Ryo Nishimura

Progesterone Is a Suppressor of Apoptosis in Bovine Luteal Cells

Abstract Progesterone is suggested to be a suppressor of apoptosis in bovine luteal cells. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells. Furthermore, apoptosis is known to be controlled by the bcl-2 gene/protein family and caspases. This study was undertaken to determine whether intraluteal progesterone (P4) is involved in Fas L–mediated luteal cell death in the bovine corpus luteum (CL) in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 and the activity of caspase-3 in the bovine CL. Luteal cells obtained from the cows in the midluteal phase of the estrous cycle (Days 8–12 of the cycle) were exposed to a specific P4 antagonist (onapristone [OP], 10−4 M) with or without 100 ng/ml Fas L. Although Fas L alone did not show a cytotoxic effect, treatment of the cells with OP alone or in combination with Fas L resulted in killing of 30% and 45% of the cells, respectively (P < 0.05). DNA fragmentation was observed in the cells treated with Fas L in the presence of OP. The inhibition of P4 action by OP increased the expression of Fas mRNA (P < 0.01); however, it did not affect bax or bcl-2 mRNA expression (P > 0.05). Moreover, OP stimulated expression of caspase-3 mRNA (P < 0.01). The overall results indirectly show that intraluteal P4 suppresses apoptosis in bovine luteal cells through the inhibition of Fas and caspase-3 mRNA expression and inhibition of caspase-3 activation.

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

Optimal Feedback Control Design Using Genetic Algorithm in Multimachine Power System

Abstract This paper introduces an application of genetic algorithm (GA) to design weighting matrices Q and R elements in Linear Quadratic Regulator (LQR) optimization process. The weighting matrices Q and R are the most important components in LQR optimization. The performance of matrices Q and R determines the output performances of the system. Commonly, a trial-and-error method has been used to construct the matrices Q and R elements. This method is very simple, but very difficult to produce good control performances. Also it takes a long time to choose the best values in its processing. Because of this, in order to improve control performances by selecting the elements of matrices Q and R, the Bryson method can be employed to give better results in shorter time than the previous method. In this paper, we use GA to construct the weighting matrices Q and R properly. We design the weighting matrices of a power system optimization by using the trial-and-error method, Bryson method, and GA method and compare the control performances. It is shown that the GA calculation is the most useful among the three methods to improve system performances via matrices Q and R design to minimize settling time of control. This idea gives a new alternative procedure in time varying feedback control to improve the stability performances.

Determining the arrangement of fictitious charges in charge simulation method using genetic algorithms

Abstract In this paper, we propose a method to decide the appropriate arrangement of fictitious charges in the charge simulation method (CSM) by using a direct search method. The appropriate arrangement is achieved by using genetic algorithms (GA) as a search method. We describe the arrangement of the charges as chromosomes and place fictitious point-charges at random on the rotational axis of a spheroidal electrode. The charge arrangement is adjusted by using GA until the potential reaches a specified value over the surface of the electrode.

Hypoxia Promotes Luteal Cell Death in Bovine Corpus Luteum

Abstract Low oxygen caused by a decreasing blood supply is known to induce various responses of cells, including apoptosis. The present study was conducted to examine whether low-oxygen conditions (hypoxia) induce luteal cell apoptosis in cattle. Bovine midluteal cells incubated under hypoxia (3% O2) showed significantly more cell death than did those incubated under normoxia (20% O2) at 24 and 48 h of culture, and had significantly lower progesterone (P4) levels starting at 8 h. Characteristic features of apoptosis, such as shrunken nuclei and DNA fragmentation, were observed in cells cultured under hypoxia for 48 h. Hypoxia increased the mRNA expressions of BNIP3 and caspase 3 at 24 and 48 h of culture. Hypoxia had no significant effect on the expressions of BCL2 and BAX mRNA. Hypoxia also increased BNIP3 protein, and activated capsase-3. Treatment of P4 attenuated cell death, caspase-3 mRNA expression, and caspase-3 activity under hypoxia. Overall results of the present study indicate that hypoxia induces luteal cell apoptosis by enhancing the expression of proapoptotic protein, BNIP3, and by activating caspase-3, and that the induction of apoptosis by hypoxia is partially caused by a decrease in P4 production. Because hypoxia suppresses P4 synthesis in bovine luteal cells, we suggest that oxygen deficiency caused by a decreasing blood supply in bovine corpus luteum is one of the major factors contributing to both functional and structural luteolysis.

Possible Role of Interleukin-1 in the Regulation of Bovine Corpus Luteum Throughout the Luteal Phase

Abstract Interleukin-1 (IL-1) is one of the principal cytokines that participate in local regulation of many reproductive functions. The present study was undertaken to determine whether mRNAs for IL-1α, IL-1β, and IL-1 type I receptor (IL-1R) are expressed in bovine corpora lutea (CL), and whether luteal cells respond to treatment with IL-1α and IL-1β during the luteal phase. Bovine CL were classified into five stages (early, Days 2–3; developing, Days 5–6; mid, Days 8–12; late, Days 15–17; and regressed, Days 19–21). IL-1α, IL-1β, and IL-1R mRNAs were detected by reverse transcription-polymerase chain reaction (PCR) in all luteal stages examined. Densitometric analysis of PCR products revealed increases of the mRNA of IL-1α and IL-1R in the CL of the regressed stage (P < 0.05). There was less mRNA for IL-1β in the regressed stage than in the developing and mid stages (P < 0.05). When developing, mid, and late luteal cells were treated with IL-1α (1–30 ng/ml) or IL-1β (1–30 ng/ml) for 24 h, IL-1α and IL-1β dose-dependently increased prostaglandin (PG) F2α and PGE2 production by the luteal cells of all stages (P < 0.05), indicating the presence of functional IL-1R in bovine CL. However, progesterone synthesis was not affected by either IL-1α or IL-1β treatment. Stimulation with IL-1α and IL-1β decreased the PGE2:PGF2α ratio in the developing stage (P < 0.05), whereas it increased the ratio in the mid stage (P < 0.05). In the late stage, the ratio of IL-1β-treated cells was greater than that of IL-1α-treated cells (P < 0.05). Overall results indicate that genes for IL-1α and IL-1β are expressed and a functional IL-1R is present in the bovine CL throughout the luteal phase, and suggest that IL-1α and IL-1β have different roles as local modulators to regulate PGF2α and PGE2 production during the luteal phase.

Positive Feedback Loop Between Prostaglandin E2 and EGF-Like Factors Is Essential for Sustainable Activation of MAPK3/1 in Cumulus Cells During In Vitro Maturation of Porcine Cumulus Oocyte Complexes

During in vitro maturation of porcine cumulus-oocyte complexes (COCs), follicle-stimulating hormone (FSH) increases both prostaglandin E2 (PGE2) production and the expression levels of EGF-like factors. The ligands act on cumulus cells by the autocrine system due to their specific receptors, EP2, EP4, or EGF receptor. When each pathway is suppressed by inhibitors, complete cumulus expansion and oocyte maturation do not occur. In this study, we examined the relationship between both of these pathways in cumulus cells of porcine COCs. When COCs were cultured with FSH, Fshr mRNA expression was immediately decreased within 5 h, whereas Ptger2, Ptger4, and Ptgs2 expression levels were significantly increased in cumulus cells in the culture containing FSH for 5 or 10 h. The PTGS2 inhibitor NS398 significantly suppressed not only PGE2 secretion at any culture time point but also Areg, Ereg, and Tace/Adam17 expression in cumulus cells at 10 and 20 h but not at 1 or 5 h. During the early culture period, phosphorylation of MAPK3 and MAPK1 (MAPK3/1) was not affected by NS398; however, at 10 and 20 h, phosphorylation was suppressed by the drug. Furthermore, down-regulations of MAPK3/1 phosphorylation and expression of the target genes by NS398 was overcome by the addition of either PGE2 or EGF. FSH-induced cumulus expansion and meiotic progression to the MII stage were also suppressed by NS398, whereas these effects were also overcome by addition of either PGE2 or EGF. These results indicated that PGE2 is involved in the sustainable activation of MAPK3/1 in cumulus cells via the induction of EGF-like factor, which is required for cumulus expansion and meiotic maturation of porcine COCs.

Anti-Apoptotic Roles of Prostaglandin E2 and F2alpha in Bovine Luteal Steroidogenic Cells

Abstract Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8–12 of the cycle) were treated for 24 h with PGE2 (0.001–1 μM) and PGF2alpha (0.001–1 μM). Prostaglandin E2 (1 μM) and PGF2alpha (1 μM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.

Automatic arrangement of fictitious charges and contour points in charge simulation method for two spherical electrodes

Abstract We propose a method to decide the appropriate arrangement of both fictitious charges and contour points in the charge simulation method for the case that the shape of electrodes can be expressed in the spherical coordinates. In this paper, we use genetic algorithm (GA), which is inspired by the mechanism of natural selection where stronger individuals are likely the winners in a competing environment, as a search method. We calculated the potential distribution around two spherical electrodes with different potentials above a grounded electrode plate. The surface potentials of the electrodes were assumed to be 5000 and 30 V , respectively, for example. The total number of the charges was 22 for the electrode system. First, we placed fictitious point-charges and contour points at random inside the electrodes and on the surfaces of electrodes, respectively. The arrangement was automatically adjusted by using GA until the potential reached the desired values at all the test points placed on the electrode surfaces. We show that the GA can be useful to determine the appropriate arrangement of both fictitious charges and contour points simultaneously.

Protein Kinase C (PKC) Increases TACE/ADAM17 Enzyme Activity in Porcine Ovarian Somatic Cells, Which Is Essential for Granulosa Cell Luteinization and Oocyte Maturation

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.