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The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor \g=b\mRNA contents were determined by semiquantitative RT\p=n-\PCR.The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor \g=b\.Furthermore, whereas IGF-I upregulated oestrogen receptor \g=b\ expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor \g=b\.Luteal cells with increased oestrogen receptor \g=b\can respond fully to oestradiol, leading to cell hypertrophy.

Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes. Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes. Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action. Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor [the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)] were found to be highly expressed in corpora lutea throughout pregnancy. In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition. Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide. In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media. Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture. The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones. Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17beta-estradiol; and PRL. Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17beta-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression. In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6. However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum. The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.

In this study, we investigated whether progesterone exerts an intraluteal action despite the lack of progesterone receptors (PR) in the rat corpus luteum and whether progesterone acts through the glucocorticoid receptor (GR) to enhance its own levels by down-regulating the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). We first established that the corpus luteum constitutively expresses the GR throughout pregnancy and after parturition. We also generated a temperature sensitive SV-40 transformed luteal cell line (GG-CL) that expresses the GR and 20alpha-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20alpha-HSD mRNA in both cultured corpora lutea and in the luteal cell line. RU486, a PR/GR antagonist, completely blocked both the progesterone and the dexamethasone mediated inhibition of 20alpha-HSD expression in GG-CL cells. In summary, this report provides the first evidence that despite the absence of the PR in the rat corpus luteum, progesterone can act through the GR to down-regulate the expression of 20alpha-HSD, an enzyme that catabolizes progesterone and reduces progesterone secretion by the corpus luteum.

Background Scant evidence has been reported on the evaluation of quality-of-life (QOL) in patients who had undergone surgical treatment due to pelvic floor prolapse including cystocele. The aim of this study is to evaluate the impact of surgical intervention on patients' QOL before and after surgery. Methods Between 1997 and 2007, 135 patients (median age: 66.6 years) with pelvic floor prolapse including cystocele underwent bladder neck suspension with anterior/posterior colporrhaphy. The follow-up period was 39.6 months. Seventy-two patients (53 %) had urinary incontinence. The cystocele was graded as mild (grade 2), moderate (grade 3), and severe (grade 4) in 35, 60, and 40, respectively, according to the Baden-Walker classification. A urodynamic study was performed in 69 patients (51 %) who had obstructive symptoms with 100 ml or more of postvoid residual urine. Postoperative QOL was longitudinally assessed in 114 patients by scoring three disease-specific items (sensation of vaginal bulging, obstructive symptoms, urinary incontinence), and one overall health-related QOL (HR-QOL), and compared with corresponding baseline scores. Results A longitudinal study demonstrated that a significant improvement in these symptoms was sustained at a median follow-up of 62.2 months. HR-QOL was significantly associated with vitality assessed by SF 36 (p = 0.036). Multivariate analysis revealed that update urinary incontinence, pre-operative HR-QOL was independent prognostic factors for predicting postoperative patient's satisfaction. Conclusions Although surgical repair of pelvic floor prolapse can achieve acceptable results with intermediate-term durability as well as improving the QOL, preoperative patients' HR-QOL may be considered in the decision making process for treatment.

We report a case of uterine angiomyolipoma confirmed with molecular-genetic analysis by fluorescence in situ hybridization (FISH). A 25-year-old nulliparous woman visited Yamaguchi University Hospital with a complaint of lower abdominal pain. Magnetic resonance imaging demonstrated an ill-bordered uterine tumor and exploratory laparotomy revealed a myometrial elastic-soft tumor at the anterior wall of the uterine corpus. Histopathologically, the tumor consisted of fascicles of smooth muscle cells with intermingled adipocytes and small to medium-sized arterial blood vessels surrounded by epithelioid cells of clear cytoplasm. FISH examination revealed chromosome X trisomy, which was comparable to a previously reported molecular-genetic finding of PEComa family tumors including angiomyolipoma. Although the tumor was immunohistochemically negative for HMB-45 antigen, the histological and FISH findings were compatible with angiomyolipoma.

We evaluated the usefulness of cefpodoxime proxetil (CPDX-PR) in the treatment of puerperal infection and obtained the following results. (i) The susceptibilities of 124 clinical isolates from 85 uterine lochia samples were determined. The MIC at which the growth of Streptococcus agalactiae, Escherichia coli, and Bacteroides fragilis isolates was inhibited by 90% (MIC90) was 0.39 micrograms/ml or less. The MIC90 for Staphylococcus aureus was 3.13 micrograms/ml. (ii) Seven puerperal women received 200 mg of CPDX-PR orally. The CPDX concentration in the lochias in the uterine cavity was not statistically different from that in the vagina, suggesting that the vaginal samples, which can be obtained more safely and aseptically, can be substituted for the uterine samples. The CPDX concentration in cubital venous blood reached a peak of 1.61 micrograms/ml at 3 hours after CPDX-PR administration. The CPDX concentration in the lochias gradually increased and reached a peak of 1.20 micrograms/ml in the uterine cavity and 1.27 micrograms/ml in the vagina at 5 h after drug administration and gradually declined thereafter. These results suggest that CPDX-PR, with its good transfer to the lochia and its potent antimicrobial activity, is a promising drug for the prophylactic and therapeutic treatment of puerperal infections caused by susceptible organisms.

Urocortin is a member of the CRF neuropeptide family and has a 43% homology to CRF in amino acid sequence. Urocortin has been found to bind with high affinity to CRF receptors. CRF has been detected in the human ovary and has been demonstrated to suppress ovarian steroidogenesis in vitro. In this study we examined urocortin and CRF receptor expression in normal cycling human ovaries, using immunohistochemistry and RT-PCR. Normal cycling human ovaries were obtained at oophorectomy and hysterectomy from patients who underwent surgery for cervical cancer or myoma uteri. Intense urocortin immunoreactivity was detected in luteinized thecal cells of regressing corpora lutea, in which only luteinized thecal cells have the capacity for steroidogenesis. Immunoreactive urocortin was also detected in luteinized granulosa and thecal cells of functioning corpora lutea, in which both cell components are capable of producing steroids. RT-PCR analyses revealed that messenger ribonucleic acid levels for urocortin, CRF, and CRF receptor type 1 and type 2alpha were significantly higher in the regressing corpus luteum than in the functioning corpus luteum. The spatial and temporal immunolocalization patterns of CRF receptor were similar to those of urocortin. These results suggest that urocortin is locally synthesized in steroidogenic luteal cells and acts on them as an autocrine and/or paracrine regulator of ovarian steroidogenesis, especially during luteal regression.

Serum melatonin concentrations were studied in normal pregnant women and in women with several types of pathologic pregnancies, e.g., twins, preeclampsia or intrauterine growth retardation (IUGR). Blood samples were collected from the maternal antecubital vein at 14:00 hr (daytime) and 02:00 hr (nighttime) during pregnancy, and also from the umbilical vein and artery immediately after delivery. Serum melatonin concentrations were measured by radioimmunoassay. Daytime serum melatonin levels in normal (single fetus; singleton) pregnancies were low. While the levels showed an increasing tendency toward the end of pregnancy, no statistically significant changes occurred. On the other hand, the nighttime serum melatonin levels increased after 24 weeks of gestation, with significantly (P0.01) high levels after 32 weeks; these values decreased to non-pregnant levels on the 2nd day of puerperium. Nighttime serum melatonin levels were significantly (P0.05) higher in twin pregnancies after 28 weeks of gestation than in singleton pregnancies, whereas the patients with severe preeclampsia showed significantly (P0.05) lower serum melatonin levels than the mild preeclampsia or the normal pregnant women after 32 weeks of gestation. Melatonin concentrations in umbilical vessels showed a higher tendency in neonates who were born during at night compared with the other neonates; moreover, those in the umbilical artery were generally higher than those in the umbilical vein. The present results indicate that in humans, the maternal serum melatonin levels show a diurnal rhythm, which increases until the end of pregnancy, reflecting some pathologic states of the feto-placental unit. Fetuses may produce melatonin with a circadian rhythm.

The present study was undertaken to investigate the role of vascular endothelial growth factor (VEGF) in luteal angiogenesis and the regulation of VEGF in the corpus luteum (CL) during mid-pregnancy in rats. Protein concentrations and mRNA levels of VEGF in the CL significantly increased from Day 9 to Day 12 and remained at the same level as Day 12 until Day 15. To study whether estradiol is involved in VEGF expression between Day 12 and Day 15, rats undergoing hypophysectomy-hysterectomy on Day 12 were treated with estradiol until Day 15. Protein concentrations and mRNA levels of VEGF in the CL were significantly decreased by hypophysectomy-hysterectomy, and this inhibitory effect was completely reversed by estradiol treatment. Changes in vascular density in the CL were parallel to those in VEGF expression. To examine whether the effect of estradiol is mediated by VEGF, anti-VEGF antibody was administered to hypophysectomized-hysterectomized rats simultaneously with estradiol. The recovery in the vascular density, CL weight, and serum progesterone concentration caused by estradiol was significantly inhibited by the anti-VEGF antibody treatment. In conclusion, the present study has demonstrated that VEGF contributes to luteal angiogenesis, CL development, and progesterone production during mid-pregnancy in rats and that luteal VEGF expression is increased by estradiol.

To investigate the relationship between apoptosis and the Bcl-2/ Bax system in the human corpus luteum (CL), the frequency of apoptosis and expression of Bcl-2 and Bax were examined in the CL during the menstrual cycle and in early pregnancy. In situ analysis of DNA fragmentation showed that the number of apoptotic cells was much greater in the regressing CL than that in the midluteal phase CL, whereas there were almost no apoptotic cells in the CL of early pregnancy. Immunohistochemistry revealed that Bcl-2 expression was observed in the luteal cells in the midluteal phase and early pregnancy, but not in the regressing CL. In contrast, Bax immunostaining was observed in the regressing CL, but not in the midluteal phase and early pregnancy. bcl-2 messenger ribonucleic acid (mRNA) levels in the CL during the menstrual cycle were highest in the midluteal phase and lowest in the regressing CL. In the CL of early pregnancy, bcl-2 mRNA levels were significantly higher than those in the midluteal phase. In contrast, bax mRNA levels were highest in the regressing CL and remarkably low in the CL of early pregnancy. Western blot analyses revealed that Bcl-2 expression was significantly lower in the regressing CL than in the midluteal phase and early pregnancy, and that Bax expression was, in contrast, significantly higher in the regressing CL than in the midluteal phase and was remarkably low in the CL of early pregnancy. When corpora lutea of the midluteal phase were incubated with hCG, hCG significantly increased the mRNA and protein levels of Bcl-2 and significantly decreased those of Bax. In conclusion, Bcl-2 and Bax may play important roles in the regulation of the life span of the human CL by controlling the rate of apoptosis. hCG may act to prolong the life span of the CL by increasing Bcl-2 expression and decreasing Bax expression when pregnancy occurs.

To investigate the possible role of vascular endothelial growth factor (VEGF) and its receptors in the human corpus luteum (CL), expression of VEGF and its receptors, the fms-like tyrosine kinase and the kinase insert domain-containing region (KDR), was analyzed in the CL during the menstrual cycle and in early pregnancy. Immunohistochemistry revealed that VEGF was localized in luteal cells and both flt-1 and KDR were also localized in luteal cells, in addition to vascular endothelial cells. Messenger RNA (mRNA) expression of VEGF, flt-1, and KDR remained constant in the CL during the luteal phase and was lower in the regression phase. In the pregnant CL, VEGF mRNA expression was higher compared with that in the midluteal phase, and mRNA expression of both flt-1 and KDR was the same as that in the midluteal phase. Western blot analyses revealed that the change in protein expression of VEGF, flt-1, and KDR was similar to that in their mRNA expression. To study the effect of human CG (hCG) on VEGF expression in the CL, corpora lutea obtained from the midluteal phase were incubated with hCG (1 IU/ml) for 6 h. hCG increased the expression of mRNA and protein of VEGF. In conclusion, VEGF and its receptors may play important roles in development and function of the CL, and VEGF may exert a paracrine-autocrine role in regulating luteal function. hCG may act to prolong the life span of the CL by stimulating VEGF expression when pregnancy occurs.

Nitric oxide (NO) is known to be generated from L-arginine and may regulate glomerular filtration, tubular sodium reabsorption, and renin secretion. Impairment of renal function might influence NO production secondary to endothelial dysfunction, decreased NO synthesis and increased activity of arginine analogues inhibiting NO synthase. In this study, we evaluated the effect of L-arginine on the blood pressure and urinary sodium excretion in patients with chronic renal failure. A 300-ml dose of 10% L-arginine solution was administered intravenously over 30 min and blood pressure was monitored every 10 min under basal conditions and for 120 min after infusion. The patients were divided into two groups based on the reduction in mean blood pressure (dMBP) following infusion, namely non-responders (dMBP10 mmHg) and responders (dMBP10 mmHg). Urine and blood samples were collected to determine electrolytes, urinary NO2 + NO3 by the Griess method, urinary cGMP, plasma renin activity (PRA), and the plasma aldosterone concentration (PAC). L-arginine significantly decreased MBP in 8 patients and caused no significant change in 10 patients. Urinary sodium excretion and the NO2 + NO3 level were significantly increased following L-arginine infusion and the increment of fractional excretion of sodium was higher in responders. However, there were no significant changes in PRA, PAC, and cGMP. Our findings suggest that a vasodilator effect of NO induced by L-arginine loading may, at least in part, be associated with increased renal sodium excretion in patients with chronic renal failure.

The superoxide radical and its scavenger, superoxide dismutase (SOD), play important roles in the regulation of corpus luteum function. The present study was undertaken to investigate whether SOD is related to pregnancy-induced maintenance of corpus luteum function. Placentae obtained from rats on Day 12 of pregnancy were incubated for 24 h, and the supernatant was used as placental luteotropins. Pseudopregnant rats were given the placental incubation medium from Day 9 to Day 12 of pseudopregnancy. The treatment significantly increased serum progesterone concentrations on Day 12 of pseudopregnancy. Both activities and mRNA levels of copper-zinc SOD (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in the corpus luteum were also increased on Day 12 of pseudopregnancy. Treating the placental incubation medium with charcoal significantly eliminated the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD expression, but not on Cu,Zn-SOD expression. The inhibitory effect of the charcoal treatment on luteal Mn-SOD expression was reversed by supplementation with testosterone or dihydrotestosterone (DHT), but serum progesterone concentrations were recovered only by DHT. Testosterone or DHT alone had no effect on serum progesterone concentrations and luteal SOD expression. In conclusion, placental luteotropins increased SOD expression in the corpus luteum and stimulated progesterone production, suggesting that SOD is involved in the maintenance of the corpus luteum function by placental luteotropins. In addition, androgen, with other placental luteotropins, acted to stimulate progesterone production and Mn-SOD expression in pseudopregnant rats.

Immature rats receiving equine chorionic gonadotropin (eCG) and human CG (hCG) were used to study the time course changes in nitric oxide synthase (NOS) activity in the ovary during ovulation. To study the role of NO in ovulation, the effects of intrabursal injection of L-N(G)-monomethylarginine (L-NMMA, 125 microg/20 microl/bursa), a NOS inhibitor, on the number of ova shed were also examined. Rats were sacrificed at -48, 0, 3, 6, 9, 12, and 24 h after hCG injection, and the ovaries were collected for the NOS activity assay, Western blotting, NADPH-diaphorase histochemistry and immunohistochemistry. Total NOS and constitutive NOS activities in the ovary increased significantly at 9 h after hCG injection and the values remained high thereafter. Inducible NOS (iNOS) activity was detectable as a small peak at 3 and 6 h after hCG injection. Endothelial NOS (eNOS) protein production increased after hCG injection with a peak at 12 h, whereas iNOS protein production decreased at 12 and 24 h after hCG injection. NADPH-diaphorase positive cells increased at the thecae of growing follicles after hCG injection, appeared at mural granulosa cells before ovulation, and were detected in newly formed corpora lutea, which coincided with the results in eNOS positive cells by immunohistochemistry. L-NMMA given to rats at 5 or 7 h after hCG was most effective in reducing the number of ova shed. These results indicate that the NOS activity and NOS positive cells increased after hCG injection, and that eNOS was likely the main NOS increasing in the ovary during ovulation. It is concluded that NO produced between 5 and 9 h after hCG might play a supportive role in ovulation.

Superoxide radicals are known to inhibit progesterone production by luteal cells and have also been reported to cause apoptosis in various cells. The corpus luteum has an antioxidant enzyme to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu, Zn-SOD). However, it remains unknown how the decrease in intracellular Cu,Zn-SOD activity influences luteal function. This study was therefore undertaken to investigate whether suppression of intracellular Cu,Zn-SOD activity inhibits progesterone production by rat luteal cells and causes apoptosis. To suppress intracellular Cu, Zn-SOD activity, dispersed rat luteal cells were incubated with Cu, Zn-SOD antisense oligonucleotides. The 48-h treatment with antisense oligonucleotides (10 microM) inhibited Cu,Zn-SOD activity by 50% and Cu,Zn-SOD mRNA level by 30%, whereas sense oligonucleotides used as the control had no effect. Progesterone concentration in the medium was significantly decreased by the 48-h treatment with antisense oligonucleotides in the presence of hCG, and this inhibitory effect was completely blocked by the simultaneous addition of N-acetyl-L-cysteine, an antioxidant. Treatment with antisense oligonucleotides caused no significant change in the percentage of apoptotic cells as morphologically evaluated by the nuclear staining with Hoechst dye. In conclusion, the decrease in intracellular Cu, Zn-SOD activities inhibits progesterone production by rat luteal cells, which may be mediated by superoxide radicals, suggesting that intracellular Cu,Zn-SOD plays important roles in the regulation of luteal function.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,Zn-SOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1alpha (IL-1alpha), IL-beta, IL-6, and tumor necrosis factor alpha. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,Zn-SOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ER alpha and ER beta) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER alpha and ER beta messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER alpha and ER beta) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER alpha mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ER beta mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER alpha and ER beta mRNA expression at the cellular level, by in situ hybridization, showed ER alpha expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ER beta mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypophysectomy, performed early in pregnancy, induced a sharp decline in ER alpha mRNA expression but a less-marked reduction in ER beta mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER alpha and ER beta mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ER beta mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ER beta mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

Ephemerality and prolongation of luteal function have been matters of great concern in reproduction for many years. However, their control mechanisms are very complex and differ among mammals. Recently, evidence has indicated that reactive oxygen species may play important roles in the regulation of luteal function. Reactive oxygen species are present in most somatic cells and are involved in apoptosis, or 'physiological cell death'. In the corpus luteum, reactive oxygen species also exert luteolytic effects as well as some paradoxical luteotrophic effects. This paper discusses the possible roles of reactive oxygen species in the control of luteal function.

To study the relationship between follicular atresia, apoptosis, and nitric oxide (NO) generation in follicular development, steroidogenesis, NO levels in follicular fluid and apoptosis were analysed in the various sized follicles of women receiving ovarian stimulation with human menopausal gonadotrophin (HMG)-human chorionic gonadotrophin (HCG) treatments for in-vitro fertilization (IVF)-embryo transfer. The follicles were divided into three groups by diameter: large follicle,or = 18 mm; medium follicle,or = 12 andor = 15 mm; small follicle,or = 10 mm. Follicular fluid was obtained from 20 women 34 h after HCG administration, and the concentrations of oestradiol, progesterone and testosterone, and nitrite, nitrate, arginine and citrulline were measured. Granulosa cells obtained from each group of follicular fluid were stained with Hoechst dye, and nuclear morphology was examined by a fluorescence microscopy. Oestradiol and progesterone concentrations in large follicles were significantly (P0.01) higher than those in medium or small follicles, and testosterone concentrations in small follicles were significantly (P0.01) higher than those in large follicles. There were no significant differences in the concentrations of nitrite, nitrate, arginine and citrulline among three groups. The percentage of apoptotic cells with nuclear fragmentation was significantly (P0.01) higher in small follicles than in large follicles. The present results suggested that small follicles with poor response to HMG may undergo atresia through apoptosis. No significant difference in the follicular NO level between large and small follicles led us to speculate on a different responsiveness to NO in these two types of follicles.

The effect of prostaglandin F2 alpha (PGF2 alpha) on superoxide radical production by macrophages was studied in pseudopregnant rats. Peritoneal macrophages prepared on day 7 or 13 of pseudopregnancy (psp) were incubated with various doses of PGF2 alpha for 90 min, and the production of superoxide radical was measured by the cytochrome C reduction method. PGF2 alpha significantly stimulated superoxide radical production by macrophages on day 13 of psp, but not on day 7 of psp. The pretreatment of macrophages with an inhibitor of protein kinase C (H7), Ca2+ channel blocker (Verapamil), Ca2+ chelators (EGTA, BAPTA), and an inhibitor of GTP-binding protein (pertussis toxin) prevented the stimulatory effects of PGF2 alpha on superoxide radical production. In conclusion, PGF2 alpha stimulated superoxide radical production by macrophages through the intracellular signal transduction pathway including activation of protein kinase C through the GTP-binding protein and Ca2+ influx, which would play important roles in the luteolytic process in psp rats.

An involvement of reactive oxygen species in CL regression has been reported. We have shown that a decrease in serum progesterone concentrations coincides with a decrease in superoxide dismutase (SOD) activities and an increase in lipid peroxide levels in the CL after Day 15 of pregnancy. Recently it has been found that ischemia-reperfusion stimulates reactive oxygen species production and causes tissue damage in various organs. We therefore tested the effect of ischemia-reperfusion in the ovary on CL function in pregnant rats. On Day 15 of pregnancy, after clamping of the bilateral ovarian vessels for 30 min, the ovaries were reperfused for 90 min by declamping. The ischemia-reperfusion decreased serum progesterone concentration and SOD activity in the CL and increased lipid peroxide in the CL 90 min after reperfusion. The effects of ischemia-reperfusion, including the decrease in serum progesterone concentrations, were completely blocked by simultaneous injection of SOD and catalase, but not by indomethacin administration. The present study shows that CL function was inhibited by reactive oxygen species produced by ischemia-reperfusion in the ovary and that the effect was not mediated through prostaglandins.

Nutritional status was assessed in 214 CAPD patients from eight centers in Japan. All patients were receiving adequate dialysis, as indicated by KT/Vurea and Karnofsky's activity scale. Subjective global assessment indicated that 25% of 83 female patients and 27% of 131 male patients were, at least, moderately malnourished. Mid-arm muscle circumference was lower in malnourished male patients than in well-nourished patients, however, in female patients none of the anthropometric variables reflected nutritional status. Lower serum albumin level was observed in malnourished male patients, but not in female patients. Daily protein intake, estimated from protein catabolic rate (PCR) was approximately 0.8 g/Kg BW in both males and females. This value is similar to those reported in literatures for CAPD patients. In our well-rehabilitated patients, PCR did not reflect the nutritional status as indicated by subjective global assessment. The data suggest that low protein intake was not always the major cause of malnutrition in these patients and that other factors, such as endocrine disorder, may also have contributed to malnutrition.

Recombinant human growth hormone (r-hGH), 1 U/kg body weight/week, was administered subcutaneously for 2-4 weeks to two end stage renal disease patients with severe malnutrition. Following r-hGH, there were significant increase in hematocrit level and serum concentration of albumin, IGF-1 and GH, and decrease of urea nitrogen. It was concluded that r-hGH exerts a therapeutic effect to nutritional status in renal failure patients by improving severe hypoalbuminemia, susceptibility to infectious disease, intractable ascites and so on.

OK-432 and etoposide were administered intravenously to a patient with liver metastasis of ovarian cancer. OK-432 induced IFN-gamma and TNF. SuPS and PPD skin reaction became positive after the therapy. The size of metastatic lesions was reduced significantly (partial response). It is suggested that the combination therapy of OK-432 and etoposide may be effective to the liver metastasis of ovarian cancer.

Stable isotope 15N-compound, 15N-urea, is useful marker to investigate nitrogen metabolism in clinical nephrology, particularly in chronic renal failure or dialysis. 15N-urea incorporation into plasma albumin in addition to plasma 15N disappearance was studied in 6 patients with endstage chronic renal failure. As a result, only minor fraction of administered 15N-urea was incorporated into albumin in this study. In addition, it was also confirmed that high energy diet may promote protein synthesis through 15N incorporation to plasma amino acids, such as alanine, in these patients with low protein meal. Therefore, administration of 15N-compound to human subjects may contribute to provide us the important informations on nitrogen metabolism. For instance, urea kinetics are described in the endstage chronic renal failure in this review. However, less expensive 15N-compounds should be provided and more simple but accurate measurement of 15N activity should be developed for the further clinical application of the stable isotope.

The effects of atrial natriuretic peptide (ANP) on the regional renal blood flow were examined in anesthetized dogs. A thermal diffusion technique was applied in an attempt to observe the intrarenal blood distribution. Tissue blood flow was continuously measured according to the changes of the thermal gradient produced by a semiconductor (Peltier effect). The data (x) obtained by these probes were closely related to those (y) of the ion gas clearance method (the probe for cortical blood flow; y = 1.08x-6.23 r = 0.99, the probe for medullary blood flow; y = 1.00x +0.26 r = 0.99). The basal values of cortical and medullary blood flows measured with the flow probe in anesthetized mongrel dogs were 85 +/- 16 and 55 +/- 9 ml/min/100 g kidney weight, respectively. ANP was infused into renal artery at doses of 0.01 (n = 6), 0.05 (n = 8), 0.3 (n = 6) micrograms/kg/min and the effect on renal hemodynamics and renal excretory functions was observed. The effect was also compared with that of furosemide. Low dose of ANP did not affect renal blood distribution. Intermediate dose of ANP selectively increased medullary blood flow from 46 +/- 8 to 62 +/- 7 ml/min/100 g kidney weight, and higher dose significantly increased both cortical and medullary blood flow. ANP produced diuresis and natriuresis in a dose-dependent manner, which was not necessarily related to the changes of intrarenal blood distribution. Furosemide did not affect the renal hemodynamics. Several parameters of renal functions such as lithium and free water clearances, showed changes different from those with ANP infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vaginal antibiotics suppository on the retroperitoneal-space infection were studied in 42 patients who had hysterectomy and lymphadenectomy with diagnosis of uterine cancer stage I or II. All patients received intravenous administration of 1 g LMOX (3 times/day) for 7 days after operation. One group of patients (21 cases) received 0.5 g of ABPC vaginal suppository (twice/day) for 10 days immediately after operation. Another group of 21 patients did not received vaginal suppository. The LMOX concentrations in the exudate decreased rapidly from the 3rd day to the 7th day. The frequencies of bacterial appearance in the retroperitoneal-space in the vaginal suppository group were 33% on 5th day, 33% on 7th day and 20% on 9th day, while those in the control group were 100%, 100% and 76%, respectively. It was concluded that the administration of vaginal antibiotics suppository was easy to handle and effective for preventing the retroperitoneal-space infection after hysterectomy and lymphadenectomy.