Rescue of the corpus luteum and an increase in luteal superoxide dismutase expression induced by placental luteotropins in the rat: action of testosterone without conversion to estrogen

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

Changes in superoxide production rate and in superoxide dismutase and glutathione peroxidase activities in subcellular organelles in mouse liver under exposure to low doses of low-intensity radiation

The functioning of the antioxidant system in mouse liver at increased stationary concentration of active oxygen species induced by whole-body chronic exposure of mice to gamma-irradiation (137Cs, 0.6 cGy/day, 9 days) was studied. Synchronous changes (growth with an extreme) in activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are found that may be considered as evidence in favor of maintenance of regulatory links in the antioxidant system of liver. The superoxide production rate in microsomes and nuclei also changed with an extreme with the rise in accumulated radiation dose. In microsomes the superoxide production rate reached a maximum at lower doses than the activity of Cu,Zn-SOD did. In nuclei the increase in superoxide production rate was not compensated by the rise in Cu,Zn-SOD activity within the studied dose range. The findings indicate some imbalance between production and consumption of superoxide radicals in microsomes and nuclei; in mitochondria these processes are balanced, leading to more resistance to low-dose irradiation.     

Cu,Zn-superoxide dismutase-dependent apoptosis induced by nitric oxide in neuronal cells

Nitric oxide (NO) challenge to human neuroblastoma cells (SH-SY5Y) ultimately results in apoptosis. Tumor suppressor protein p53 and cell cycle inhibitor p21 accumulate as an early sign of S-nitrosoglutathione-mediated toxicity. Cytochrome c release from mitochondria and caspase 3 activation also occurred. Cells transfected with either wild type (WT) or mutant (G93A) Cu, Zn-superoxide dismutase (Cu,Zn-SOD) produced comparable amounts of nitrite/nitrate but showed different degree of apoptosis. G93A cells were the most affected and WT cells the most protected; however, Cu, Zn-SOD content of these two cell lines was 2-fold the SH-SY5Y cells under both resting and treated conditions. We linked decreased susceptibility of the WT cells to higher and more stable Bcl-2 and decreased reactive oxygen species. Conversely, we linked G93A susceptibility to increased reactive oxygen species production since simultaneous administration of S-nitrosoglutathione and copper chelators protects from apoptosis. Furthermore, G93A cells showed a significant decrease of Bcl-2 expression and, as target of NO-derived radicals, showed lower cytochrome c oxidase activity. These results demonstrate that resistance to NO-mediated apoptosis is strictly related to the level and integrity of Cu,Zn-SOD and that the balance between reactive nitrogen and reactive oxygen species regulates neuroblastoma apoptosis.     

The effect of natural dicarbonyls on activity of antioxidant enzymes in vitro and in vivo

Natural dicarbonyls, which may be accumulated during oxidative stress in atherosclerosis (e.g. malondialdehyde) or carbonyl stress in diabetes mellitus (glyoxal and methylglyoxal) effectively inhibited activities of commercial preparations of the antioxidant enzymes: Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and Se-contained glutathione peroxidase from human and bovine erythrocytes, and also rat liver glutathione-S-transferase. After incubation of human erythrocytes with 10 mM of each investigated dicarbonyls the decrease of intracellular Cu,Zn-SOD was observed. The decreased activity of erythrocyte Cu,Zn-SOD was also detected in patients with diabetes mellitus type 2 with carbohydrate metabolism impairments but effective sugar-lowered therapy was accompanied by the increase of this enzyme activity. The increase of erythrocytes Cu,Zn-SOD activity in diabetic patients treated with metformin (which may utilize methylgly-oxal) was higher than in erythrocytes of diabetic patients subjected to traditional therapy.     

细胞渗透性神经酰胺可抑制大鼠离体黄体细胞甾体激素生成并诱导细胞凋亡

本文旨在观察神经酰胺对离体孵育的大鼠黄体细胞孕酮分泌及细胞凋亡的影响,以PMSG－hCG处理的雌性Wistar大鼠为模型,分离制备黄体细胞,将外源性细胞渗透性神经酰胺与黄体细胞共同孵育,分别用放免法和流式细胞仪分析神经酰胺对黄体细胞孕酮生成和凋亡的影响,同时还检测了一氧化氮合酶（NOS）活性和一氧化氮（NO）水平的变化,结果显示,神经酰胺可以剂量相关方式抑制hCG－诱导的孕酮分泌,而对基础孕酮没有显著影响,离体孵育12h的大鼠黄体细胞存在自发性凋亡,5umol/L神经酰胺能显著增加亡率（P＜0．05）,流式细胞仪分析可见增强的凋亡蜂,实验还发现,50umol/L神经酰胺能明显促进NOS活性（P＜0．01）和NO生成（P＜0．01）,结果提示,神经酰胺可能通过调节甾体激素生成和细胞凋亡而作为一种重要的信息分子参与黄体退化等卵巢的生理过程。     

Increased Superoxide Dismutase Activity Improves Survival of Cultured Postnatal Midbrain Neurons

Abstract: Copper/zinc superoxide dismutase (Cu/Zn-SOD) is a major free radical scavenging enzyme. Increased Cu/Zn-SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn-SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn-SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn-SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn-SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn-SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn-SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn-SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn-SOD activity does not seem to play a critical role in determining the mode of cell death in this culture system.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

Detection of manganese superoxide dismutase mRNA in the theca interna cells of rat ovary during the ovulatory process by in situ hybridization

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).     

H2O2 but not $${\hbox{O}_{2}^{-}}$$ elevated by oxidized LDL enhances human aortic smooth muscle cell proliferation

The oxidation of low density lipoprotein (LDL) as a key event in atherosclerosis suggests that antioxidant interventions may reduce the risk of atherosclerosis. However, the better strategies among antioxidant remedies for atherosclerosis remains difficult to be determined. Here, we show that oxidized LDL increases the steady-state level of intracellular hydrogen peroxide through stimulating the protein expressions of Nox1 and Cu/Zn superoxide dismutase (SOD) in human aortic smooth muscle cells (SMCs). The intracellular content of hydrogen peroxide rather than superoxide is a key modulator for vascular SMC (VSMC) proliferation, implying that without co-expression of catalase, increased Cu/Zn-SOD activity alone may not be beneficial to reduce the growth of VSMC in an atherosclerotic plaque.     

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.     

Superoxide dismutase activity, lipid peroxide production and corpus luteum steroidogenesis during natural luteolysis and regression induced by oestradiol deprivation of the ovary in pseudopregnant rabbits.

The relationship of oxygen free radicals to corpus luteum function in rabbits was explored during various stages of pseudopregnancy, including natural and induced luteal regression. Induced luteolysis was achieved during mid-pseudopregnancy by removal of an oestradiol capsule placed at the onset of pseudopregnancy, which suppressed ovarian oestradiol production. Activity of manganese superoxide dismutase (Mn SOD) was significantly and positively correlated with ovarian progesterone production (P < 0.01) throughout pseudopregnancy and during natural regression. Oestradiol deprivation for 12, 24 or 72 h resulted in declines in Mn SOD activity and progesterone secretion, although Mn SOD rose and corpus luteum steroidogenesis was restored to normal when the capsule was replaced for 48 h before assessment, having been removed for 24 h. Lipid peroxide and progesterone concentrations were not correlated, although a significant rise in lipid peroxides in the luteal tissue was detected after deprivation of oestradiol for 72 h. Changes in progesterone production and Mn SOD activity were not associated with alterations in concentration of prostaglandin F metabolite. These data suggest that Mn SOD may be involved in regulating function of the corpus luteum during pseudopregnancy in rabbits and that oxygen free radicals may play a role in regression of corpus luteum in this species.     

c—erbB2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法,研究反义ｃ－ｅｒｂＢ２寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｃ－ｅｒｂＢ２ ＯＤＮ）对大鼠黄体细胞ｈＣＧ诱导的孕酮分泌的影响,及其与外源性ｃＡＭＰ和Ｃａ＾２＋以及蛋白抑制剂放线菌酮（ＣＹＸ）之间的关系。结果表明,反义ｃ－ｅｒｂＢ２以剂量相关方式抑制黄体细胞ｈＣＧ诱导的孕酮的产生,同时使ｃ－ｅｒｂＢ２蛋白染色阳性的黄体细胞百分数下降,无义ｔａｔ ＯＤＮ没有相应的作用。１０＾－４     

反义c-fos寡脱氧核苷酸对hCG诱导大鼠黄体细胞孕酮和雌二醇生成的影响

采用离体孵育大鼠黄体细胞的方法,观察了反义c-fos寡脱氧核苷酸(反义c-fos ODN)对hCG诱导的黄体细胞孕酮(P)和雌二醇(E     

Increased oxidative stress induces apoptosis in human cystic fibrosis cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis

The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p <.05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.     

Role of ERK1/2 in the differential synthesis of progesterone and estradiol by granulosa cells.

A major concept in mammalian ovarian physiology is that follicle-stimulating hormone (FSH) activates the granulosa cells (GCs) in the Graafian follicle to selectively produce estradiol, but not progesterone, during the follicular phase of the menstrual or estrous cycle. However, given the fact that FSH can induce production of both estradiol and progesterone by GCs cultured in vitro, it has been postulated for a long time that there is a factor present in the ovary that selectively prevents FSH-induced progesterone production. Here, we provide evidence that two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase-1 and -2 (ERK1/2) can differentially regulate FSH-stimulated estradiol and progesterone production. Using primary rat GCs from early antral follicles cultured in serum-free medium for 48 h, we found that the addition of a specific inhibitor of ERK1/2 activation, U0126, caused the attenuation or enhancement of FSH-induced progesterone or estradiol production, respectively, in a dose-dependent manner. Throughout the 48-h culture period in this culture system ERK1/2 molecules in their activated state (phospho-ERK1/2) were clearly detectable in GCs exposed to FSH. The addition of U0126 caused a decrease in the levels of phosphorylated but not unphosphorylated ERK1/2 which was maintained throughout the 48-h culture, suggesting that U0126 was continuously active to inhibit the phosphorylation of ERK1/2. The divergent regulation of FSH-induced progesterone and estradiol synthesis by U0126 was further supported by demonstrating that U0126 inhibits and stimulates FSH-induced mRNA levels of steroidogenic acute regulatory protein and P450 aromatase, respectively. Collectively, this study clearly identified ERK1/2 as the first intracellular signaling molecules that differentially regulate FSH-induced progesterone and estradiol synthesis in GCs.     

Progesterone promotes survival of the rat corpus luteum in the absence of cognate receptors

Progesterone production by the corpus luteum (CL) is essential for preparation of the endometrium for implantation and for the maintenance of gestation. Progesterone modulates its own production and opposes functional luteal regression induced by exogenous agents, such as prostaglandin F(2alpha). In the present study, we evaluated whether progesterone is also capable of interfering with the process of structural luteal regression, which is characterized by a decrease in weight and size of the gland because of programmed cell death (i.e., apoptosis). We have found that a low number of luteal cells undergo apoptosis throughout gestation. On the day of parturition, but following the initial decline in endogenous progesterone production, a small increase in the number of luteal cells undergoing cell death was observed. This increase in apoptotic cells continued postpartum, reaching dramatic levels by Day 4 postpartum, and was accompanied by a marked decrease in average luteal weight. We have established that the exogenous administration of progesterone significantly reduces the decline in luteal weight observed during structural luteal regression postpartum. This effect was associated with a decrease in the number of cells undergoing apoptosis and with enhanced circulating levels of androstenedione. Furthermore, in vivo administration of progesterone delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that neither the CL of gestation nor the newly formed CL after postpartum ovulation express the classic progesterone-receptor mRNA. In summary, the present results support a protective action of progesterone on the function and survival of the CL through inhibition of apoptosis and stimulation of androstenedione production. Furthermore, this effect is carried out in the absence of classic progesterone receptors.