an adult granulosa cell tumour. It was composedof thin, fibrous septa separating sheets of small cellscontaining nuclei with prominent grooving. OccasionalCall–Exner bodies were present (Fig. 1). The granulosacells in the tumour were strongly positive for testosterone(Fig. 2). Peritoneal washings were negative for tumourcells. On the first post-operative day, the APTT was inAB

In our recent reports, the novel isoform cDNAs of the ER alpha (ER alpha isoform S cDNA), ER beta (ER beta isoform M cDNA) and PR (PR isoform S and PR isoform T cDNAs) have been identified. These isoform cDNAs contained the previously unidentified 5'-sequences on exons 4-8 (ER alpha isoform S cDNA), exons 5-8 (ER beta isoform M cDNA) or exons 4-8 (PR isoform S and PR isoform T cDNAs). The genomic DNA analysis revealed that the 5'-sequences were derived from the novel independent exons, the ER alpha exon S, ER beta exon M, PR exon S and PR exon T, respectively. Furthermore, the existence of the novel variant mRNA, termed the i45 PR mRNA variant, with the insertion of the previously unidentified exons, termed the exons i45a and i45b, has been demonstrated by the reverse transcription-polymerase chain reaction on the RNA of the human uterine endometrium. From these results, we have concluded that the genes for the human female sex steroid hormone receptors contain the novel intronic exons, that the novel isoform mRNAs are transcribed using the intronic exon and exons 4-8 (or exons 5-8) of the gene, and that the novel variant mRNA is generated by the insertion of the intronic exons in the PR. In the present communication, our recent data along with others on the novel isoform/variant mRNAs for the human female sex steroid hormone receptors will be summarized.

The presence of human progesterone receptor (PR) mRNA variants has been demonstrated in uterine endometrium and breast tissues as well as in cancer cells of these tissues. While exon deletions by the alternative splicing in these variants have been reported, there are few reports available on the PR mRNA variants with exon insertion. In the present study, we attempted to detect a PR mRNA variant containing the exon insertions in normal uterine endometrium. Endometrial tissues were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with PCR primers which were located in exons 3 and 8. Analysis of the RT-PCR products revealed the presence of a novel PR mRNA variant which contained a 232 bp inserted nucleotide sequence between exons 4 and 5. We termed this transcript the "i45 PR mRNA variant". Genomic analysis indicated that the inserted sequence was derived from two novel independent exons of 123 bp and 109 bp, termed "exon i45a" and "exon i45b", respectively, which are located between exons 4 and 5 of the human PR gene. The i45 PR mRNA variant was further detected in uterine endometrial cancer tissues as well as in the normal uterine endometrium. These results demonstrate the presence of a novel PR mRNA variant with exon insertions in the human tissue for the first time. The i45 PR variant protein, possibly transcribed from this i45 PR mRNA variant, may play physiological and/or pathological roles in the human uterine endometrium.