Toshifumi Takahashi

Induction of hTERT expression and phosphorylation by estrogen via Akt cascade in human ovarian cancer cell lines

We examined the mechanism by which estrogen regulates telomerase activity in Caov-3 human ovarian cancer cell lines, which express ER, to determine whether the regulation affects the expression and/or phosphorylation of the telomerase catalytic subunit (hTERT). 17β-Estradiol (E2) induced telomerase activity and hTERT expression. Transient expression assays using luciferase reporter plasmids containing various fragments of hTERT promoter showed that the estrogen-responsive element appeared to be partially responsible for the E2-induced activation of the hTERT promoter. Either pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, or transfection with a dominant-negative Akt attenuated the E2-induced activation of the hTERT promoter. In addition, estrogen induced the phosphorylation of IκB inhibitor protein via the Akt cascade, and cotransfection with a dominant-negative subunit of NFκB attenuated the response of the ERE-deleted hTERT promoter to E2. Moreover, E2 induced the phosphorylation of hTERT, the association of 14-3-3 protein and NFκB with hTERT, and nuclear accumulation of hTERT in an Akt-dependent manner. These results indicate that E2 induces telomerase activity not only by transcriptional regulation of hTERT via an ERE-dependent mechanism and a PI3K/Akt/NFκB cascade, but also by post-transcriptional regulation via Akt-dependent phosphorylation of hTERT. Thus, the phosphorylation of Akt is a key event in the induction of telomerase activity by E2 in human ovarian cancer cells.

The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs

OBJECTIVE To investigate the relationship between the incidence of apoptotic bodies in membrana granulosa and follicular steroid concentrations in human follicles. DESIGN Case-controlled prospective study for 132 individual follicles. SETTING Procedures were performed in Yamagata University Hospital. PATIENT(S) Thirty-six normo-ovulatory women with tubal infertility underwent ovulation induction for IVF-ET with a conventional hyperstimulation method. INTERVENTION(S) Patients underwent follicle aspiration after the administration of hCG. MAIN OUTCOME MEASURE(S) The nuclei of recovered granulosa cells were examined by fluorescence microscopy, and the incidence of apoptotic bodies was tabulated. Intrafollicular steroids were evaluated mainly by RIA. These data were analyzed with respect to oocyte-retrieval, oocyte maturity, fertilization, and embryo quality. RESULT(S) Membrana granulosa cells in the follicles from which oocytes were subsequently fertilized showed a significantly lower incidence of apoptotic bodies than those in follicles from which the oocytes did not fertilize. Membrana granulosa cells in the follicles from which oocytes were developed into good quality showed a significantly lower incidence of apoptotic bodies than those in the follicles from which oocytes developed into fair and poor quality. The incidence of apoptotic bodies was significantly higher in the mural granulosa cell region than in the cumulus cell region in most cases. Intrafollicular E2, P, and free T levels were not different between the oocyte groups. CONCLUSION(S) These results indicate that lower incidence of apoptotic bodies in individual follicles is associated with better outcomes for oocytes. Also, mural granulosa cells and cumulus cell in each follicle may show differentiation during follicular maturation.

Fasudil inhibits vascular endothelial growth factor–induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF)–induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer. [Mol Cancer Ther 2007;6(5):1517–25]

Poor Embryo Development in Mouse Oocytes Aged In Vitro Is Associated with Impaired Calcium Homeostasis

Abstract We examined whether impairment of intracellular Ca2+ homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca2+ oscillations at fertilization: these Ca2+ oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca2+ store was decreased in in vitro-aged oocytes. A decrease in the Ca2+ store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca2+-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca2+ oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca2+ homeostasis (such as a decrease in the ER Ca2+ store) caused an alteration in Ca2+ oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H2O2 or thapsigargin. Acting together, alteration in Ca2+ oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.

Aged Mouse Oocytes Fail to Readjust Intracellular Adenosine Triphosphates at Fertilization

Abstract Postovulatory aging of oocytes significantly affects embryonic development. Also, altered Ca2+ oscillation patterns can be observed in fertilized, aged mouse oocytes. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum, and the latter relies on ATP availability, we simultaneously measured changes in intracellular ATP concentration ([ATP]i) and Ca2+ oscillations in fresh and aged mouse oocytes. We continuously assessed changes in [ATP]i from intracellular free Mg2+ concentration measured by fluorescent dye Magnesium Green (MgG) while intracellular Ca2+ concentration ([Ca2+]i) was monitored by Fura-PE3. At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation in [Ca2+]i, indicating a relative decrease in [ATP]i. In fresh oocytes, it was quickly followed by a significant decrease below baseline, indicating a relative increase in [ATP]i. In contrast, in aged oocytes, such a decrease in MgG fluorescence was not observed. In a separate experiment, ATP content in fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in unfertilized fresh and aged oocytes. Intracellular ATP content at 5 h after fertilization was increased in both oocytes, where fresh oocytes showed a significantly higher intracellular value than aged oocytes. These findings suggest that aged mouse oocytes fail to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization might lead to an altered Ca2+ oscillation pattern and poor developmental potency, which is commonly noted in aged oocytes.

Application of Vitrification to Human Embryo Freezing

To solve the problem of multiple pregnancies during the in vitro fertilization (IVF) and embryo transfer procedure, excess embryos must be cryopreserved for embryo transfer in future. We applied the vitrification method to cryopreservation of human embryos. A total of 31 frozen-thawed embryo transfer cycles were analyzed at the Yamagata University Hospital, Yamagata, Japan. The patients were introduced to IVF treatment and had an excess of valuable embryos to be frozen after the transfer of three fresh embryos that did not result in establishing a pregnancy. Excess human 8- to 16-cell stage embryos were exposed to vitrification solution and then frozen in liquid nitrogen. The cryoprotectant was removed by washing the embryos in media containing different concentrations of cryoprotectant. Three days after LH surge and/or 2 days after ultrasonographic ovulation the embryos were transferred. The rate of poor quality embryos significantly increased and the rate of good quality embryos decreased after thawing the embryos frozen by the vitrification method. In menstrual cycles with good quality embryo transfer, a higher rate of pregnancies was established than in the cycles in which fair or poor quality embryos were the highest grade of embryos transferred into the uterus. In total, 5 pregnancies were established from 31 embryo tansfers; 4 pregnancies were in cycles associated with the transfer of good quality embryos, and 1 pregnancy was in a cycle in which the highest grade of embryo was fair. When compared with slow embryo freezing methods, vitrification has marked advantages for clinical application in terms of cost and time. Vitrification will be an alternative method for embryo freezing.

Effects of aging on inositol 1,4,5-triphosphate-induced Ca2+ release in unfertilized mouse oocytes.

We previously demonstrated in the mouse oocyte that in vivo postovulatory aging significantly suppresses activity of the endoplasmic reticulum (ER) Ca2+‐ATPase (Igarashi et al. 1997. Mol Reprod Dev 48:383–390). We undertook the present study to further examine the effects of oocyte aging on Ca2+ release from the inositol 1,4,5‐triphosphate (InsP3)‐sensitive Ca2+ channels of the ER membrane, because not only Ca2+ reuptake, but also Ca2+ release from the ER, substantially affect Ca2+ oscillations in fertilized oocytes. A transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) was induced by photolysis of caged InsP3 microinjected into the cytoplasm in both fresh (14 hr post hCG) and aged (20 hr or 24 hr post hCG) oocytes, where the maximum rate of increase in [Ca2+]i significantly decreased in the aged oocytes. Reduced ER Ca2+ release in the aged oocyte may not be attributable to aging‐related desensitization of the InsP3‐sensitive Ca2+ channels in the ER because concentrations of caged InsP3 for half maximal [Ca2+]i increase were identical for fresh and aged oocytes. The peak [Ca2+]i response following administration of 5 μM thapsigargin, a specific ER Ca2+‐ATPase inhibitor, was significantly reduced in the aged oocyte, suggesting reduction of the ER Ca2+ stores. We conclude from these results that reduction of Ca2+ release from the InsP3‐sensitive Ca2+ stores in the aged oocyte arises from depletion of the ER Ca2+ stores with aging. These aging‐related changes in Ca2+ release and reuptake may account for alterations in Ca2+ oscillations in aged fertilized oocytes. Mol. Reprod. Dev. 55:299–306, 2000.

Hyaluronan in follicular fluids and fertilization of oocytes

OBJECTIVE To determine the concentrations of hyaluronan, E(2), and progesterone in follicular fluids (FFs) and the incidence of apoptotic granulosa cells. Also, to examine the relationship between the concentration of hyaluronan and follicular steroids, the incidence of apoptotic cells, and the fertilizability of the oocyte in the same follicle. DESIGN Samples of 130 follicles were retrospectively analyzed for hyaluronan and steroids and the incidence of apoptotic cells. SETTING The reproductive center in Yamagata University Hospital. PATIENT(S) Forty women infertile because of tubal damage or unknown causes undergoing IVF treatment were selected. INTERVENTION(S) The samples were collected from follicle aspirations. MAIN OUTCOME MEASUREMENT(S) The concentrations of hyaluronan and steroids in FFs, the incidence of apoptotic granulosa cells, and oocyte fertilizability. RESULT(S) The levels of hyaluronan in FF were found to correlate positively with P (r=0.444, P<0.0001) and the incidence of apoptotic cumulus granulosa cells (r=0.387, P=0.002) and inversely with E(2) (r = -0.601, P<0.0001) and free T (r = -0.344, P=0.001). The concentration of hyaluronan in FFs containing a subsequently fertilized oocyte after insemination was significantly lower than that in FFs containing a subsequently unfertilized oocyte (P=0.0005) (fertilized, 50.0 +/- 2.6 ng/mL; triploidy, 59.1 +/- 6.8; and unfertilized, 66.9 +/- 5.9). CONCLUSION(S) The concentration of hyaluronan in FF is an indicator for estimation of oocyte viability for fertilization.

Fasudil-induced hypoxia-inducible factor-1α degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)–dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1α induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1α expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1α protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1α mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1α expression without altering HIF-1α mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1α expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1α expression is through preventing HIF-1α degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1α signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1α signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis. [Mol Cancer Ther 2008;7(6):1551–61]

Effects of Controlled Ovarian Hyperstimulation on Oocyte Quality in Terms of the Incidence of Apoptotic Granulosa Cells

AbstractPurpose: The aim was to investigate which ovarian hyperstimulation protocol performed in the same patients causes development of oocytes of good quality. Methods: Twenty normo-ovulatory women underwent three different controlled ovarian hyperstimulation protocols for in vitro fertilization–embryo transfer. Patients underwent follicle aspiration after administration of human chorionic gonadotropin (hCG). The total number of retrieved oocytes, the number of mature oocytes, and the rate of mature oocytes were examined. Recovered granulosa cells were stained with Hoechst 33258 and examined by fluorescence microscopy to estimate the incidence of apoptotic cells. Results: The total number of oocytes and the number of mature oocytes in gonadotropin-releasing hormone agonist (GnRHa) + human menopausal gonadotropin (hMG) + hCG and hMG + hCG cycles were higher than those in the natural cycle (P < 0.0001). The rate of mature oocytes in hMG + hCG cycle was the highest among the three protocols (P < 0.04). In the mural granulosa cells, the incidence of apoptotic cells in the GnRHa + hMG + hCG cycle was significantly higher than those of the natural (P < 0.002) and hMG + hCG cycles (P = 0.0002). The incidence of apoptotic cumulus granulosa cells in the GnRHa + hMG + hCG cycle was significantly higher than those of natural and hMG + hCG cycles (P < 0.002). Moreover, the incidence of apoptotic cumulus granulosa cells in the hMG + hCG cycle was significantly lower than that in the natural cycle (P < 0.01). Conclusions: These results indicated that hMG + hCG is the most appropriate controlled ovarian hyperstimulation protocol among the three examined with regard to oocyte quality.