Neonatal immune challenge affects the regulation of estrus cyclicity and feeding behavior in female rats.

A single immune challenge with lipopolysaccharide (LPS) in the neonatal period has a long-lasting influence on immune response. Using female Sprague-Dawley rats, we examined whether neonatal LPS challenge influences the life-long neuroendocrine sensitivity of reproductive function and feeding behavior to LPS, and whether stress-related neuropeptides and their receptors are involved in neonatal LPS-induced physiological change. On day 10 after birth, all pups were injected with LPS (100 microg/kg, i.p.) or saline. Then, in Experiment 1, LPS (100 microg/kg, i.p.) or saline was injected at diestrous in adulthood, and the length of the estrous cycle, 24h food intake and body weight change were recorded. In Experiment 2, the mRNA expression levels of corticotropin-releasing hormone (CRH), urocortin (UCN), urocortin 2 (UCN2), CRH receptor type 1 (CRH-R1) and CRH receptor type 2 (CRH-R2) in the hypothalamus were measured using real-time PCR. LPS injection in adulthood prolonged the estrous cycle in neonatal LPS-injected rats. LPS injection in adulthood decreased food intake and body weight in both neonatal LPS- and saline-injected rats, more so in the latter. Basal expressions of UCN2 and CRH-R2 mRNA were higher in neonatal LPS-injected rats than in saline-injected rats. These findings indicate that neonatal immune challenge influences the anti-stress regulation of the estrous cycle and feeding behavior in adulthood. Increased expression of UCN2 and CRH-R2 might enhance the sensitivity of the estrous cycle in suppressing the effects of LPS.

International Journal of Developmental Neuroscience


Associations of endogenous sex hormones and sex hormone-binding globulin with lipid profiles in aged Japanese men and women.

BACKGROUND: The associations of endogenous estrogen with lipid profiles and insulin resistance were conflicting in both men and women. We determined the associations of serum estradiol level and free estradiol index (FEI) with lipid profiles and insulin resistance in aged Japanese men and women. METHODS: One hundred seventy-four men and 208 postmenopausal women aged from 50 to 85 y were included in this study. Lipid profiles, fasting glucose and insulin as well as estradiol, testosterone, dehydroepiandrosterone-sulfate (DHEA-S) and sex hormone-binding globulin (SHBG) in serum were measured. RESULTS: Serum estradiol concentration in men showed a significant and negative correlation with high density lipoprotein-cholesterol (HDL-C) concentration and a significant and positive correlation with triglyceride (TG) after adjustment for age and body mass index (BMI). In addition, estradiol level was positively correlated with insulin and HOMA index. In women, serum levels of estradiol and testosterone were not correlated with lipid profiles and insulin resistance. SHBG concentration was negatively correlated with TG after adjustment for age and BMI in women. CONCLUSION: Endogenous estradiol level is associated with unfavorable lipid profiles and insulin resistance in aged men. In aged women, SHBG level is associated with favorable lipid profiles.


Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats.

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.


Associations of circulating adiponectin with estradiol and monocyte chemotactic protein-1 in postmenopausal women.

OBJECTIVE: The aim of the present study was to clarify the association of serum adiponectin concentrations with serum 17beta-estradiol concentrations in pre-, peri-, and postmenopausal women. In addition, the associations of serum adiponectin with serum concentrations of proinflammatory and anti-inflammatory cytokines were examined in women during the menopausal transition. DESIGN: A total of 197 women were enrolled in this study: 33 premenopausal women, 80 perimenopausal women, and 84 postmenopausal women. Serum adiponectin concentration was measured by an enzyme-linked immunosorbent assay. Serum concentrations of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha, anti-inflammatory cytokine IL-10, and the chemokines IL-8, macrophage inflammatory protein-1beta and monocyte chemotactic protein-1 were measured by using a multiplexed human cytokine assay. RESULTS: Serum adiponectin concentration showed a significant negative correlation with serum estradiol concentration (r= -0.400, P=0.001) in postmenopausal women but not in pre- and perimenopausal women, and this correlation was significant after adjustment for age and body mass index. Serum adiponectin concentration also showed a significant negative correlation with serum monocyte chemotactic protein-1 concentration (r= -0.244, P=0.05) in postmenopausal women. CONCLUSION: An increase in adiponectin level due to a decrease in estradiol results in a reduction in monocyte chemotactic protein-1 level in postmenopausal women, suggesting that adiponectin may be associated with a protective role against insulin resistance and atherosclerosis, which occur in the postmenopausal stage.


Transition of leptin receptor expression during pubertal development in female rat pituitary.

Leptin is thought to play important roles in pubertal development and reproductive function in the female. Leptin receptor is expressed in various tissues including the pituitary and hypothalamus. We investigated the transition of leptin receptor (Ob-R) expression in female rat pituitary during pubertal development. Real-time RT-PCR was performed for long-form leptin receptor (Ob-Rb) and short-form leptin receptor (Ob-Ra) in the pituitary and cerebrum of the rats aged 4 (juvenile), 6 (pubertal), and 8 weeks (adult). Double immunohistochemical colocalization of Ob-R and luteinizing hormone (LH) was performed in pituitaries from 4-week-old female rats. Serum leptin concentrations of 4-, 6-, and 8-week-old rats were measured by radioimmunoassay. In the pituitary, expression of Ob-Rb mRNA in 4-week-old rats (1.00 +/- 0.16) was significantly higher than in 8-week-old rats (0.61 +/- 0.07, p<0.05), although expression of Ob-Ra mRNA did not differ among 4-, 6-, and 8-week-old rats. In cerebrum, Ob-Ra and Ob-Rb mRNA expressions did not differ significantly among 4-, 6-, and 8-week-old rats. Intense staining of Ob-R and colocalization of Ob-R and LH were seen in 4-week-old rat pituitary. On the other hand, serum concentrations of leptin in 6- and 8-week-old rats were significantly higher than those in 4-week-old rats (p<0.05, p<0.01, respectively). In conclusion, since the pituitary gonadotroph is a potential target of leptin, the juvenile rat pituitary might prepare for a subsequent increase of serum leptin concentration by expressing Ob-Rb.


Circulating profiles of osteoprotegerin and soluble receptor activator of nuclear factor kappaB ligand in post-menopausal women.

OBJECTIVE: The aim of this study was to elucidate the detail profiles of circulating osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in post-menopausal women. METHODS: Eighty Japanese post-menopausal women were enrolled in this cross-sectional study. Circulating OPG and free fraction of sRANKL (free sRANKL), PTH, calcium and phosphorus, age, years since menopause, body mass index, bone mineral density of the vertebral bodies (LBMD) and bone turnover markers were determined in each subject. RESULTS: In rank order correlation analysis, serum OPG concentrations had a significant positive correlation with age (r=0.291, p=0.024) and a marginal significant negative correlation with LBMD (r=-0.247, p=0.062). However they did not have correlations with LBMD or other parameters after adjustment for age. Serum free sRANKL concentrations had a significant positive correlation with age (r=0.332, p=0.010) and a significant negative correlation with LBMD (r=-0.608, p<0.001). This correlation with LBMD persisted after adjustment for age. In a multiple regression analysis with a stepwise model, the main determinants of LBMD were age and serum free sRANKL (p=0.015 and p=0.006, respectively). CONCLUSIONS: We found the increase in circulating OPG and sRANKL with age and a robust negative correlation between circulating free sRANKL and LBMD after adjustment for age. The increase in circulating free sRANKL may reflect directly or indirectly the conditions coexistent with bone loss in post-menopausal women.

Journal of Endocrinological Investigation


Associations of interleukin-6 with interleukin-1beta, interleukin-8 and macrophage inflammatory protein-1beta in midlife women.

OBJECTIVE: The aim of the present study was to determine the associations of interleukin (IL)-6 with other cytokines and chemokines and to compare these associations in peri- and postmenopausal women. METHODS: Ninety-nine perimenopausal and 92 postmenopausal women were enrolled in this study. Serum concentrations of IL-6, IL-1beta, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, tumor necrosis factor (TNF)-alpha, interferon gamma, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, macrophage inflammatory protein (MIP)-1beta and monocyte chemotactic protein (MCP)-1 were measured simultaneously using a multiplexed cytokine assay. RESULTS: Among the 17 cytokines, IL-6, IL-1beta, IL-5, IL-7, IL-8, IL-10, MCP-1 and MIP-1beta were detected in serum in more than 50% of the women. Serum levels of IL-4 and MCP-1 in postmenopausal women were significantly higher than those in perimenopausal women. Serum IL-6 concentrations showed significant and positive correlations with serum concentrations of IL-1beta, IL-8, MIP-1beta, IL-7 and MCP-1 in women regardless of menopausal status, and these correlations were still significant after adjustment for age and body mass index. CONCLUSION: Serum IL-6 concentration was found to be closely associated with serum concentrations of IL-1beta, IL-8, MIP-1beta, IL-7 and MCP-1 in women regardless of menopausal status, suggesting that these cytokines act in concert with the progression of several symptoms and various diseases.


FKBP5, SERT and COMT mRNA expressions in the peripheral leukocytes during menstruation cycle in healthy reproductive females.

There have been several evidences that the mRNA expressions in the peripheral leukocytes may indicate not only physical but also psychological states. The purpose of this study is whether the mRNA expressional changes in the leukocytes are related to the mental states across the menstrual cycle in reproductive healthy female subjects. Thirty-eight female subjects (22.4+/-1.4 year-old) were participated in this study at three menstruation cycle periods (menstrual, follicular and luteal phase). The FKBP5 (FK506-binding protein gene), SERT (serotonin transporter gene) and COMT (catechol-o-methyltransferase gene) mRNA expressions in the leukocytes were determined with hormonal data. The psychological changes were assessed with self-rating hospital anxiety and depression scale (HADS). Only one thirds of subjects (n=12) had regular menstrual cycles during the experiment. So we analyzed the data from these 12 subjects. The anxiety score of each subject was changed across the menstrual cycle (Friedman test: P<0.05). The FKBP5 mRNA expression was significantly lower in the follicular phase than in the other phases but no changes were seen in either SERT or COMT mRNA expressions among the phases. In conclusion, there are differences of HADS anxiety score and FKBP5 mRNA expression in the leukocytes across the menstrual cycle but there is no correlation between anxiety scores and FKBP5 mRNA.


Circulating osteoprotegerin is associated with age and systolic blood pressure, but not with lipid profile or fasting glucose, in postmenopausal women.

OBJECTIVE: Osteoprotegerin (OPG), an inhibitor of osteoclastogenesis and osteoclast activation, has been reported to be linked to vascular biology. The aim of this study was to clarify the relationships between circulating OPG and the risk factors for vascular disorders in postmenopausal women. DESIGN: Eighty Japanese postmenopausal women were enrolled in this cross-sectional study. Clinical parameters (age, number of years since menopause, body mass index, systolic and diastolic blood pressure); serum concentrations of OPG, creatinine, calcium, and phosphorus; serum lipid profile; plasma glucose; and bone mineral density of the L2-4 vertebral bodies were determined for each woman. RESULTS: In rank-order correlation analysis, serum OPG concentrations had significant positive correlations with age (r = 0.29, P = 0.03), systolic blood pressure (r = 0.45, P < 0.01), diastolic blood pressure (r = 0.34, P < 0.01), and serum creatinine (r = 0.29, P = 0.04). Serum OPG concentration also had a marginally significant negative correlation with bone mineral density of the L2-4 vertebral bodies (r = -0.25, P = 0.06). However, serum OPG did not correlate with body mass index, serum lipid profile, or plasma glucose. The correlation of serum OPG with systolic blood pressure persisted after adjustment for both age and serum creatinine. CONCLUSIONS: These results suggest that increased circulating OPG in postmenopausal women is closely related to higher systolic blood pressure, which could cause atherosclerosis.


Decreased expression of kisspeptin mediates acute immune/inflammatory stress-induced suppression of gonadotropin secretion in female rat.

Kisspeptin and its corresponding receptor, the G protein-coupled receptor 54, play an important role in reproductive systems. It has been suggested that reproductive disorders in metabolically disrupted animals are caused by the alteration of hypothalamic KiSS-1 systems. Immune/inflammatory challenge is also known to disrupt reproductive function. However, the effects of immune/inflammatory challenge on KiSS-1 systems have not been investigated. In this study, we showed that lipopolysaccharide (LPS) injection decreased hypothalamic KiSS-1 mRNA expression as well as plasma LH levels in ovariectomized rats. Indomethacin completely blocked the suppressive effects of LPS on LH secretion and KiSS-1 mRNA level. Furthermore, we showed that i.v. injection of kisspeptin increased plasma LH levels in LPS-administrated rats to the same degree as in saline-injected rats. These results suggest that KiSS-1 systems are sensitive to immune/inflammatory challenge conditions and transmit these signals into the central reproductive system.


Differences in sensitivity to cold in Japanese men and postmenopausal women aged > or =50 years.

Sensitivity to cold is associated with several factors, such as aging, sex, and body composition. However, no previous studies have examined the differences in sensitivity to cold in men and women or the association of hormonal levels with sensitivity to cold. The aim of the present study was to clarify both the change in sensitivity to cold with aging and the difference in sensitivity to cold between men and women. Associations were also examined between circulating hormonal concentrations and the changes with aging and differences in sensitivity. This population-based cohort study enrolled healthy Japanese men and women aged > or = 50 years. A standardized 210-item health questionnaire was used to obtain information on symptoms of sensitivity to cold. Serum concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), estradiol, testosterone, dehydroepiandrosterone sulfate, and sex hormone-binding globulin (SHBG) were measured. Of the 154 men and 180 women enrolled in this study, more women than men had sensitivity to cold. Whereas the percentage of men who had sensitivity to cold significantly increased with aging (P < 0.05), the percentage of women who had sensitivity to cold was already high (23.7%) at 50 to 60 years of age and did not change with aging. In men, advancing age and low body mass index (BMI) were significantly associated with sensitivity to cold (P < 0.05); however, age and BMI in women were not similarly associated. In addition, the effect of sex after adjustment for age was significant (P < 0.05), and there was also a numeric but nonsignificant effect of sex after adjustment for BMI. In men, low serum levels of the gonadal hormone FSH were significantly associated with sensitivity to cold in logistic analysis, but this association was nonsignificant after multivariate analysis. Serum concentrations of gonadal hormones and SHBG in women were not associated with sensitivity to col. The association of age with sensitivity to cold was different in men and women; the association of BMI with sensitivity to cold might be different in men and women. In addition, these changes in sensitivity to cold were not associated with circulating hormonal concentrations.


Estradiol suppresses NMU mRNA expression during sexual maturation in the female rat pituitary.

Neuromedin U (NMU) suppresses food intake and gonadotropin secretion. However, the developmental transition of NMU expression in the pituitary gland and the regulation of NMU expression are unclear. The objective of this study was to examine the transition of the expression of NMU mRNA in the pituitary glands of female rats from the juvenile period to the mature period of development. Furthermore, factors such as estradiol, insulin, leptin, and inhibin A, whose expressions change throughout puberty and which affect gonadotropin secretion in pituitary cell culture, were examined. In the pituitary gland, the expression of NMU mRNA was significantly lower in 8-week-old rats than in 4- and 6-week-old rats. In the pituitary cell culture, the expressions of NMU mRNA in the estradiol- and insulin-treated groups were significantly lower than in the control group. These results suggest that the expression of NMU mRNA in the female rat pituitary is reduced as the rats develop from the pubertal to the mature period. Additionally, the reduction of NMU expression in the pituitary may be related to the increases in serum estradiol and insulin levels that occur during the pubertal period, which may negate NMU suppression of gonadotropin secretion.

International Journal of Developmental Neuroscience


閉経後婦人の血中osteoprotegerin濃度と心血管病変の危険因子との関係の検討


Associations of adiponectin with sex hormone-binding globulin levels in aging male and female populations.

Changes with aging in serum gonadal hormones, sex hormone-binding globulin (SHBG) and adiponectin, which are involved in insulin resistance and metabolic syndrome, are different in men and women. We examined the relationships of serum adiponectin concentrations with serum concentrations of hormones and SHBG in men and women. One hundred fifty-four men and 180 women aged from 50 to 85 years were included in the cross-sectional study. Serum concentrations of testosterone, estradiol, dehydroepiandrosterone-sulfate, SHBG and adiponectin were measured in men and women. Serum adiponectin concentration showed a significant positive correlation with serum SHBG concentration in men and women (r=0.404, p<0.0001 and r=0.348, p<0.0001, respectively). Multiple regression analysis also showed that SHBG was the significant predictor for adiponectin in men and women. Serum adiponectin concentration in men showed a significant positive correlation with total testosterone concentration, but that in women was not significantly correlated with total testosterone concentration. Both adiponectin and SHBG concentrations in men (r=-0.222, p=0.0051 and r=-0.334, p<0.0001, respectively) and women (r=-0.281, p=0.0014 and r=-0.251, p=0.0048, respectively) showed significant negative correlations with the homeostasis model assessment (HOMA) index. Correlations between adiponectin and SHBG were significantly positive in both men and women, and both adiponectin and SHBG might be involved in insulin resistance in both sexes.


切迫早産管理が骨代謝に与える影響の検討(第1報)

切迫早産管理が骨代謝に与える影響の検討(第1報)


Association of serum cytokine concentrations with psychological symptoms in midlife women.

The purpose of the present study was to clarify the association of serum cytokine concentrations, determined using a multiplexed cytokine assay, with psychological symptoms in midlife women. Fifty-three peri- and post-menopausal women with and without psychological symptoms in Greene's climacteric scale were enrolled in this study. Levels of 17 cytokines in serum samples were measured simultaneously using a multiplexed human cytokine assay. Serum interleukin (IL)-6 concentration in women with psychological symptoms (2.71+/-047 pg/ml) was significantly (p=0.009) higher than that in women without psychological symptoms (0.98+/-0.18 pg/ml). Serum IL-8 concentration in women with psychological symptoms (33.4+/-8.17 pg/ml) was also significantly (p=0.022) higher than that in women without psychological symptoms (7.87+/-1.64 pg/ml). In addition, serum IL-10 concentration in women with psychological symptoms (0.74+/-0.26 pg/ml) was significantly (p=0.048) higher than that in women without psychological symptoms (0.07+/-0.04 pg/ml). Tumor necrosis factor (TNF)-alpha in serum was detected only in women with psychological symptoms. Serum IL-2 concentration in women with psychological symptoms tended (p=0.066) to be higher than that in women without psychological symptoms. No significant differences were found between levels of other cytokines in women with and without psychological symptoms. Psychological stress manifested as climacteric symptoms in midlife women may be associated with increases in serum concentrations of IL-6, IL-8, IL-10, and TNF-alpha.


Diagnostic performance of serum total testosterone for Japanese patients with polycystic ovary syndrome.

It is reported that the incidence of clinical and biochemical hyperandrogenism may be lower in Japanese patients with PCOS. Hyperandrogenism is included as a referential but not as an essential factor in the diagnostic criteria of the Japanese Society of Obstetrics and Gynecology (JSOG 1993). However, some patients with the typical clinical features of PCOS are not diagnosed with PCOS using JSOG 1993 criteria because they do not have a high LH level, which is defined as essential for diagnosis. In this study, we compared total testosterone (T) levels between Japanese patients with PCOS diagnosed using the JSOG 1993 criteria and normal menstrual women (controls). Fifty controls and 46 patients with PCOS were enrolled in this study. Furthermore, we evaluated the sensitivity and specificity of each cut-off value of T. The mean T level of patients with PCOS was significantly higher than that of the control (86 +/- 48 vs 68 +/- 46, P<0.01), and the prevalence rates of hyperandrogenism (T >114 ng/dL; defined as the mean +2SD of the control) were 10.2% in patients with PCOS and 4% in controls. The area under the ROC curve of T was 0.72, and there was no decision threshold to diagnose PCOS by T alone with both high sensitivity and high specificity. If the threshold is set as 110 ng/dL in order to gain high specificity, 94% of women whose serum level passed the threshold will be patients with PCOS. Although T should not be used as an independent essential factor of Japanese PCOS, it might be useful as a complementary factor in order to diagnose patients who have typical clinical features of PCOS but does not fulfill the JSOG 1993 criteria for PCOS.


Effect of bed rest during pregnancy on bone turnover markers in pregnant and postpartum women.

The aims of our study were to evaluate the changes in bone turnover markers during pregnancy and puerperium as a longitudinal study and to elucidate the effect of bed rest during pregnancy on bone turnover markers in pregnant and postpartum women. The study population comprised 27 Japanese pregnant women aged 23-40 years. All women were recruited for the longitudinal study from the outpatients clinic of the Department of Obstetrics and Gynecology, Tokushima University Hospital. Concentrations of serum bone-specific alkaline phosphatase (BAP), urinary cross-linked type I collagen N-telopeptides (NTx), serum NTx and urinary C-terminal telopeptide of type I collagen (CTx) were measured at 10, 26, 30 and 36 weeks of pregnancy and at 4 days and 1 month postpartum. In addition, we recruited 15 pregnant women (aged 25-35 years) who were treated by bed rest before 30 weeks of pregnancy for threatened premature delivery and compared bone turnover markers in these women with those in 22 normal pregnant women (aged 22-39 years). Concentrations of serum BAP, serum NTx, urinary NTx and urinary CTx were measured at 30 and 34 weeks of pregnancy and at 4 days and 1 month postpartum. In the longitudinal study, serum BAP concentration at 1 month postpartum was significantly higher than that at any stage of pregnancy and that at 4 days postpartum. Urinary concentration of NTx increased gradually during pregnancy and showed a peak at 36 weeks of pregnancy, followed by a decrease in the postpartum period. Serum NTx concentration and urinary CTx concentration showed the same patterns of change as that of urinary NTx concentration. In the comparison study, urinary concentrations of NTx and CTx at 30 and 34 weeks of pregnancy in women with bed rest were significantly (p<0.0001 and p<0.001, respectively) higher than those in normal pregnant women. Serum NTx concentration at 34 weeks of pregnancy in women with bed rest was also significantly (p=0.0029) higher than that in normal pregnant women. Serum BAP concentration at 34 weeks of pregnancy in women with bed rest was significantly (p=0.0038) higher than that in normal pregnant women, and these high levels were maintained during puerperium. Serum BAP concentration at 34 weeks of pregnancy was significantly correlated with duration of bed rest (r=0.767, p=0.0041). Immobilization due to bed rest during pregnancy is associated with increases in bone turnover markers in pregnant and postpartum women. Concentrations of bone resorption markers increased rapidly at the start of bed rest, while the concentration of a bone formation marker gradually increased toward puerperium.


The type 2 corticotrophin-releasing hormone receptor mediates orexin A-induced luteinising hormone suppression in ovariectomised rats.

Orexins are thought to be regulatory factors of the arousal and sleep patterns. They also affect immune, feeding, autonomic and neuroendocrine systems. We have previously shown that intracerebroventricular (i.c.v.) injection of orexin decreases pulsatile luteinising hormone (LH) secretion in ovariectomised (OVX) rats. However, the details of this mechanism have not been fully examined. Intracerebroventricular injection of orexin A also stimulates corticotrophin-releasing hormone (CRH) systems, which have been implicated in the stress-induced suppression of reproductive function. In the present study, we investigated the role of CRH systems in orexin-induced LH suppression. OVX rats were implanted with i.c.v. and intravenous (i.v.) cannulae. After i.c.v. injection of orexin and/or CRH receptor antagonists, blood samples were collected through the i.v. cannula at 6-min intervals for 120 min for LH measurement. Intracerebroventricular injection of orexin A or B (3 nmol/2.5 microl) suppressed pulsatile LH secretion. Coadministration of orexin A and alpha-helical corticotrophic-releasing factor (CRF), a nonselective CRH receptor antagonist (13 nmol/2.5 microl), or astressin(2)B, a selective type2 (CRH-R2) CRH receptor antagonist (28 nmol/2.5 microl), partly restored pulsatile LH secretion. Orexin B-induced LH suppression was not restored by alpha-helical CRF. In addition, i.c.v. injection of orexin A increased CRH and urocortin II (UcnII), but not Ucn mRNA levels, in the hypothalamus. These findings suggest that CRH-R2 mediates orexin A-induced LH suppression and it is possible that CRH and UcnII in the hypothalamus are involved in this pathway.


Changes in serum cytokine concentrations during the menopausal transition.

The purpose of the present study was to clarify the changes in serum concentrations of 17 cytokines in healthy women during the menopausal transition by using a multiplexed cytokine assay and to clarify the associations of these cytokines with serum estradiol concentration. Sixteen premenopausal, 54 perimenopausal and 52 postmenopausal women were enrolled in this study. Seventeen cytokines in serum samples were measured simultaneously using a Bio-Plex human cytokine 17-Plex assay. Serum IL-6 concentration showed a weak positive correlation with age (r=0.196, p<0.05). Postmenopausal women for whom less than 5 years had passed since menopause showed significant (p<0.05) increase in serum concentrations of IL-2, GM-CSF and G-CSF, while serum IL-4 concentration was significantly (p<0.05) increased in postmenopausal women for whom more than 5 years had passed since menopause. Serum estradiol concentration showed a significant negative correlation with serum IL-6 concentration and weak negative correlations with serum concentrations of IL-2, IL-8 and GM-CSF. We were able to simultaneously measure the levels of 17 cytokines using a highly sensitive cytokine assay, and we found that the changes in serum cytokine concentrations during the menopausal transition differed. We also found that serum IL-6 concentration during the menopausal transition was negatively correlated with serum estradiol concentration.


Association of serum undercarboxylated osteocalcin with serum estradiol in pre-, peri- and early post-menopausal women.

We investigated changes in serum concentration of undercarboxylated osteocalcin (ucOC), which is a sensitive marker of vitamin K status, and association of ucOC concentration with estradiol concentration in pre-, peri- and early post-menopausal women. The study population consisted of 193 pre-, peri- and post-menopausal Japanese women aged 39-66 yr. Serum ucOC concentration was measured to assess vitamin K status; serum concentrations of intact osteocalcin (OC) and bone-specific alkaline phosphatase (BAP) were measured as bone formation markers; and urine concentration of N-telopeptide was measured as a bone resorption marker. Serum estradiol and estrone concentrations were measured by a highly sensitive radioimmunoassay. Bone mineral density (BMD) was measured at the lumbar spine. Serum concentration of ucOC in peri-menopausal women was significantly (p=0.0005) higher than that in pre-menopausal women, while serum OC concentration in post-menopausal women for whom 1 yr had passed since menopause was significantly (p=0.0003, p=0.024, respectively) higher than the concentrations in pre-menopausal and peri-menopausal women. Serum ucOC concentration showed a significant negative correlation with estradiol concentration (r=-0.372, p<0.0001) and a significant positive correlation with serum FSH concentration (r=0.324, p<0.0001). Serum OC concentration was positively correlated with serum FSH concentration (r=0.317, p<0.001). The results showed that the change in ucOC concentration during the menopausal transition is different from that in OC concentration. In addition, serum ucOC concentration is closely associated not only with FSH concentration but also estradiol concentration.

Journal of Endocrinological Investigation


Change in serum undercarboxylated osteocalcin concentration in bilaterally oophorectomized women.

We investigated changes in serum undercarboxylated osteocalcin (ucOC) concentrations, bone turnover markers and spine bone mineral density (BMD) in women who had undergone bilateral oophorectomy during the premenopausal period. The study population comprised 141 bilaterally oophorectomized and 32 premenopausal women for a cross-sectional study. The longitudinal study consisted of 21 bilaterally oophorectomized women. Serum ucOC concentration, serum concentrations of intact osteocalcin (OC) and bone-specific alkaline phosphatase (BAP) as bone formation markers, urine N-telopeptide (NTx) concentration as a bone resorption marker and serum parathyroid hormone (PTH) concentration were measured. Serum concentration of ucOC in women at 1 month after bilateral oophorectomy was significantly (p<0.05) higher than that in premenopausal women and the high level was sustained after surgical menopause. On the other hand, serum OC concentration at 1 month after surgical menopause was not different from that in premenopausal women. In the longitudinal study, serum ucOC concentration at 1 month after surgical menopause was significantly (p<0.05) increased compared to that before bilateral oophorectomy, while serum OC concentrations before and at 1 month after surgical menopause were not significantly different. The results of this study showed that serum ucOC concentration rapidly increases in women after bilateral oophorectomy and that change in serum ucOC concentration after surgical menopause is different from change in serum OC concentration.


Association of interleukin-8 with hot flashes in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women.

The purpose of this study was to identify serum cytokine concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes. Serum concentrations of 17 cytokines were simultaneously measured using a multiplexed human cytokine assay in 129 premenopausal, perimenopausal, and postmenopausal women and 50 bilateral oophorectomized women. Serum IL-8 concentrations in midlife women and bilateral oophorectomized women with severe hot flashes were significantly higher than the concentrations in women without hot flashes and women with mild and moderate hot flashes. Serum macrophage inflammatory protein-1beta concentration in women with severe hot flashes was significantly higher than those in women without hot flashes and women with mild and moderate hot flashes. Serum IL-8 concentrations in premenopausal, perimenopausal, and postmenopausal women and bilateral oophorectomized women with hot flashes were significantly higher than those in women without hot flashes. IL-8 may be associated with peripheral vasodilation in women with hot flashes.

The Journal of Clinical Endocrinology and Metabolism


Effect of vitamin K2 treatment on carboxylation of osteocalcin in early postmenopausal women.

We examined the serum level of undercarboxylated osteocalcin (uc OC), which is a sensitive marker of vitamin K status, and levels of bone turnover markers in early postmenopausal women receiving vitamin K2 treatment with or without vitamin D3. Thirty-four postmenopausal women with a mean age of 53 years whose bone mineral density (BMD) was less than 0.809 g/cm2 (osteopenia and osteoporosis) were treated with vitamin K2 or with a combination of vitamin K2 and vitamin D3. Seventeen women received daily oral administration of 45 mg vitamin K2 and 17 women received daily oral administration of 45 mg vitamin K2 plus 0.75 microg 1alpha-hydroxyvitamin D3. Serum levels of uc OC, intact osteocalcin (OC) and bone alkaline phosphatase (BAP), urinary deoxypyridinoline (DPD) levels and BMD at the lumbar spine were measured before and at 1 and 2 years after the start of treatment. Serum uc OC levels in women treated with vitamin K2 alone and with both vitamin K2 and vitamin D3 decreased significantly (p < 0.05). Serum levels of intact OC and BAP in women treated with vitamin K2 did not show significant changes, while those in women who received the combined treatment decreased significantly (p < 0.05). On the other hand, urinary DPD level in women treated with vitamin K2 did not change, while that in women who received the combined treatment tended to decrease (p < 0.1). Serum uc OC levels in early postmenopausal women who received vitamin K2 decreased due to carboxylation of uc OC. Combined treatment with vitamin K2 and vitamin D3 may be effective for sustaining BMD in early postmenopausal women whose bone turnovers are highly activated.


Different effects of oral conjugated equine estrogens and transdermal estrogen on undercarboxylated osteocalcin concentration in postmenopausal women.

Undercarboxylated osteocalcin (ucOC) is a sensitive marker of vitamin K status, and triglyceride (TG) has been shown to be the main transporter of vitamin K. In the present study, we examined the difference between ucOC concentrations in postmenopausal women receiving hormone therapy (HT) with oral conjugated equine estrogens (CEE) and transdermal estradiol (TE2). We also examined the associations of ucOC concentration with estradiol concentration and TG. Ninety-two postmenopausal women were recruited for this study. Serum concentrations of ucOC, intact osteocalcin, estradiol, and TG were measured before and after 12 months of HT. Forty-six women received oral administration of 0.625 mg of CEE and 2.5 mg of medroxyprogesterone acetate daily, and 46 women received transdermal administration of 50 mug of 17beta-estradiol twice weekly and 2.5 mg of medroxyprogesterone acetate daily. The ucOC concentration in women during HT with oral CEE was significantly (P < 0.01) lower than that in women during HT with TE2. Serum estradiol concentrations during HT with CEE showed a significant inverse correlation with ucOC concentrations and the ratio of ucOC/OC during HT (P < 0.05 and P < 0.01, respectively). In addition, the serum ucOC concentration in women with an increased percentage of change in TG was significantly (P < 0.01) lower than that in women with a decreased percentage of change in TG during HT with oral CEE. The effect of HT with TE2 on ucOC concentration in women is weaker than the effect of HT with oral CEE. Suppression of ucOC concentration in postmenopausal women during HT with oral CEE might be associated with the effect of vitamin K through increased TG induced by oral CEE.


塩酸ラロキシフェンが骨・脂質代謝と内分泌系に及ぼす作用の検討

塩酸ラロキシフェンが骨・脂質代謝と内分泌系に及ぼす作用の検討


更年期障害に対する漢方治療中のサイトカインの動態. 23, 76-79, 2006.04.

産婦人科漢方研究のあゆみ

産婦人科漢方研究のあゆみ


Undercarboxylated osteocalcin concentration in postmenopausal women receiving hormone therapy daily and on alternate days.

Undercarboxylated osteocalcin (ucOC) is a sensitive marker of vitamin K status. The authors examined the difference in serum ucOC concentrations in postmenopausal women receiving hormone therapy (HT) daily and on alternate days, and assessed the association between ucOC and triglyceride concentrations, which are related to the transport of vitamin K. Seventy-three postmenopausal women were recruited for this study. Thirty-seven women received 0.625 mg of conjugated equine estrogens (CEE) and 2.5 mg of medroxyprogesterone acetate (MPA) daily, and 36 women received 0.625 mg of CEE and 2.5 mg of MPA on alternate days. The concentrations of serum ucOC, bone turnover markers, lipid profiles, and hormones were measured before and after 12 months of HT. The ucOC concentration in women taking HT daily was significantly (P < 0.01) lower than that in women taking HT on alternate days. Serum ucOC concentrations during HT showed a significant (P < 0.01) inverse correlation with estradiol concentrations during HT. Serum estradiol concentrations during HT showed a significant (P < 0.01) positive correlation with triglyceride concentrations during HT. Furthermore, ucOC concentrations during HT showed a significant (P < 0.05) inverse correlation with triglyceride concentrations in women receiving HT. The effect of HT on alternate days on ucOC concentration was weaker than the effect of HT daily. In addition, ucOC concentration after 12 months of HT daily might be decreased due to the conversion of ucOC to carboxylated OC by the effect of vitamin K through increased triglyceride levels induced by oral CEE.


Comparison and problems of measured values of LH, FSH, and PRL among measurement systems.

Several measurement systems are used for LH, FSH, and PRL in Japan, but the comparison of the measured values is difficult in some cases. In this study, we compared the measured values of 3 types of measurement systems widely used in Japan, SPAC-S, ARCHITECT, and Centaur, in females with a normal menstrual cycle and with various ovulatory disorders. Variant LH was discriminated by the criterion, SPAC-S LH/ARCHITECT LH ratio <0.5. Excluding the variant LH, the correlation of the measured LH values among the measurement systems was high, 0.94-0.99, and the value measured by ARCHITECT LH and Centaur LH was slightly higher than that measured by SPAC-S LH. As for FSH, the correlation was also high, 0.94-1.00, and the value measured by ARCHITECT FSH and Centaur FSH was slightly lower than that measured by SPAC-S FSH. The important judgment criterion of polycystic ovary syndrome (PCOS), a high level of LH relative to FSH, has been set to 1.0 or higher LH/FSH using SPAC-S. When the regression line of the LH/FSH ratio among the measurement systems was investigated, it can be said that 1.25 or higher by ARCHITECT and 1.40 or higher by Centaur are appropriate for the judgment criteria of a high level of LH relative to FSH. As for PRL, the correlation was high, 0.96-0.99, but the value showed large differences among the systems. For the diagnosis of hyperprolactinemia, the higher limit of the normal range may be 15 ng/mL for SPAC-S, 30 ng/mL for ARCHITECT, and 24 ng/mL for Centaur.


Regulatory factors on parathyroid hormone-related peptide production by primary culture of lactating rat mammary gland.

Parathyroid hormone-related protein (PTHrP) is a major cause of humoral hypercalcemia of malignancy, but has also been widely found in fetal and adult non-neoplastic tissues. Lactating mammary gland has been shown to produce large amounts of PTHrP, and high levels of PTHrP have been measured in milk. We have examined the influences of several substances on the secretion of two different forms of PTHrP by primary cultures of mammary cells isolated from lactating rats to examine the regulatory mechanisms of PTHrP production by mammary cells. Primary cultures of mammary cells seeded at a density of 10(5) cells per 35 mm culture dish were grown on collagen gels. First, after cells were left 24 hours for attachment and incubated in 2 % FCS containing medium with for 12 hours, PTHrP (1 - 87) secretions were measured in conditioned medium with hormone supplementation for 1, 24 and 48 hours. Progesterone (10(-7) - 10(-5) mol/l) significantly suppressed PTHrP (1 - 87) secretion in a dose-dependent manner (p < 0.01), while 17beta-estradiol had no influence on PTHrP (1 - 87) secretion. Prolactin, a known stimulator of PTHrP expression in vivo, had no effect in this in vitro model. Second, PTHrP (1 - 34) secretion levels from confluent lactating mammary cells for 24 hours were evaluated. The same results were obtained in the case of PTHrP (1 - 87) secretion from non-confluent cells. Furthermore, dexamethasone (10(-6) mol/l) significantly suppressed PTHrP (1 - 34) secretion (p < 0.01). These results suggest that PTHrP production from the lactating mammary gland is suppressed by progesterone as well as dexamethasone. Progesterone dramatically falls after delivery, thus possibly accelerating PTHrP production by lactating mammary glands and resulting in considerable amounts of PTHrP secreted into the milk.


閉経後婦人の可溶型破骨細胞分化因子(Soluble RANKL)と破骨細胞分化抑制因子(Osteoprotegerin)の血中動態に関する検討


Effect of hormone replacement therapy on the production of bone-resorbing cytokines by peripheral blood cells in postmenopausal women.

We studied the effects of hormone replacement therapy (HRT) with estrogen on postmenopausal changes in the production of bone-resorbing cytokines interleukin 1 beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Both cytokines were measured in the supernatants of lipopolysaccharide (LPS)-stimulated whole-blood cells from 72 untreated and 44 HRT-treated women by ELISA. The levels of IL-1beta were significantly higher in women in their 40s and 50s and in postmenopausal women than in women in their teens, 20s and 30s, while the levels of TNFalpha did not show any changes related to age. Both levels in HRT-treated women were significantly lower than those in untreated women at almost every postmenopausal stage. In a prospective study, HRT induced significant declines in both levels. These results show that estrogen decreases the accelerated production of IL-1beta and reduces the production of TNFalpha in postmenopausal women at each postmenopausal stage, even in late-postmenopausal women.


Effects of postmenopausal hormone therapy every day and every other day on lipid levels according to difference in body mass index

The objective of this study was to determine the effects of postmenopausal estrogen and progestogen therapy (EPT) every day and every other day on lipid levels, particularly triglyceride (TG) levels, according to difference in body mass index (BMI). Ninety-nine postmenopausal women (mean age, 53.9 +/- 5.6 years; mean BMI, 22.8 +/- 2.8 kg/m) were randomly treated with EPT every other day or every day for 1 year. Fifty women received oral administration of 0.625 mg of conjugated equine estrogen (CEE) and 2.5 mg of medroxyprogesterone acetate (MPA) every other day, and 49 women received oral administration of 0.625 mg of CEE and 2.5 mg of MPA every day. Blood samples were collected at baseline and after 1 year of therapy for measurement of fasting TG, total cholesterol, high-density lipoprotein-cholesterol (HDL-C), and apolipoproteins. Data from 88 of the 99 postmenopausal women were used for analysis. In women whose BMI was 25 kg/m or higher, TG levels during EPT every day increased by 26.8%, while TG levels during EPT every other day decreased by 12.3%. There was a significant (P < 0.05) difference between percentage changes in TG during EPT every day and every other day. In women whose BMI was less than 25 kg/m, TG levels during EPT every day increased by 21.7%, while during EPT every other day TG levels did not change. The mean levels of estradiol during EPT every day in women whose BMI was less than 25 kg/m and in women whose BMI was 25 kg/m or higher were 28.5 and 38.7 pg/mL, respectively, the difference between these levels was significant (P < 0.01). On the other hand, there was no significant difference between levels of estradiol during EPT every other day in these two BMI groups. Triglyceride levels during EPT every day with conventional doses of CEE and MPA increased more in overweight and obese postmenopausal women in association with increased estrogen levels.


Serum estrogen level after hormone replacement therapy and body mass index in postmenopausal and bilaterally ovariectomized women

The objective of this study was to determine the relationships of serum estrogen levels after hormone replacement therapy (HRT) every other day and every day with body mass index (BMI) in postmenopausal and bilaterally ovariectomized women. Eighty-six postmenopausal and 51 bilaterally ovariectomized women who had been suffering from vasomotor symptoms such as hot flush or atrophy of the vagina were randomly treated with HRT every other day or every day. Seventy-four patients received oral administration of 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) every other day, and 63 patients received oral administration of 0.625 mg CEE and 2.5 mg MPA every day as conventional HRT. Eighty-four postmenopausal and 50 bilaterally ovariectomized women completed this study. Serum estradiol levels after HRT every day in postmenopausal and bilaterally ovariectomized women were significantly (P <0.05 and <0.01, respectively) correlated with BMI, while those after HRT every other day were not correlated with BMI. The differences between estradiol levels after 12 months of treatment and initial estradiol levels were also significantly (P <0.01) correlated with BMI in both postmenopausal and bilaterally ovariectomized women who received HRT every day but not in women who received HRT every other day. Serum estrone level after HRT every day and the difference between estrone level after 12 months of treatment and initial estrone level were significantly (P <0.05 and <0.01, respectively) correlated with BMI only in bilaterally ovariectomized women. Serum estradiol levels after HRT every day increase more in overweight women than in non-overweight postmenopausal and bilaterally ovariectomized women. The results of the present study regarding the relationship between serum estradiol levels after HRT and BMI should be useful for selecting dosages of drugs to be used in HRT.


低用量HRT施行中の腰椎骨密度の長期的推移 --- 結合型エストロゲンと酢酸メドロシキプロゲステロンの隔日投与の場合


隔日投与によるホルモン補充療法の再考

隔日投与によるホルモン補充療法の再考


Serum estradiol concentration as measured by HPLC-RIA and bone mineral density in postmenopausal women during hormone replacement therapy

To determine the relationship between the serum estradiol concentration and bone mineral density (BMD) of the lumbar spine in postmenopausal women treated with conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) every other day and every day. Eighty-four postmenopausal women were randomly treated with hormone replacement therapy (HRT) every other day and every day. Forty-seven women received oral administration of 0.625 mg CEE and 2.5 mg MPA every other day, and 37 women received oral administration of 0.625 mg CEE and 2.5 mg MPA every day. BMD of the lumbar spine at 12 months and serum concentrations of estradiol and estrone at 6 and 12 months after treatment were examined. The estradiol concentration in subjects treated every other day showed a significant (p < 0.001) positive correlation with the percentage change in lumbar BMD, while that in subjects treated every day was not correlated with the percentage change in BMD. The differences between serum estradiol concentrations after 12 months of treatment and initial serum estradiol values in women treated every other day and every day also showed a significant (p < 0.01 and 0.05, respectively) positive correlation with percentage changes in BMD. In women treated every other day, body mass index (BMI) in the subjects in whom BMD did not increase was significantly (p < 0.01) lower than that in the subjects in whom BMD did increase. The serum estradiol concentration in women treated every other day has a strong positive correlation with the percentage change in lumbar BMD, but a higher estradiol concentration may be needed for women in whom BMD did not increase with HRT every other day after due consideration of individual characteristics such as BMI.


Circulating osteoprotegerin in women during GnRH-agonist treatment and their relationships with mineral components and biomarkers of bone turnover

A novel cytokine termed osteoprotegerin (OPG) that is also called osteoclastogenesis-inhibitory factor, which inhibits osteoclast maturation and activity, was recently isolated. In order to determine the influence of estrogen deficiency on the levels of circulating OPG in women, we studied the changes in the levels of circulating OPG in 10 Japanese women ages 25-49 (mean +/- SD, 34.0 +/- 6.9) years with endometriosis receiving gonadotropin-releasing hormone agonists (GnRH-a) therapy. We further analyzed whether the levels of circulating OPG have relations with the levels of the biomarkers of bone turnover or those of circulating mineral components in these patients during GnRH-a treatment. The patients were treated with a monthly injection of 3.75 mg leuprolide acetate depot for 6 months. In all patients, the concentrations of serum estradiol decreased after 6 months of GnRH-a treatment. The bone mineral density of the lumber spines in these patients significantly (P < 0.01) decreased (percentage change: mean +/- SD, -5.4 +/- 2.1%), while circulating OPG levels significantly (P < 0.01) increased after 6 months of treatment. The values of circulating OPG had significant correlations with those of urinary pyridinoline (r = 0.59, P < 0.01), urinary deoxypylridinoline (Dpd) (r = 0.46, P < 0.05), and serum alkaline phosphatase (r = 0.66, P < 0.01) but not with those of serum carboxy-terminal propeptide of type I procollagen during GnRH-a treatment. The values of circulating OPG also correlated significantly with those of serum calcium (Ca) and phosphorus (P) (r = 0.65 and 0.72, P < 0.01). Further analyses revealed that the percentage change in the value of circulating OPG had a significant correlation with that of urinary Dpd (r = 0.84, P < 0.01). These results suggest that circulating OPG levels rise against the increase in osteoblastic bone resorption and circulating Ca levels in the case of estrogen deficiency, possibly as a compensatory mechanism serving to limit circulating Ca levels and bone density.


Hormone replacement therapy in postmenopausal women.

Hormone replacement therapy (HRT) is effective for relieving vasomotor symptoms such as hot flash and vaginal atrophy and for preventing bone loss in postmenopausal and bilaterally ovariectomized women. However, compliance with HRT was reported to be low despite the benefits of HRT. In addition, results of several recent large-scale randomized clinical trials have demonstrated that protection from cardiovascular disease is not an indication for treatment with estrogen and progestin in postmenopausal women. Recent studies have demonstrated that low-dose HRT is safe and effective for prevention of postmenopausal bone loss. Low-dose HRT has also been shown to be effective for reducing the number and severity of hot flashes, improving vaginal atrophy, and inducing favorable changes in lipids, lipoproteins and hemostatic factors. Moreover, low-dose regimens of CEE (conjugated equine estrogen) and MPA (medroxyprogesterone acetate) result in higher rates of amenorrhea and endometrial protection compared with the conventional dose of HRT. Low-dose HRT may improve the compliance rate and may be more effective than conventional-dose HRT for reducing the risk of breast cancer. On the other hand, it has been shown that transdermal estrogen treatment reduces the incidence and severity of hot flashes and that long-term treatment with transdermally administered estrogen is effective for protection against osteoporosis. Transdermal administration of estrogen is recommended in postmenopausal women with hypertriglycemia because this treatment has little effect on lipid metabolism. The serum estradiol level was reported to be closely related to estrogenic effects on various tissues. An HRT regimen should be based on the needs of each patient. Serum estradiol levels in women should be maintained at appropriate levels for benefits and not be excessively high in order to prevent side effects. Selection of the most appropriate regimen of HRT (dose, route of administration and schedule) for the needs of the individual are important factors to increase the rate of continuation with HRT.

The Journal of Medical Investigation : JMI


ラット卵巣細胞培養系におけるCINC産生に及ぼす温経湯の作用機序に関する検討

ラット卵巣細胞培養系におけるCINC産生に及ぼす温経湯の作用機序に関する検討

産婦人科漢方研究のあゆみ

産婦人科漢方研究のあゆみ


自然閉経後ならびに両側卵巣摘出後婦人における内因性エストロゲン濃度と腰椎骨密度の比較検討


隔日投与によるホルモン補充療法施行中の腰椎骨密度の長期的変化

隔日投与によるホルモン補充療法施行中の腰椎骨密度の長期的変化


血中エストロゲン濃度の変動による血中osteoprotegerin(OPG)濃度の推移の検討


Stimulatory Effect of the Herbal Medicine Keishi-bukuryo-gan on a Cytokine-induced Neutrophil Chemoattractant,in Rat Ovarian Cell Culture

We investigated the effects of Keishi-bukuryo-gan, a Japanese herbal medicine, and its crude ingredients in relation to the production of cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), which are known to stimulate the secretion of CINC/gro in the ovulatory process, and the effects of Keishi-bukuryo-gan with those of Toki-shakuyaku-san, which has been shown to have an effect on the ovary. We cultured whole ovarian dispersates from immature (3-week-old) female rats with Keishi-bukuryo-gan, Toki-shakuyaku-san and crude ingredients of Keishi-bukuryo-gan. The contents of CINC/gro, IL-1beta and TNFalpha in the cultured media were determined by enzyme-linked immunosorbent assay (ELISA). Keishi-bukuryo-gan stimulated the secretion of CINC/gro in a dose-dependent manner, and the secretion of CINC/gro into the culture medium increased significantly at concentrations of Keishi-bukuryo-gan of 10 and 100 microg/mL (P < 0.001). The stimulatory effect of Keishi-bukuryo-gan on the production of CINC/gro is significantly (P < 0.001) stronger than that of Toki-shakuyaku-san at the same concentrations of 100 microg/mL. In addition, Keishi-bukuryo-gan stimulated the secretion of IL-1beta in a dose-dependent manner, while it did not stimulate the secretion of TNFalpha even at a concentration of 100 microg/mL. Moutan Cortex, Paeoniae Radix and Persicae Semen, which are crude ingredients of Keishi-bukuryo-gan, enhanced the secretion of CINC/gro significantly (P < 0.01) in cultured whole ovarian dispersates. These results show that Keishi-bukuryo-gan can stimulate the secretion of CINC/gro as well as the production of IL-1beta and that this stimulatory effect of Keishi-bukuryo-gan was significantly stronger than that of Toki-shakuyaku-san in immature rat ovarian cell culture.

American Journal of Reproductive Immunology


ホルモン補充療法中の骨密度の増加に必要な血中エストラジオール濃度の検討

ホルモン補充療法中の骨密度の増加に必要な血中エストラジオール濃度の検討


The herbal medicine Unkei-to stimulates the secretion of a cytokine-induced neutrophil chemoattractant, CINC/gro, in the rat ovarian cell culture

We investigated the ovulation-inducing effects of Unkei-to, a Japanese herbal medicine, in relation to the production of sex steroid hormones (17beta-estradiol and progesterone), cytokine-induced neutrophil chemoattractant (CINC/gro), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) in the rat ovarian cell culture. Unkei-to at a concentration of 100 microg/mL significantly stimulated the secretions of 17beta-estradiol and progesterone (P < 0.01) in cultured whole ovarian dispersates. Unkei-to also enhanced the secretion of CINC/gro in a dose-dependent manner, and the secretions of CINC/gro increased significantly at concentrations of 10 and 100 microg/mL (P < 0.01). These stimulatory effects of Unkei-to on steroidgenesis and CINC/gro production are very similar to those of another Japanese herbal medicine, Toki-Shakuyaku-san. In addition, Unkei-to significantly (P < 0.01) enhanced the secretions of both IL-1beta and TNF-alpha, which are known to stimulate the secretion of CINC/gro in the ovulatory process, at concentrations of 10 and 100 microg/mL. The stimulatory effect of Unkei-to at a concentration of 100 microg/mL on IL-1beta/was significantly (P < 0.01) lower than that of Toki-Shakuyaku-san, while the stimulatory effects of these two herbal medicines at a concentration of 100 microg/mL on TNF-alpha were similar. These results show that Unkei-to can stimulate ovarian steroidgenesis and the ovulatory process by inducing the secretion of CINC/gro with IL-1beta and TNF-alpha in vitro. Unkei-to has stimulatory effects on both steroidgenesis and the ovulatory process in the ovary as well as a stimulatory effect on the hypothalamus-pituitary axis, and it may be useful for treating patients with ovulatory disorders.


Serum Leptin Levels in Women throughout Life;Relationship to Body Mass Index and Serum Estradiol Levels

Japanese Journal of Reproductive Endocrinology


Serum osteoprotegerin/osteoclastogenesis-Inhibitory factor during pregnancy and lactation and their relationships with calcium regulating hormones and bone turnover markers

Pregnancy and lactation induce dynamic changes in maternal bone and calcium metabolism. A novel cytokine termed osteoprotegerin (OPG)/osteoclastogenesis-inhibitory factor (OCIF) was recently isolated; this cytokine inhibits osteoclast maturation. To define the effects of pregnancy and lactation on circulating OPG/OCIF in mothers, we studied the changes in the levels of OPG/ OCIF as well as those of calcium-regulating hormones and biochemical markers of bone turnover in the maternal circulation during pregnancy (at 8-11 weeks, at 22-30 weeks, at 35-36 weeks and immediately before delivery) and lactation (at 4 days and at 1 month postpartum). Serum intact parathyroid hormone levels did not change and were almost within the normal range in this period. In contrast, serum 1,25-dihydroxyvitamin D levels increased with gestational age and were above the normal range during pregnancy. After delivery, they fell rapidly and significantly (P<0.01) to the normal range. The levels of serum bone-specific alkaline phosphatase, one of the markers of bone formation, increased with gestational age. After delivery, these levels were further increased at 1 month postpartum. The levels at 1 month postpartum were significantly higher than those at 8-11 and 22-30 weeks of pregnancy (P<0.01 and P<0.05 respectively). The levels of serum C-terminal telopeptides of type I collagen, one of the markers of bone resorption, did not change during pregnancy. After delivery, they rapidly and significantly (P<0.01) rose at 4 days postpartum, and had then fallen by 1 month postpartum. Circulating OPG/OCIF levels gradually increased with gestational age and significantly (P<0.01) increased immediately before delivery to 1.40+/-0.53 ng/ml (means+/-S.D.) compared with those in the non-pregnant, non-lactating controls (0.58+/-0.11 ng/ml). After delivery, they fell rapidly to 0.87+/-0.27 ng/ml at 4 days postpartum and had fallen further by 1 month postpartum. These results suggest that the fall in OPG/OCIF levels may be partially connected with the marked acceleration of bone resorption after delivery.


閉経後婦人における内因性エストロゲン濃度と腰椎骨密度の検討

閉経後婦人における内因性エストロゲン濃度と腰椎骨密度の検討


血中エストロゲン濃度の変動による血中osteoprotegerin(OPG)濃度の推移および骨代謝との関係の検討


ラット卵巣細胞培養系におけるケモカイン産生に及ぼす温経湯の効果に関する検討

ラット卵巣細胞培養系におけるケモカイン産生に及ぼす温経湯の効果に関する検討

産婦人科漢方研究のあゆみ

産婦人科漢方研究のあゆみ


The Obesity in Bilateral Ovariectomized Rats is Related to a Decrease in the Expression of Leptin Receptors in the Brain

We investigated the expression levels of leptin receptors in the brain of ovariectomized (OVX) rats. The mean expression level of ob mRNA in adipose tissues of OVX rats was significantly (P < 0.01) lower than that in the SHAM operation group rats, and the mean body weight of OVX rats was significantly (P < 0.01) greater than that in the SHAM group rats. However, there were no differences between serum leptin concentrations in these two groups. The mean level of leptin receptor (OB-R) mRNA expression in the brain tissue and the mean level of long form type OB-R (OB-RL) mRNA expression in the hypothalamus of the OVX rats were significantly (P < 0.05) lower than those in the SHAM group rats. These changes were cancelled by supplementation with 17 beta-estradiol in OVX rats. These results suggested that not only changes in the expression level of ob mRNA in adipose tissue and the serum leptin concentration but also changes in the OB-R mRNA in the brain are involved in the body weight increase in OVX rats and that a decrease in OB-R makes transmission of signals to suppress the amount of food intake difficult, thus leading to an increase in body weight.

Biochemical and Biophysical Research Communications


Effects of leptin on gonadotropin secretion in juvenile female rat pituitary cells


Efficacy of every-other-day administration of conjugated equine estrogen and medroxyprogesterone acetate on gonadotropin-releasing hormone agonists treatment in women with endometriosis

We performed a randomized controlled study to determine the efficacy of add-backed therapy by every-other-day administration of 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) on GnRH agonists (GnRH-a) treatment in Japanese women with symptomatic endometriosis. At the end of treatment, serum estrone and estradiol levels in the add-back group (n = 11) were significantly higher than those in the control group (n = 10). The assessment of Beecham classification by bimanual examination, serum CA-125 levels, and the frequency of genital bleeding revealed no significant differences between the two groups. The add-back group showed reduced Kupperman indices relative to those of the control group, and could prevent the loss of bone density. These findings led to a conclusion that GnRH-a therapy added back by every-other-day administration of 0.625 mg CEE and 2.5 mg MPA was a safe and effective treatment for Japanese women with endometriosis.


Postmenopausal changes in production of type 1 and type 2 cytokines and the effects of hormone replacement therapy

An appropriate defense against infective agents or malignant cells is attributed to the exquisitely balanced T helper 1 type (cellular) and T helper 2 type (humoral) immune reactions. We investigated the effect of hormone replacement therapy (HRT) on postmenopausal changes in the production of interferon (IFN)-gamma and interleukin (IL)-10, a type 1 and a type 2 cytokine, respectively. Both cytokines were measured by ELISA in the supernatant of lipopolysaccharide-stimulated whole blood cells from 72 untreated and 44 HRT-treated women. Thirteen women were examined before and during HRT. The production of IFN-gamma in women in their 40s and in postmenopausal women was significantly higher compared with that of younger women. However, IFN-gamma fell to the lowest level in the late postmenopausal stage, whereas the production of IL-10 increased gradually with age and in parallel with the postmenopausal period. Thus, in women in the mid-and late postmenopausal period, excessive production of type 2 cytokine (IL-10) compared with type 1 cytokine (IFN-gamma) occurred. The IFN-gamma levels of women on HRT were significantly lower than those of untreated women in the early and mid-postmenopausal stages, and IL-10 levels of women on HRT were significantly lower than those of untreated women in the mid-and late postmenopausal stages. HRT induced a significant decrease in the production of IL-10 and tended to lower the level of IFN-gamma. Production of IL-10 is augmented in postmenopausal women. HRT probably prevents postmenopausal women from an aberration of the immune system by improving the balance of type 1 and type 2 immune reactions.


Effects of leptin on secretion of LH and FSH from primary cultured female rat pituitary cells

Leptin, which is the product of the obese gene, is believed to play important roles in pubertal development and reproductive function in females. In a study using adult male rats, it was found that leptin stimulated secretion of gonadotropin from the pituitary in a dose-related manner. However, there has been no such study in female rats. To investigate the effects of leptin on the production of LH and FSH from the pituitary in female rats, using primary cultured pituitary cells. In this study, we determined body weight, serum leptin concentration and serum estradiol (E(2)) concentration in female Wistar rats at 3, 5, 6, 7, 9 and 11 weeks of age, and cultured pituitary cells from 6-week-old female Wistar rats with leptin (0--10(-7) mol/l) and GnRH (0 or 10(-8) mol/l). Then basal and GnRH-stimulated extra- and intracellular LH and FSH were assayed by RIA. Serum leptin concentration increased with increases in body weight and E(2) concentration. The pubertal serum leptin concentration was about 10(-10) mol/l. At a lower or moderate concentration, leptin produced dose-related increases in both basal and GnRH-stimulated extra- and intracellular LH and FSH in pituitary cells. At a concentration of 10 mol/l, leptin significantly (P<0.05) stimulated both basal and GnRH-stimulated extra- and intracellular LH and FSH. However, at greater concentrations, these effects diminished. These results indicated that leptin induced pituitary cells to produce and secrete both LH and FSH, with or without GnRH. The concentration of leptin that induced the greatest production of gonadotropins by pituitary cells was 10(-10) mol/l, which was the same as the physiological pubertal concentration. Leptin may be involved in the onset of puberty. It is also conceivable that leptin may be a cause of ovulatory failure, not only in weight loss but also in weight gain.


ホルモン補充療法中の骨密度の増加にエストロンは関与するか?

ホルモン補充療法中の骨密度の増加にエストロンは関与するか?


妊娠·産褥期の血中Osteoprotegerin(OPG)/Osteoclastgenesis Inhibitory Factor(OCIF)濃度の推移及び骨代謝との関係の検討 Osteoporosis Japan


Orexin-A Suppresses the Pulsatile Secretion of Luteinizing Hormone via β-Endorphin

Orexins, the novel hypothalamic neuropeptides that stimulate feeding behavior, have been shown to suppress the pulsatile secretion of LH in ovariectomized rats. However, the mechanism of this action is still not clear. We examined the effect of naloxone, a specific opioid antagonist, on the suppression of the pulsatile secretion of LH by orexins to determine whether beta-endorphin is involved in this suppressive effect. We administered orexins intracerebroventricularly and injected naloxone intravenously in ovariectomized rats, and we measured the serum LH concentration to analyze the pulsatile secretion. Administration of orexin-A significantly reduced the mean LH concentration and the pulse frequency, but coadministration of naloxone significantly restored the mean LH concentration and the pulse frequency. Administration of orexin-B also significantly reduced the mean LH concentration and the pulse frequency, and coadministration of naloxone did not restore them. These results indicate that orexin-A, but not orexin-B, suppresses GnRH secretion via beta-endorphin.


Postmenopausal changes in serum cytokine levels and hormone replacement therapy

Our purpose was to investigate the effect of hormone replacement therapy on the postmenopausal changes in serum cytokine levels. Fifteen cytokines were measured by an enzyme-linked immunosorbent assay in 97 untreated and hormone replacement-treated women. Thirteen women were examined before and during hormone replacement therapy. Serum concentrations of macrophage colony-stimulating factor were significantly (P < .05) lower during the early postmenopausal period (< or = 10 years) than the values in premenopause and the elevated levels in the late postmenopausal period (< or = 30 years). A significant increase in tumor necrosis factor alpha and a decline in transforming growth factor beta1 were found in late postmenopausal women. Serum levels of macrophage colony-stimulating factor in women receiving hormone replacement therapy were significantly higher than those in untreated postmenopausal women. Furthermore, hormone replacement therapy induced a significant (P < .01) increase in serum levels of macrophage colony-stimulating factor, whereas serum levels of other cytokines were not affected. It is well documented that macrophage colony-stimulating factor lowers serum cholesterol concentrations and prevents atherosclerosis. Inducing the production of macrophage colony-stimulating factor is a possible additional mechanism of hormone replacement therapy in mediating the antiatherogenic effect.

American Journal of Obstetrics and Gynecology


B cell subsets in postmenopausal women and the effect of hormone replacement therapy

In elderly subjects the capacity for antibody production is depressed. This immunosenescence state of humoral immunity is associated with the occurrence of autoimmune disorders involving CD5+ B (B-1) cells. Since estrogen is capable of stimulating the production of autoantibodies, this sex steroid hormone may be a contributing cause of the higher incidence of autoimmune diseases in women. In the present study, B cell subsets in women during the postmenopausal period was determined. The effect of hormone replacement therapy (HRT) on B cell subsets was examined to establish whether the administration of gonadal hormones influence humoral immunity in postmenopausal women. Forty six untreated pre- and postmenopausal women and 39 women on HRT were studied. The proportion of B-1 (CD5+) and conventional CD5- B (B-2) lymphocytes was determined by two-color flow cytometry. Serum autoantibodies to a nuclear antigen and to interleukin (IL)-1alpha were measured by immunofluorescence and by radioimmunoassay, respectively. Thirteen women were examined prospectively before and during HRT. In late postmenopausal women (> or = 30 years postmenopausal period), the proportion of B-2 cells was significantly reduced (p<0.01) compared to those of premenopausal and perimenopausal women. HRT induced a significant (p<0.01) increase in the percentage of B-2 cells, while that of B-1 cells remained unchanged. HRT did not affect autoantibody production. HRT may retard the progress of immunosenescence by increasing the production of B-2 cells. Moreover, HRT appears not to increase the risk of autoimmune diseases developing in postmenopausal women.


Expression of Ovarian Prolactin Receptor in Relation to Hormonal Changes during Induction of Ovulation in the Rat

We examined the relationships between the expression of the short and long forms of the prolactin (PRL) receptor (PRLR) mRNA in the ovary and changes in the levels of serum hormones such as sex steroid hormones and PRL during induction of ovulation in the rat. The expression of both forms of PRLR mRNA in the ovary was examined by Northern blot analysis in immature female rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovarian tissues and blood samples were obtained before treatment, 24 and 48 h after PMSG injection and 4, 6, 8, 12, 24 and 48 h after hCG treatment. Serum levels of 17beta-estradiol, progesterone and PRL were determined by radioimmunoassay. Serum levels of 17beta-estradiol rapidly increased to a maximal level 48 h after PMSG injection and then rapidly declined until 4 h after hCG injection. Serum levels of progesterone gradually increased after PMSG treatment, markedly increased to 114.2 nmol/l 8 h after hCG treatment and remained high until 48 h after hCG treatment. The serum level of PRL peaked at 66.2 microg/l (p < 0.01) 48 h after PMSG injection, and a temporary decrease after hCG treatment was followed by a continuously high level from 8 to 48 h. The expression of the long form of PRLR mRNA increased significantly (p < 0.01) to 688% of the control level after PMSG treatment, while that of the short form increased to only 184% of the control level. The expression of the long form of PRLR-mRNA rapidly declined until 6 h and then gradually increased until 48 h after hCG treatment. On the other hand, the expression of the short form of PRLR mRNA decreased to a nadir 12 h after hCG injection and then increased significantly (p < 0.01) to 142% of the control level. Our results showed that the changes in the short and long forms of PRLR mRNA differed in a time-specific manner and that these two forms are involved in different functions in the rat ovary during induction of ovulation. It is thought that the long form of PRLR mRNA is involved in folliculogenesis, while the short form of PRLR mRNA may play an important role in the formation and maintenance of the corpus luteum in the rat ovulatory cycle.


Transient Increase in the Levels of T-Helper 1 Cytokines in Postmenopausal Women and the Effects of Hormone Replacement Therapy

The aim of this study was to determine, at least in part, T-cell function in postmenopausal women and the effects of hormone replacement therapy (HRT). Levels of T-helper 1 (Th1) cytokines (IL-2 and IFN-gamma) and T-helper 2 (Th2) cytokines (IL-4 and IL-10) produced by phytohemagglutinin-stimulated whole blood cells from 72 untreated and 44 HRT-treated women were measured by ELISA. Thirteen of the 44 HRT-treated women were examined before and during HRT. The production of IL-2 increased gradually with advance of the postmenopausal period. The levels of IL-2 in women in the early (< or =10 years) and mid (>10 and <30 years) postmenopausal stages were significantly higher than those in women in their second, third and fourth decades. The level in women in the late (> or =30 years) postmenopausal stage, however, was significantly lower than those in women in the early and mid postmenopausal stages. The level of IFN-gamma was highest in women in the mid postmenopausal stage. On the other hand, the levels of Th2 cytokines did not change with age or after menopause until the mid postmenopausal period but were significantly lower in women in the late postmenopausal stage. IFN-gamma levels in women on HRT were significantly lower than those in untreated postmenopausal women at all postmenopausal stages. HRT induced a significant decrease in the production of IL-2 and IL-4. In conclusion, production of Th1 cytokines is augmented in women after menopause. HRT prevents this increase, thereby improving the aberration of Th1/Th2 balance that is implicated in an inadequate immune response and pathological conditions.


Biological Effects of Hormone Replacement Therapy in Relation to Serum Estradiol Levels

Tissues in various parts of the body have different sensitivities to estradiol. However, it is very difficult to measure the serum estradiol levels precisely in women receiving oral conjugated equine estrogen, which is a mixture of estrogens. In the present study, we precisely measured the serum levels of estradiol in postmenopausal women undergoing hormone replacement therapy (HRT), and we clarified the relationships between serum estradiol levels and the effects of HRT on the Kupperman index, bone mineral density (BMD), serum gonadotropin, lipid metabolism and unscheduled bleeding as the clinical endpoints. Sixty-eight postmenopausal or bilaterally ovariectomized women, aged 30-64 years, who had been suffering from vasomotor symptoms such as hot flush or atrophy of the vagina were randomly assigned to two groups: one group of 34 patients who received oral administration of 0.625 mg conjugated equine estrogen (CEE, Premarin, Wyeth) and 2.5 mg medroxyprogesterone acetate (MPA, Provera, Upjohn) every other day, and another group of 34 patients who received oral administration of 0.625 mg CEE and 2.5 mg MPA every day. All subjects were re-classified into three groups according to the serum estradiol level after 12 months of treatment: (1) low estradiol group (<15 pg/ml, n = 25); (2) middle estradiol group (> or =15 and <25 pg/ml, n = 27), and (3) high estradiol group (> or =25 pg/ml, n = 16). We examined the relationships between serum estradiol level and the effects of estradiol on the Kupperman index, BMD, serum gonadotropin levels, lipid profile and unscheduled bleeding in these three groups. RESULTS obtained by using our newly developed high-performance liquid chromatography (HPLC)-radioimmunoassay (RIA) system clearly showed that the effects on each tissue in postmenopausal women receiving oral CEE and MPA is closely related to estradiol level. The effects of HRT on BMD, serum gonadotropin levels and lipid profile were shown to be clearly dependent on the serum estradiol levels, while the effect of HRT on the Kupperman index was independent of the serum estradiol level. Furthermore, it was also found that a very low concentration of estradiol (<15 pg/ml) was sufficient to suppress the serum LH and FSH levels and to relieve vasomotor symptoms, and that the minimum concentration of estradiol required to increase BMD was 15 pg/ml. On the other hand, the level of estradiol required to reduce total cholesterol, low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (Apo B) was found to be more than 25 pg/ml, while the level required to increase high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 (Apo A1) was at least 15 pg/ml. The incidence of unscheduled bleeding was also lower in the low estradiol group than in the other estradiol level groups. These results suggest that the different clinical endpoints have different response thresholds and thus reflect tissue sensitivity to estradiol levels achieved by HRT.


Evidence that Toki-shakuyaku-san and its Ingredients Enhance the Secretion of a Cytokine-Induced Neutrophil Chemoattractant (CINC/gro) in the Ovulatory Process

We investigated the effects of Toki-shakuyaku-san and its crude ingredients in relation to the secretion of a cytokine-induced neutrophil chemoattractant, CINC/gro, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) in the ovulatory process. Toki-shakuyaku-san significantly (p < 0.01) stimulated the secretion of 17 beta-estradiol but did not stimulate the secretion of progesterone in cultured whole ovarian dispersates. Toki-shakuyaku-san enhanced the secretion of CINC/gro in a dose-dependent manner and the production of CINC/gro at concentrations of 10 and 100 micrograms/ml of Toki-shakuyaku-san increased significantly (p < 0.01). Toki-shakuyaku-san also enhanced secretions of both IL-1 beta and TNF alpha, which are known to stimulate the secretion of CINC/gro in the ovulatory process. The production of TNF alpha increased significantly (p < 0.05) with 10 and 100 micrograms/ml of Toki-shakuyaku-san. Atractylodis Lanceae Rhizoma, Cnidii Rhizoma, Angelicae Radix, Paeoniae Radix and Alismatis Rhizoma, which are crude ingredients of Toki-shakuyaku-san, significantly (p < 0.01) enhanced the secretion of CINC/gro at concentrations of 100 micrograms/ml. The results of this study show that Toki-shakuyaku-san can stimulate the secretion of 17 beta-estradiol and stimulate the ovulatory process by stimulating the production of CINC/gro, IL-1 beta and TNF alpha in vitro. As a treatment for ovulatory disorders, Toki-shakuyaku-san may have stimulatory effects on both steroidogenesis and the ovulatory process.

Methods and Findings in Experimental and Clinical Pharmacology


Effect of hormone replacement therapy on post-menopausal changes of lymphocytes and T cell subests

Immunosenescence is associated with the occurrence of lethal diseases, such as infection and malignancy. Since endocrinosenescence occurs simultaneously with immunosenescence, we determined whether or not lymphocytes and T cell subsets were altered in post-menopausal women. The ability of hormone replacement therapy (HRT) to reverse or modify the aberrations of the cell populations observed in elderly women was also examined. Thirty-nine untreated post-menopausal women and 39 women on HRT were studied. The proportions of lymphocytes and T cell subsets (helper, cytotoxic and immature T cells, and naive and memory/activated T cells) were determined by two color flow cytometry. Thirteen women were examined before and during HRT. At late post-menopause (> or = 30 years post-menopausal period), the proportion of peripheral blood lymphocytes showed a tendency to decline (p=0.06) compared with that at early (< or = 10 years) post-menopause. Significant (p<0.05) decrease in naive T cells and an increase in memory/activated T cells occurred at late post-menopause compared to those at early post-menopause. The percentage of lymphocytes in women on HRT was significantly (p<0.05) higher than that in untreated women at late post-menopausal stage. Furthermore, in a prospective study, HRT induced a significant (p<0.02) increase in the percentage of lymphocytes but showed no effect on the aberrations of naive and memory/activated T cells. HRT prevents the decline in the lymphocytes observed in post-menopausal women. However, HRT appears not to influence the observed alteration in T cell subsets.

Journal of Endocrinological Investigation


Close correlation between estrogen treatment and renal phosphate reabsorption capacity

To determine the influence of estrogen on the activity of renal proximal tubular reabsorption of inorganic phosphate (Pi) in women, we examined the changes of the renal threshold phosphate concentration (also denoted as TmP/GFR), as well as the changes in the concentrations of mineral components in the circulation in two groups of women--one receiving hormone replacement therapy (HRT) and one receiving gonadotropin-releasing hormone agonists (GnRH-a) therapy. We also examined the changes in the concentrations of serum PTH in the GnRH-a group. The patients in the HRT group were continuously treated with 0.625 mg conjugated equine estrogens plus 2.5 mg medroxyprogesterone acetate per day. The patients in the GnRH-a group were treated with a monthly injection of 3.75 mg leuprolide acetate depot for 6 months. The values of TmP/GFR decreased in all of the patients who received HRT. The mean percentage change in TmP/GFR was -14.5% (range, -24.3% to -9.6%). In contrast, in all of the patients treated with GnRH-a, the values of TmP/GFR increased after 6 months of treatment (the mean percentage change was 28.5%; range, 18.2-78.3%) and returned to the preadministration level at 12 weeks after stopping therapy. In these patients, both the values of TmP/GFR and the concentrations of serum Pi correlated significantly with circulating estradiol levels (r = -0.767, P < 0.01 and r = -0.797, P < 0.01, respectively), but the concentrations of serum corrected calcium did not correlate. Moreover, in the same patients, the levels of serum intact PTH decreased significantly (P < 0.05) after 6 months of treatment, but at 12 weeks after stopping therapy the trends of these levels varied among individual patients. These results suggest that estrogen could act directly to suppress sodium-dependent Pi reabsorption in the renal proximal tubules.

The Journal of Clinical Endocrinology and Metabolism


ホルモン補充療法中のエストロゲン濃度と骨代謝マーカーに関する検討

ホルモン補充療法中のエストロゲン濃度と骨代謝マーカーに関する検討


骨減少症ならびに骨粗鬆症に対するエストロゲン·プロゲスチンの連日投与法と隔日投与法の比較 --- 血中エストラジオール濃度との関係


Involvement of gonadal steroid hormone disturbance in altered prolactin receptor gene expression in the liver of diabetic mice

The levels of mRNA for long and three short forms of prolactin receptor (PRLR) were examined in the livers of normal (db+/db-) and insulin-resistant diabetic (db+/db+) mice to assess the role of gonadal steroid hormones in the regulation of PRLR gene expression in diabetes mellitus. In females, plasma levels of testosterone in diabetic mice were higher, and those of 17beta-estradiol were lower when compared with levels in normal mice. By contrast, diabetic male mice had lower plasma levels of testosterone than normal males and showed no significant difference in the low circulating level of 17beta-estradiol compared with normal males. The short 3 form of PRLR (PRLR3) mRNA was the most abundant in the liver of both normal and diabetic mice. In addition, the level of PRLR3 mRNA in normal females was 8-fold higher than in normal males. The level of PRLR3 mRNA in diabetic females was approximately a quarter lower than in normal females, whereas the level of PRLR3 mRNA in diabetic males was approximately 2-fold higher than in normal males. During postnatal development, the level of PRLR3 mRNA increased during puberty in normal females, while the level in diabetic females decreased to a nadir at 7 weeks of age followed by a progressive rise. On the other hand, the levels of PRLR3 mRNA in both normal and diabetic males decreased gradually during 5 to 14 weeks of age. Testosterone treatment of diabetic males and females resulted in a 49.1 and 49.8% decrease of PRLR3 mRNA respectively. 17beta-Estradiol treatment slightly (18%) increased levels of PRLR3 mRNA in diabetic males. These results suggest that the hepatic level of PRLR mRNA is regulated by the inhibitory effect of testosterone and the stimulatory effect of estrogen in both normal and diabetic mice.


Combination of automatic HPLC-RIA method for determination of estrone and estradiol in serum

We developed a highly sensitive assay for estrone and 17 beta-estradiol in serum. Estrone and 17 beta-estradiol, obtained by solid-phase extraction using a Sep pak tC18 cartridge, were purified by high-performance liquid chromatography (HPLC). Quantitation of estrone and 17 beta-estradiol were carried out by radioimmunoassay. Not insignificantly, this automatic system of extraction and HPLC succeeded in analyzing 80 samples a week. Intra-assay coefficients of variation (CV) for estrone and 17 beta-estradiol ranged from 19.5 to 28.7%, and from 8.5 to 13.7%, respectively. The minimum detectable dose for estrone and 17 beta-estradiol were 1.04 pg/ml and 0.64 pg/ml, respectively. The serum levels of 17 beta-estradiol using our method strongly correlated with those by Gas chromatography mass spectrometry (GC-MS). The serum levels of estrone and 17 beta-estradiol in 154 peri- and postmenopausal women were estimated to be between 15 and 27 pg/ml and between 3.5 and 24.0 pg/ml, respectively, while the serum level of 17 beta-estradiol in postmenopausal women, in particular, was estimated to be from 3.5 to 6.3 pg/ml. For postmenopausal women who suffered from vasomotor symptoms, the mean levels of estrone and 17 beta-estradiol at 12 to 18 hours after treatment with daily 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) were 135.0 and 21.3 pg/ml at 12 months, respectively. On the other hand, levels of estrone and 17 beta-estradiol at 12 to 18 hours after treatment with CEE and MPA every other day, were 73.4 and 15.3 pg/ml, respectively. These highly sensitive assays for estrone and 17 beta-estradiol are useful in measuring low levels of estrogen in postmenopausal women, and monitoring estrogen levels in women receiving CEE as hormone replacement therapy.


Measurement of N- and C-terminal-region fragments of parathyroid hormone-related peptide in milk from lactating women and investigation of the relationship of their concentrations to calcium in milk.

Parathyroid hormone-related peptide (PTHrP) is found in very high concentrations in the milk of various mammals. However, little is known about its physiological role in this fluid. To obtain detailed profiles of PTHrP in milk, we measured the concentrations of PTHrP in human milk by two different region-specific assays, PTHrP(1-87) (N-PTHrP) and PTHrP(109-141) (C-PTHrP). We also examined the correlations between PTHrP and Ca concentrations in milk as well as the correlations between PTHrP and secreted milk volume. The levels of N-PTHrP and C-PTHrP were relatively low after delivery and gradually increased to 13.87 +/- 2.40 nmol/l (mean +/- S.E.M.) and 56.39 +/- 11.31 nmol/l respectively on the 10th day postpartum. N-PTHrP concentration remained steady until the 6th month postpartum when weaning starts, at which point it decreased slightly. C-PTHrP levels changed in a similar way to N-PTHrP levels but were 2- to 5-fold higher. Milk Ca concentration, and content, correlated with C-PTHrP concentration, and content (r = 0.422 and r = 0.769 respectively; P < 0.0001) but not with N-PTHrP. N-PTHrP concentration in the milk samples on the 4th day postpartum correlated with the volume of milk secreted during the 24 h before the samples were taken (r = 0.524, P < 0.01), but C-PTHrP concentration did not. These results suggest that PTHrP in human milk may play some role in the maintenance of lactation through the N-terminal region and in promoting Ca transfer into milk via the C-terminal region.


Effects of transforming growth factor-β on the production of parathyroid hormone-related peptide in a human ovarian cancer cell line in vitro

Journal of Obstetrics and Gynaecology Research


[Assessment of serum estrogen levels by RIA with HPLC during hormone replacement therapy].

We studied the serum estradiol and estrone levels in 146 peri and postmenopausal women, and in 38 women who had complained of various climacteric disturbance symptoms during daily hormone replacement therapy (HRT) with conjugated equine estrogen (CEE) 0.625 mg and medroxyprogesterone acetate (MPA) 2.5 mg. Serum estradiol and estrone were measured before treatment, and at 6 months, and after one year of the HRT therapy by HPLC-radioimmunoassay. In 146 peri and postmenopausal women, the serum level of estradiol was from 3 to 6pg/ml. The serum level of estradiol in 38 women after HRT significantly increased (p < 0.01) from 3.34 to 23.6 pg/ml at 6 months, and 21.5 pg/ml at 12 months. The serum level of estrone significantly increased (p < 0.01) from 26.6pg/ml to 156.7pg/ml at 6 months, and 137.2pg/ml at 12 month. These results are very useful for deciding on the doses of hormones and the expected serum estradiol level in HRT for Japanese women.

Acta Obstetrica et Gynaecologica Japonica


Assessment of Serum Estrogen Levels by RIA with HPLC during Hormone Replacement Therapy

ホルモン補充療法における血中エストロゲン値の高感度測定法による検討


Studies on the Combination Treatment with Clomiphene Citrate and Toki-shakuyaku-san

排卵障害患者に対するクロミフェン·当帰芍薬散併用療法の有用性の検討


Mechanism of anovulation in hyperprolactinemic amenorrhea determined by pulsatile gonadotropin-releasing hormone injection combined with human chorionic gonadotropin


骨粗鬆症に対するホルモン補充療法および活性型ビタミンDの有効性に関する検討

骨粗鬆症に対するホルモン補充療法および活性型ビタミンDの有効性に関する検討


The effect of continuous combined regimen of estrogen progestin on the postmenopausal woman

中高年婦人および両側卵巣摘出婦人に対するエストロゲン·プロゲスチン持続併用療法の骨および脂質に対する検討

The Journal of the Japan Menopause Society


Inhibitory effect of Sho-saiko-to on the growth of gynecological cancer cell lines by autocrine mechanism of transforming growth factor-β

婦人科癌における小柴胡湯の抗腫瘍効果及び作用機序に関する検討


Interaction of Activin A and Gonadal Steroids on FSH Secretion from Primary Cultured Rat Anterior Pituitary Cells

Biochemical and Biophysical Research Communications


Maintenance of Androgen-,Glucocorticoid-or Estrogen-responsive Growth in Shionogi Carcinoma 115 Subline Subline Sustained in Castrated Mice with High Dose of Estrogen for 30 Generations(3 Years)

Gann : Japanese Journal of Cancer Research


Roles of Transforming Growth Factor β in Inhibition of Androgen-induced Growth of Shionogi Carcinoma Cellsin Serum-Free Medium


The mechanism of the effect of combination treatment with clomiphene and bromocriptine in patients with normoprolactinemic anovulation

Journal of Endocrinological Investigation


Studies of the Combination Treatment with Unkei-to and Clomiphene Citrate

クロミフェン無効の正プロラクチン血性排卵障害症例に対するクロミフェンー温経湯併用療法の検討


Effects of Androgen,Fibroblast Growth Factors or Other Various Growth Factors on Growth of Shinogi Carcinoma Cells in a Protein-Free Medium


Clinical Implication of Serum Antibody to Thomsen-Friednreich Antigen in Patients with Gynecological Malignancies

婦人科悪性腫瘍組織に発現するThomsen-Friedenreich抗原とその血中抗体価に関する臨床的研究


HRTの慎重投与例への対応 -7.慢性肝疾患，胆嚢炎および胆石症の既往

HRTの慎重投与例への対応 -7.慢性肝疾患，胆嚢炎および胆石症の既往


婦人科癌における小柴胡湯の抗腫瘍効果に関する検討

婦人科癌における小柴胡湯の抗腫瘍効果に関する検討


ラット下垂体腫瘍細胞のプロラクチン分泌に及ぼす漢方薬の効果に関する検討

ラット下垂体腫瘍細胞のプロラクチン分泌に及ぼす漢方薬の効果に関する検討

産婦人科漢方研究のあゆみ

産婦人科漢方研究のあゆみ


モノクロナール抗体を用いた逆受身赤血球凝集反応によるプロラクチン測定法の基礎的検討とその臨床的応用 --- 赤血球凝集反応による新しいプロラクチン測定試薬


治療に抵抗を示し，著明な血小板減少を来した再生不良性貧血合併妊娠の1症例

治療に抵抗を示し，著明な血小板減少を来した再生不良性貧血合併妊娠の1症例