Glucose transporter 1 is important for the glycolytic metabolism of human endometrial stromal cells in hypoxic environment.

AIM: The study aimed to elucidate the glycolytic metabolism of human endometrial stromal cells (hESCs) in hypoxic environment. MAIN METHODS: The hESCs were cultured in hypoxic environment, and their metabolic pathways were analyzed using metabolomics. We assessed glucose uptake using 2-deoxyglucose (2-DG) assay. The expression of glucose transporters (GLUTs) required for glucose uptake was determined using real-time quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, we knocked down GLUT1 and examined the uptake of 2-DG. KEY FINDINGS: Under hypoxia, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate were significantly elevated in hESCs (P < 0.05). This finding indicated enhancement in glycolysis. The volume of glucose uptake increased significantly under hypoxia (P < 0.05). Hypoxia simultaneously induced the expression of GLUT1 and GLUT3 mRNA (P < 0.05) and attenuated the expression of GLUT8 (P < 0.05). Glucose uptake was significantly inhibited upon knockdown of GLUT1 (P < 0.0001). SIGNIFICANCE: These results demonstrated a very important role of glucose transport under hypoxia. Also, hESCs utilize glycolysis to adapt to hypoxic conditions that could occur in menstrual and implantation period. These findings pave the way to study implantation failure and tumors originating from the endometrium.

Progestin-induced heart and neural crest derivatives-expressed transcript 2 inhibits angiopoietin 2 via fibroblast growth factor 9 in human endometrial stromal cells.

Heart and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). In the mouse, HAND2 plays an important role in uterine receptivity by suppressing several fibroblast growth factors (FGFs). However, the regulation of FGF family members by progestin-induced HAND2 and the role of FGF in vascular regeneration in the endometrium remains poorly understood. To investigate these molecular mechanisms, primary human ESCs were cultured with estradiol (E2), medroxyprogesterone acetate (MPA), progesterone receptor (PR) antagonist RU486, HAND2-specific small interfering RNA (siRNA), and recombinant FGF. The expression levels of FGF family members, HAND2, angiopoietin (ANGPT), and vascular endothelial growth factor (VEGF) were assessed by real-time PCR and ELISA. Out of six FGF genes known to be expressed in the human endometrium, only one, FGF9, was significantly downregulated in human ESCs after 3 days of progestin treatment. E2 + MPA attenuated the mRNA and protein levels of FGF9 during decidualization of ESCs, and this effect was blocked by RU486. Silencing of HAND2 significantly increased FGF9 expression in ESCs treated with E2 + MPA. Moreover, FGF9 activated FGF receptor in human ESCs, triggering ANGPT2 production, which resulted in enhancement of the ANGPT2/ANGPT1 protein ratio. Taken together, progestin-PR signaling and its target HAND2 play an essential role in FGF9 suppression in the human endometrium. In addition, progestin-induced HAND2 inhibits ANGPT2 production by suppressing FGF9 in ESCs. These results suggest that HAND2 may contribute to endometrial vascular maturation by regulating FGF9 during decidualization.

Decidualization of the human endometrium.

BACKGROUND: Decidualization of the human endometrium, which involves a dramatic morphological and functional differentiation of human endometrial stromal cells (ESCs), is essential for the establishment of a successful pregnancy. Decidualization results from a complex interplay of transcription factors, morphogens, cytokines, cell cycle regulators, and signaling pathways. METHODS: Based on a literature review, the regulation of, and the molecular mechanisms involved in, the decidualization of the endometrium are described. MAIN FINDINGS: Progesterone, together with proteins that are regulated by progesterone and/or cyclic adenosine monophosphate, including homeobox A10, forkhead box O1, signal transducers and activators of transcription, and heart and neural crest derivatives expressed transcript 2, forms a critical network for ESC decidualization and is a prerequisite to successful implantation. Decidualized ESCs contribute to the microenvironment at the feto-maternal interface and its direct or indirect influence on extracellular matrix remodeling, regulation of the local immune response, anti-oxidative stress, and angiogenesis (vascular maturation). Impairment of this process is associated with a variety of pregnancy disorders, including infertility, recurrent miscarriages, and uteroplacental disorders. CONCLUSION: A deeper understanding of the process of decidualization is expected to provide new insights into the fields of reproductive biology and reproductive medicine.

Effects of the hypoxia-inducible factor-1 inhibitor echinomycin on vascular endothelial growth factor production and apoptosis in human ectopic endometriotic stromal cells.

Recent evidence points to a possible role for hypoxia-inducible factor (HIF)-1 in the pathogenesis and development of endometriosis. The objectives of this study were to investigate the critical role of HIF-1 in endometriosis and the effect of the HIF-1 inhibitor echinomycin on human ectopic endometriotic stromal cells (eESCs). Ectopic endometriotic tissues were obtained from 20 patients, who received an operation for ovarian endometriomas. We examined vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) production, HIF-1 expression, cell proliferation and apoptosis of eESCs. Cobalt chloride (CoCl2) significantly induced expression of HIF-1α protein and VEGF production in a time-dependent manner in eESCs, but reduced SDF-1 production. VEGF production was significantly suppressed by treatment of 100 nM echinomycin without causing cell toxicity, but 0.1-10 nM echinomycin or 100 nM progestin had no significant effect. SDF-1 production was not affected by echinomycin treatment at any dose. Echinomycin inhibited cell proliferation and induced apoptotic cell death of the eESCs, and significantly inhibited expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Echinomycin inhibits VEGF production and induces apoptosis of eESCs by suppression of Bcl-2 and Bcl-xL. These findings suggest the unique therapeutic potential for echinomycin as an inhibitor of HIF-1 activation for endometriosis treatment.

Lipid-soluble fraction of Shakuyaku-kanzo-to inhibits myometrial contraction in pregnant women.

AIM: Shakuyaku-kanzo-to, a Kampo medicine composed equally of shakuyaku and kanzo, is an antispasmodic drug that can inhibit contraction of uterine smooth muscles in pregnant women and rats. We aimed to test the inhibitory effects of water- and lipid-soluble extracts of shakuyaku-kanzo-to, shakuyaku, and kanzo in order to identify the fraction responsible for inhibiting uterine smooth muscle contraction in pregnancy. MATERIAL AND METHODS: Myometrial tissues were obtained from pregnant women and rats. The water- and lipid-soluble fractions of shakuyaku-kanzo-to, shakuyaku, and kanzo were obtained using the method of Bligh and Dyer. Lipid-soluble fractions were also partially purified using thin-layer chromatography (TLC) with a chloroform : methanol : water (65:25:4 by volume) solvent system to yield four TLC fractions. The effect of each fraction on oxytocin-induced myometrial contraction was examined in vitro. RESULTS: Lipid-soluble fractions obtained from shakuyaku-kanzo-to and kanzo inhibited myometrial contraction; water-soluble fractions had no effect. Of the four TLC fractions, the inhibitory effect was greatest with TLC fraction 1 (0.75 < Rf value ≤ 1.0). Neither the water-soluble nor the lipid-soluble fraction from shakuyaku inhibited myometrial contraction. CONCLUSIONS: These results suggest that lipid-soluble substances with low polarity derived from kanzo are responsible for the inhibitory effect of shakuyaku-kanzo-to on myometrial contraction.

Effect of cigarette smoking on mRNA and protein levels of oxytocin receptor and on contractile sensitivity of uterine myometrium to oxytocin in pregnant women.

OBJECTIVES: Although smoking is the most important modifiable risk factor associated with preterm delivery, the underlying mechanism by which smoking stimulates premature uterine contractions is still poorly understood. In the present study, we investigated whether cigarette smoking affects the contractile sensitivity of uterine myometrium to oxytocin in pregnant women. STUDY DESIGN: Cigarette smoking habits of pregnant women were evaluated by direct interviews and by measuring exhaled carbon monoxide (CO). We isolated myometrial strips from pregnant smokers and non-smokers and evaluated uterine contractile sensitivity to oxytocin. Gene expression levels of oxytocin receptors (OTR) were compared between myometrial strips obtained from smokers and non-smokers by real-time PCR. OTR protein levels in the myometrium were evaluated by Western blotting. RESULTS: The reported number of cigarettes smoked per day by the interviewee significantly correlated with the concentration of exhaled CO. Oxytocin sensitivity increased significantly in smokers (n=6) compared with non-smokers (n=11). Real-time PCR analysis did not reveal any significant difference in OTR mRNA expression between smokers and non-smokers. Western blotting revealed that OTR level was significantly increased in smokers compared with non-smokers. Both number of cigarettes smoked per day and the concentration of exhaled CO correlated with oxytocin sensitivity. CONCLUSION: Our findings suggest that smoking increases oxytocin sensitivity of pregnant myometrium by increasing OTR levels even though OTR mRNA expression remains unaltered, thereby increasing the risk of preterm delivery in women who smoke during pregnancy. The sensitivity is dependent on number of cigarettes smoked per day.

Regulation of decidualization and angiogenesis in the human endometrium: mini review.

AIM: The human endometrium is a dynamic tissue that undergoes regular cycles of menstruation, menstrual repair, proliferation and secretory differentiation in response to hypoxia and the female sex hormones. METHODS: We identified new target genes that are regulated by progesterone during the decidualization of human endometrial stromal cells (ESC), including interleukin-15 (IL-15), fibulin-1 (FBLN-1), and heart and neural crest derivatives expressed transcript 2 (HAND2). RESULTS: IL-15 is deeply involved in the hormonal control of the human endometrium by progesterone and may be important in embryo implantation. FBLN-1 has been shown to be an important extracellular matrix that mediates progesterone action in human ESC differentiation toward implantation. Moreover, progestin-induced HAND2 is a transcription factor that contributes to the increased levels of FBLN-1 in human ESC. Several mediators, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT) and stromal cell-derived factor 1 (SDF-1), regulate human endometrial angiogenesis. Hypoxia increased the expression of VEGF and decreased the expression of SDF-1 in ESCs. Furthermore, hypoxia reduced ANGPT1 levels in ESC; however, ANGPT2 levels were unaffected. Estradiol simultaneously induced the expressions of VEGF and SDF-1, suppressing ANGPT1 production. Therefore, hypoxia and estradiol caused an increase in the ANGPT2/ANGPT1 ratio. CONCLUSION: Hypoxia and female sex hormones are involved in the regulation of angiogenic factors in an independent manner in human ESC. Analysis of the process of decidualization and angiogenesis in the human endometrium would provide useful information for the fields of reproductive biology, regenerative medicine and tissue engineering.

Two-step inhibitory effect of kanzo on oxytocin-induced and prostaglandin F2α-induced uterine myometrial contractions.

We previously reported that shakuyaku-kanzo-to, a kampo medicine consisting of shakuyaku and kanzo, has an inhibitory effect on myometrial contractions in pregnant women. In this study, we evaluated the effects of kanzo, glycyrrhizin (a major component of kanzo), glycyrrhetinic acid (GA; a major metabolite of glycyrrhizin), shakuyaku, and paeoniflorin (a major component of shakuyaku) on agonist-induced contractions of the uterus of pregnant humans and rats. We prepared myometrial strips from the uterus of pregnant humans and rats and induced contractions with oxytocin (50 µU/mL) or prostaglandin F2α (PGF2α) (10(-7) or 10(-6) M). Kanzo (250 µg/mL) and GA (5 × 10(-6) M) inhibited the oxytocin-induced and PGF2α-induced contractions in pregnant human and rat myometrium, but shakuyaku (250 µg/mL), paeoniflorin (10(-5) M), and glycyrrhizin (10(-5) M) did not inhibit contractions in either. Interestingly, kanzo and GA showed an inhibitory effect after temporarily enhancing the PGF2α-induced contractions in the rat myometrium, but not in the human myometrium. These results suggest that kanzo has at least a two-step inhibitory effect on the myometrial contractions that originate from the kanzo itself and a metabolite of glycyrrhizin in kanzo. Furthermore, kanzo was found to be safe for inhibiting PGF2α-induced contractions in humans because it did not temporarily enhance PGF2α-induced contractions.

Requirement of heart and neural crest derivatives-expressed transcript 2 during decidualization of human endometrial stromal cells in vitro.

OBJECTIVE: To investigate the role of heart and neural crest derivatives-expressed transcript 2 (HAND2) during decidualization of human endometrial stromal cells (ESCs). DESIGN: In vitro experiment. SETTING: Research laboratory. PATIENT(S): Twenty-six patients undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were cultured for 12 days with HAND2 small interfering RNA (siRNA) or nonsilencing RNA during decidualization by medroxyprogesterone acetate (MPA) and E2. MAIN OUTCOME MEASURE(S): Decidualization was monitored by changes in cellular morphology and the expression of several decidual-specific genes. RESULT(S): HAND2 siRNA effectively suppressed HAND2 levels in ESCs after 12 days of E2 + MPA treatment. ESCs cultured with HAND2 siRNA retained a long fibroblast-like shape, whereas the cells cultured with control siRNA transformed into enlarged polygonal cells. Silencing of HAND2 expression significantly reduced connexin-43 involved in the morphologic changes. HAND2 silencing significantly reduced the mRNA levels of fibulin-1, prolactin, tissue inhibitor of metalloproteinase 3, interleukin-15, and forkhead box O1A (FOXO1A), but had no effect on the mRNA levels of dickkopf-1, serum glucocorticoid kinase 1, and insulin-like growth factor-binding protein 5. HAND2 siRNA effectively suppressed the levels of nuclear FOXO1A protein as a regulator of decidualization. CONCLUSION(S): These results suggest that HAND2 plays a key role in the regulation of progestin-induced decidualization of human ESCs.

Effects of ovarian hormone treatment on the gene expression of muscarinic acetylcholine receptors in the ovariectomized rat myometrium.

We investigate the effects of ovarian hormone on the gene expression of muscarinic acetylcholine receptors (M1-M5) in the myometrium using real-time PCR and evaluate the relationships between their expression and that of ovarian hormone receptors (ERα, ERß, and PgR). Wistar rats were sham operated (SO) or ovariectomized (OVX) and treated with vehicle, estradiol (E2), progesterone (P4), or both E2 and P4 for 2 days beginning on postoperative day 33. M1 and M4 mRNA expressions were not detected in the myometrium. M2 mRNA expression did not change significantly in the OVX and OVX+P4 groups compared to the SO group, but increased significantly in the OVX+E2 group and was normalized in the OVX+E2P4 group. M3 mRNA expression increased significantly in the OVX and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2 and OVX+E2P4 groups. M5 mRNA expression did not change significantly in all experimental groups. ERα mRNA expression increased significantly in the OVX, OVX+E2, and OVX+P4 groups compared to the SO group, but was normalized in the OVX+E2P4 group. The changes in ERß mRNA expression were similar to those of M3 mRNA expression in all experimental groups. In contrast, the changes in PgR mRNA expression did not correspond with that of M2, M3, or M5 mRNA expression in any of the experimental groups. Additionally, we evaluated the relationship between the expression of muscarinic acetylcholine receptors and ovarian hormone receptors in estrus cycle. M2 mRNA expression increased significantly in diestus and metaestrus compared in proestrus and estrus. M3 mRNA expression increased significantly in only diestrus compared in the other stages. In contrast, M5 mRNA expression did not change in estrus cycle. The changes in ERα mRNA expression appeared to be similar to those of M2 in estrus cycle, but no significant difference was found. The changes in ERß mRNA expression were similar to those of M3 mRNA expression. The change in PgR mRNA expression increased significantly in diestrus compared in metaestrus, but did not correspond with that of M2, M3, or M5 mRNA expression in estrus cycle. When acetylcholine sensitivity in the myometrium was compared between diestrus and estrus, the sensitivity is significantly lower in estrus than in diestrus. These results suggest that ovarian hormones influence the expression of M2 and M3 in the myometrium by regulating the expression of hormone receptors. E2 may upregulate M2 via ERα, but P4 may downregulate M2 by inhibiting ERα via PgR. E2 may downregulate M3 by inhibiting ERß, but P4 may not regulate the expression of M3 and ERß. M5 may be a constitutive muscarinic receptor in the myometrium because neither E2 nor P4 influence the expression of M5. The combination of E2 and P4 may contribute the reproduction by quieting down the acetylcholine-induced myometrial contraction.

Divergent regulation of angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor by hypoxia and female sex steroids in human endometrial stromal cells.

OBJECTIVE: To investigate whether hypoxia or the female sex steroids exert direct effects on angiopoietin-1 (ANGPT1), ANGPT2, and vascular endothelial growth factor (VEGF) in human endometrial stromal cells (ESCs) to clarify the regulatory function of these local angiogenic factors in the endometrium. STUDY DESIGN: Human endometrial tissues were obtained from 18 patients aged 34-47 years undergoing hysterectomy for benign reasons. ESCs were cultured under hypoxic condition or treated with 17ß-estradiol (E) and/or medroxyprogesterone acetate (MPA). The mRNA levels and production of ANGPT1, ANGPT2, and VEGF were assessed by real-time RT-PCR and ELISA, respectively. Analysis of hypoxia-inducible factor 1α (HIF-1α) and estrogen receptor α (ERα) protein levels were evaluated by Western blot analysis. RESULT(S): Hypoxia reduced the mRNA expression and protein production of ANGPT-1 in ESCs, whereas those of ANGPT2 were unaffected, resulting in an increase of the ANGPT2/ANGPT1 ratio. Hypoxia induced mRNA expression and protein production of VEGF. E simultaneously induced VEGF production and suppressed ANGPT1 production, resulting in an increase of the ANGPT2/ANGPT1 ratio. MPA or E+MPA reduced ANGPT2 production and sustained the levels of ANGPT1, resulting in a decrease of the ANGPT2/ANGPT1 ratio. With regard to the interaction of E and hypoxia, E did not affect the regulation of angiogenic factors, HIF-1α, and ERα under hypoxic conditions. CONCLUSIONS: Hypoxia and female sex hormones independently regulate the ANGPT2/ANGPT1 ratio and VEGF expression in human ESCs. These results may indicate a potential mechanism for hypoxia or female sex steroids influencing angiogenesis in the human endometrium.

Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells.

OBJECTIVE: To investigate whether heart and neural crest derivatives expressed transcript 2 (HAND2) regulates fibulin-1 (FBLN1) expression during decidualization of human endometrial stromal cells (ESCs). DESIGN: In vitro experiment. SETTING: Research laboratory. PATIENT(S): Twenty-four patients undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were cultured with various progestins (medroxyprogesterone acetate [MPA], norethisterone, levonorgestrel, dienogest, and P), E(2), dexamethasone, and/or 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP). HAND2 and FBLN1 were silenced by small interfering RNA technology. MAIN OUTCOME MEASURE(S): HAND2 and FBLN1 expression levels were assessed by real-time polymerase chain reaction and Western blot analysis. RESULT(S): MPA or E(2) + MPA increased HAND2 mRNA levels in ESCs in a time- and dose-dependent manner, and this stimulatory effect was blocked by RU-486 (P receptor antagonist). HAND2 was increased by E(2) + MPA earlier than FBLN1. Simultaneous MPA and 8-Br-cAMP treatment synergistically enhanced HAND2 mRNA levels. P and all the progestins significantly increased HAND2 mRNA levels, whereas E(2), 8-Br-cAMP, or dexamethasone alone had no effect. Silencing of HAND2 expression significantly reduced FBLN1 expression, whereas FBLN1 silencing had no effect on HAND2 expression. CONCLUSION(S): These results suggest that progestin-induced HAND2 contributes to FBLN1 expression in human ESCs.

Shakuyaku-kanzo-to inhibits smooth muscle contractions of human pregnant uterine tissue in vitro.

AIM: Shakuyaku-kanzo-to (SK) is a herbal medicine and is known to possess an antispasmodic effect on skeletal muscle and intestinal smooth muscle. However, it is unclear whether SK is effective in antagonizing uterine smooth muscle contractions. Herein, we investigated the effects of SK on smooth muscle contractions of human pregnant uterine samples. MATERIAL AND METHODS: We prepared myometrial strips from uterine tissues of pregnant women who underwent cesarean section for obstetrical indications, and examined the inhibitory effects of SK and its components, shakuyaku (S) and kanzo (K), on agonist-induced and spontaneous contractions in vitro. Oxytocin, prostaglandinF(2α) , and high KCl were utilized as agonists in this study. RESULTS: SK inhibited agonist-induced and spontaneous contractions in a dose-dependent manner. Inhibition of SK on oxytocin-induced contractions occurred at a concentration of 100 µg/mL and reached maximum effect at a concentration of more than 1000 µg/mL. The half max inhibitory concentration of SK was approximately 440 µg/mL in oxytocin-induced contractions. SK at 1000 µg/mL completely inhibited the oxytocin- and prostaglandinF(2α)-induced contractions but not the high KCl-induced contractions. The inhibitory effects on agonist-induced contractions of K, but not S, matched those of SK. CONCLUSION: These results suggest that the inhibitory effect of SK on smooth muscle contractions is due to K. The mechanism of the inhibitory effects of SK on oxytocin- and prostaglandinF(2α) -induced contractions may differ from that on KCl-induced contractions.

Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

BACKGROUND: Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. METHODS: Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl2; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. RESULTS: Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl2 in a dose-dependent manner. CoCl2 significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P < 0.01). CONCLUSIONS: Hypoxia simultaneously acts to increase VEGF via HIF-1α and to decrease SDF-1 in a HIF-1α-independent manner in ESCs. These results indicate a potential mechanism for the action of hypoxic conditions that could influence angiogenesis in the human endometrium.

Angiopoietin 1 and angiopoietin 2 in follicular fluid of women undergoing a long protocol.

OBJECTIVE: To determine the concentrations of angiopoietin 1 (ANGPT1) and ANGPT2 in individual human preovulatory follicles in relation to their diameter or volume to clarify the role of these molecules in folliculogenesis. DESIGN: Prospective study. SETTING: Research laboratory at Kansai Medical University, Osaka, Japan. PATIENT(S): Twenty-three women undergoing IVF. INTERVENTION(S): On the day of oocyte retrieval, serum samples and follicular fluid (FF) from individual follicles were collected. We analyzed 348 follicles. MAIN OUTCOME MEASURE(S): ANGPT1 and ANGPT2 concentrations in FF and serum and oocyte recovery rates. RESULT(S): On average, ANGPT1 concentrations in FF were 150 times lower than those in serum, whereas ANGPT2 concentrations in FF were 8 times higher than those in serum. The concentrations of ANGPT1 in follicles with a diameter ≤17 mm were significantly higher than those in follicles with a diameter ≥18 mm. On the other hand, the concentrations of ANGPT2 in follicles with a diameter ≤17 mm were significantly lower than those in follicles with a diameter ≥18 mm. The ANGPT2/ANGPT1 ratio increased with enlargement of follicular diameter. ANGPT1 concentrations in FF decreased with follicular volume. ANGPT2 concentrations and the ANGPT2/ANGPT1 ratio in FF rose with follicular volume. The ANGPT2/ANGPT1 ratio in FF from the oocyte recovery group was significantly higher than that from the nonrecovery group. CONCLUSION(S): Our data suggested that the change in ANGPT1 and ANGPT2 levels may be associated with follicular growth and angiogenesis during the preovulatory period.

Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.

OBJECTIVE: To investigate whether 17ß-estradiol (E(2)) and progestins exert direct effects on vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1/CXCL12) in human endometrial stromal cells (ESCs) and thereby to clarify the regulatory function of these local angiogenic factors in the endometrium. DESIGN: In vitro experiment. SETTING: Research laboratory at Kansai Medical University. PATIENT(S): Fourteen patients undergoing hysterectomy for benign reasons. INTERVENTION(S): ESCs were cultured with E(2) and/or various clinically relevant progestins (medroxyprogesterone acetate [MPA], norethisterone [NET], levonorgestrel [LNG], dienogest [DNG], and progesterone [P]). MAIN OUTCOME MEASURE(S): The mRNA levels and production of VEGF and SDF-1 were assessed by real-time reverse-transcription polymerase chain reaction and ELISA, respectively. RESULT(S): E(2) significantly induced the mRNA levels and protein production of VEGF and SDF-1 in ESCs. MPA could antagonize the E(2)-stimulated effects in a time- and dose-dependent manner, and this effect could be reversed by RU-486 (P receptor antagonist). All of the progestins (MPA, NET, LNG, and DNG; 10(-9) to 10(-7) mol/L) attenuated E(2)-induced VEGF and SDF-1 production, whereas P showed these inhibitory effects only when present in a high concentration (10(-7) mol/L). CONCLUSION(S): Progestins have inhibitory effects on E(2)-induced VEGF and SDF-1 in ESCs. These results may indicate a potential mechanism for action of the female sex steroids in the human endometrium that can be helpful for various clinical applications.

Vaginal Lymphoma with Immune Thrombocytopenic Purpura: An Unusual Case Report.

The female genital tract is rarely the initial site of presentation in lymphoma or leukemia. We report a case of non-Hodgkin's lymphoma (NHL) presenting initially in the vagina. The patient, a 75-year-old woman, had a history of immune thrombocytopenic purpura (ITP). She presented with a chief complaint of genital bleeding and introital pain. On transvaginal ultrasonography, a vaginal tumor with an irregular wall was detected, and the internal echo showed a hypoechoic and echogenic pattern. Ultrasonography and magnetic resonance imaging (MRI) suggested that the vaginal tumor was likely to be a hematoma or a hemorrhagic tumor arising from ITP. Incision and resection for a hematoma or a hemorrhagic tumor were carried out in response to genital bleeding, introital pain, and pathological diagnosis. Postoperative microscopic examination confirmed that the tumor was a vaginal NHL. The final diagnosis using the Ann Arbor staging system was high-stage (stage IV) NHL. The patient received chemotherapy, and she remains in remission for 42 months after treatment.

The PDGF B-chain is involved in the ontogenic susceptibility of the developing rat brain to NMDA toxicity.

Hypoxic-ischemic (H-I) injury to neonatal brains can cause a life-long neuronal deficit because of increased susceptibility in the neonatal period. Excitotoxicity due to overstimulation of the N-methyl-d-aspartate receptor (NMDAR) is assumed to be the basis of the injury. However, the ontogenic profile of the susceptibility does not directly correlate with the levels of NMDAR expression. Platelet-derived growth factor B-chain (PDGF-B) has been reported to protect neurons by suppressing the NMDA-evoked current and translocating the glutamate transporter to the cell membrane. Thus, we assessed the relationship between the susceptibility to H-I injury and the expression of PDGF-B in neonatal rat brain. PDGF-B infusion before and after an intrastriatal NMDA injection significantly reduced the size of the lesions in 7-day-old rats, when they are most susceptible and the neuronal expression of PDGF-B is low. Fourteen-day-old neonatal rats were found to be resistant to NMDA injury, even though NMDARs are expressed at high levels in the brain at this age. Inhibition of PDGF-B protein synthesis by antisense oligodeoxynucleotides increased the size of the NMDA-induced lesions up to 6-fold at postnatal day 14, when PDGF-B is expressed at high levels in neurons. These data suggest that PDGF-B is an important physiological modulator of NMDAR excitability in the developing brain, and that the balance between the expression of NMDAR and PDGF-B partly determines the ontogenic susceptibility to brain injury. Enhancement of the PDGF-B/receptor signal pathway might rescue neonatal brains at risk of H-I injury.

Apoptosis in cerebrum of macular mutant mouse.

Neuronal cell death in the brain of macular mutant mouse, a model of copper metabolism abnormality, has features of both apoptosis and necrosis. Apoptotic cells were morphologically identified by the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and electron microscopy. Numerous TUNEL-positive cells were identified in the cerebral cortex, hippocampus and thalamus of the hemizygotes after postnatal day 11. Ultramicroscopic studies confirmed that a number of cells had apoptotic features characterized by condensation and segregation of the nuclei. Furthermore, genomic DNA gel electrophoresis revealed a laddering pattern in the hemizygous brain. Starvation, which produced a low body weight in normal mice similar to that seen in the hemizygotes, did not result in an increase of TUNEL-positive cells. We also found that there was no increase of apoptotic cells in the brains of heterozygotes and copper-supplemented hemizygotes. Immunocytochemical analysis revealed that the distribution of copper/zinc superoxide dismutase-containing cells differed from that of TUNEL-positive cells. These findings suggest that copper deficiency is a key factor triggering apoptosis in the brain of macular mutant mouse through a mechanism different from suppression of antioxidant action of the dismutase. The improved survival period of the copper-supplemented hemizygotes may be attributed, in part, to inhibition of excessive neuronal apoptosis identified in the late stage of the disease.