Hisashi Narahara

Effect of endotoxin-induced reactive oxygen species on sperm motility

OBJECTIVEnTo define the mechanism of infection-induced damage of sperm.nnnDESIGNnThe effect of lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) on sperm motility and its modification by scavengers were investigated.nnnSETTINGnResearch laboratory of a university hospital.nnnPATIENT(S)nNormozoospermic semen samples were obtained from 37 healthy volunteers.nnnINTERVENTION(S)nThe sperms were incubated in the presence of LPS with or without scavengers.nnnMAIN OUTCOME MEASURE(S)nSperm motility was evaluated by a sperm quality analyzer (SQAIIB). ROS formation in semen samples was measured by a Berthold luminometer (LB953).nnnRESULT(S)nMotility of spermatozoa was decreased in the LPS-treated samples compared with that in the control groups. ROS was significantly higher in the LPS-treated groups than in the control groups. The addition of ROS scavengers restored the motility index and suppressed ROS production in the LPS-treated semen samples.nnnCONCLUSION(S)nThese data suggest that endotoxin-induced excessive production of ROS is responsible for the decrease in sperm motility and that antioxidant therapy may be a therapeutic option for infertile men with bacterial genital tract infection.

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids.

Abstract. In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12-o-tetradecanoylphorbol 13-acetate for 24–48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin(P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.

Inhibitory effect of interleukin-8 on the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages.

Objective: To determine whether interleukin-8 (IL-8) regulates platelet-activating factor acetylhydrolase (PAF-AH) secretion by decidual cells in vitro. Methods: Decidual macrophages were obtained from human term decidual tissue by enzymic digestion and Ficoll-Paque centrifugation. The effect of IL-8 and phorbol esters on the secretion of PAF-acetylhydrolase by these cells was examined. Results: IL-8 inhibited PAF-AH secretion by decidual macrophages in a dose-dependent manner. The inhibition was reversed partially by antibodies against tumor necrosis factor-α and IL-1β. IL-8-induced inhibition was blocked partially by the protein kinase C (PKC) inhibitors, sphingosine, and H-7 1-(5-isoquinoline sulfonyl)-2-methylpiperazine. A PKC activator, 12-O-tetradecanoyl phorbol 13-acetate, decreased PAF-AH secretion. Conclusion: IL-8 may increase the PAF concentration in the decidua via its inhibitory effect on PAF-AH secretion by decidual macrophages. IL-8-induced inhibition of enzyme secretion may have been mediated in part by PKC-dependent signal transduction.

The role of PAF in reproductive biology

Abstract Platelet-activating factor (PAF) has been shown to play an important role in both fetal lung maturation and in parturition and it has been demonstrated that part of the PAF present in the amniotic fluid is associated with surfactant and, therefore, of lung origin. PAF receptors are present in type II pneumonocytes and the addition of the autacoid promotes both glycogenolysis and increased surfactant secretion from these cells. Moreover, type II pneumonocytes and a human amnion-derived cell line (WISH) have the capability of metabolizing PAF to ethanolamine phospholipids. It has also been established that PAF will cause intracellular Ca 2+ mobilization and promote myosin phosphorylation and increased contractility in myometrial strips. Increased PAF biosynthesis in fetal tissues takes place during the latter stages of pregnancy, simultaneously with a decrease in the activity of PAF-acetylhydrolase (PAF-AH) in maternal plasma. The activity of PAF-AH is decreased by the administration of estrogens in vivo and is increased by glucocorticoids and progestins. A number of agents lower the secretion of PAF-AH by decidual macrophages, including 1,25-(OH) 2 D 3 , bacterial lipopolysaccharides, and some cytokines (IL1, TNF-α). This inhibitory action may result in higher levels of PAF and increased myometrial contractility as a consequence of infectious processes. PAF causes a profound intestinal necrosis when administered to rats by intravenous injection. Pretreatment with dexamethasone protects against this disease. The presence of PAF-AH in human milk may explain the protective effect of mothers milk against necrotizing enterocolitis.

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells

Abstract Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined: Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P< 0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation.

Platelet-activating factor-acetylhydrolase activity in follicular fluid of patients undergoing in vitro fertilization and embryo transfer *

OBJECTIVEnTo examine the role of platelet-activating factor (PAF) metabolism in the periovulatory processes.nnnDESIGNnThe PAF-acetylhydrolase activity in the follicular fluid (FF) obtained in conjunction with IVF-ET procedure was assayed and its activity was related to oocyte maturation. The PAF-acetylhydrolase activity also was related to the concentration of various ovarian hormones.nnnSETTINGnAll patients were managed and treated at Oita Medical University Hospital, Oita, Japan.nnnPATIENTSnThe study concerned 30 women between 28 and 36 years of age with tubal infertility.nnnMAIN OUTCOME MEASUREnThe activity of PAF-acetylhydrolase in FF was assayed as well as E2 and P. Oocyte maturation also was evaluated.nnnRESULTSnPlatelet-activating factor-acetylhydrolase activity was decreased significantly in the FFs of patients with a successful outcome of their pregnancies compared with the nonsuccessful group. Estradiol levels were negatively correlated with PAF-acetylhydrolase activities in the FFs. No correlation was found between the PAF-acetylhydrolase activity and P concentration in the FF. Significantly more mature and less immature oocytes were recovered in the group who subsequently became pregnant compared with the nonpregnant group.nnnCONCLUSIONSnIt is suggested that the decrease in PAF-acetylhydrolase activity may result in an increase of PAF in the FFs, which in turn may contribute to a successful pregnancy. The determination of PAF-acetylhydrolase activity in FF may serve as a prognostic marker for the evaluation of oocytes that are utilized in IVF-ET procedure.

The effect of dexamethasone on expression of mitogen-induced cyclooxygenase-2 mRNA by amnion-derived (WISH) cells

OBJECTIVESnIt has been reported that prostaglandin E(2) (PGE(2)) is synthesized in the amnion and that this synthesis increases during labor. The purpose of this study was to clarify the mechanism for the expression of cyclooxygenase-2 (COX-2) mRNA and the PGE(2) synthesis of amnion-derived (WISH) cells.nnnSTUDY DESIGNnCells were cultured and treated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dexamethasone (DEX). PGE(2) in the culture medium was measured by ELISA. Total RNA was extracted from the cells, and COX-2 mRNA expression was analyzed by Northern blot analysis.nnnRESULTSnDuring the time course of PGE(2) production in response to TPA stimulation, the PGE(2) production could not be detected until incubation had continued for 2h, but this production appeared to continue after 4h of incubation. PGE(2) production was significantly increased by TPA and suppressed by treatment with TPA and DEX. During the time course of COX-2 mRNA expression in response to treatment with TPA, the COX-2 mRNA band was detected after 1.5h. The strongest expression of COX-2 mRNA was observed at 2h incubation. After pre-treatment with TPA for 1h, the TPA-induced COX-2 mRNA was suppressed by treatment with DEX for 1 or 2h incubation in a dose-dependent manner.nnnCONCLUSIONSnThese results suggest that COX-2 mRNA is induced by TPA which activate protein kinase C, and suppressed by DEX in WISH cells.

Platelet-activating factor in human decidua: its role in parturition and preterm labor

Summary We have demonstrated that decidual macrophages produce and secrete PAF-AH of the plasma type. It is suggested that in addition to the PAF inactivation by plasma PAF-AH in the decidua, a localized PAF-AH secretion by decidual macrophages may exist for the autocrine or paracrine regulation of PAF concentration at the maternal-fetal interface. We have also demonstrated that LPS, TNF-α, IL-1α, and IL-1β inhibited the PAF-AH secretion by decidual macrophages and suggested a synergy among these bioactive molecules in the regulation of PAF metabolism in the decidua. These observations lend additional support to the concept that PAF is pathophysiologically involved in human parturition and provide further insights into the mechanism underlying the pathogenesis in preterm labor or premature rupture of membranes associated with bacterial infections.