Shuko Murakami

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.

The influence of tumor necrosis factor α (TNF) on the secretory function of bovine corpus luteum: TNF and its receptors expression during the estrous cycle

Tumor necrosis factor alpha (TNF) inversely regulates the function of bovine corpus luteum (CL). Whereas the low doses of TNF induce luteolysis, the high doses prolong CL lifespan and prevent luteolysis in vivo. We suggest that the varying effects of TNF may be caused by its action exerted on CL via multiple signaling pathways involving two distinct receptors: TNFR-I (responsible for induction of the cell death) and TNFR-II (implicated in cell proliferation). In the study, we determined CL expressions of TNF, TNFR-I and TNFR-II mRNAs during the bovine estrous cycle using semi-quantitative RT-PCR. Specific transcripts for TNF, TNFR-I and TNFR-II were found in the CL with the highest (p<0.05) expression in the regressed CL. We also examined the TNF influence on the bovine CL function in vivo. On Day 15 of the estrous cycle, cows were infused (via aorta abdominalis) with saline, TNF (1 or 10 microg) or analogue of prostaglandin (PG)F(2alpha) (aPGF(2alpha) , 500 microg; a positive control). Four hours after infusions, CLs were collected by colpotomy and luteal contents of progesterone (P(4)), stable metabolites of nitric oxide (NO; nitrite/nitrate), leukotriene (LT)C(4), luteolytic PGF(2alpha),and luteotropic PGE(2) were determined. Moreover, caspase-3 activity was measured in the CLs as an indicator of apoptosis induction. The luteal content of P(4) decreased (p<0.05) after infusion of 1 microg of TNF. TNF inversely affected PGs content in CL: the low dose increased (p<0.01) the PGF(2alpha) level and the high dose increased (p<0.05) PGE(2) level. Contents of LTC(4) and nitrite/nitrate increased (p<0.01) after the low dose of TNF. Moreover, 1 microg of TNF induced apoptosis and increased (p<0.05) caspase-3 activity in the CLs collected during the late luteal phase. In conclusion, the high expressions of TNF and TNF receptors mRNAs were observed during or just after the luteolysis. A low concentration of TNF stimulated in vivo luteolytic factors such as PGF(2alpha), LTC(4) and NO as well as induced apoptosis; whereas the high concentration of TNF stimulated a survival pathway in the bovine CL increasing luteal content of P(4) and PGE(2).

Effects of tumor necrosis factor-α on secretion of prostaglandins E2 and F2α in bovine endometrium throughout the estrous cycle

To determine the physiological significance of tumor necrosis factor-alpha (TNFalpha) in the regulation of endometrial prostaglandin (PG) release in cattle, we investigated the effects of TNFalpha on the secretion of PGE2 and PGF2alpha by bovine endometrium during the estrous cycle. Bovine uteri were classified into six stages (estrus: Day 0, early luteal 1: Days 2 to 3, early luteal 11: Days 5 to 6, mid-luteal: Days 8 to 12, late luteal: Days 15 to 17 and follicular: Days 19 to 21). After 1 h of pre-incubation, endometrial tissues (20 to 30 mg) were exposed to 0 or 0.6 nM TNFalpha for 4 h. The PGE2 concentrations in the medium were higher in the luteal stages than in the follicular stage and in estrus. In contrast, PGF2alpha concentrations were higher in the follicular stage and in estrus than in the luteal stages. The ratio of the basal concentrations of PGE2 and PGF2alpha (PGE2/PGF2alpha ratio) was higher in the luteal stages than in the follicular stage and in estrus. Although TNFalpha stimulated both PGE2 and PGF2alpha secretion during the entire period of the estrous cycle, the level of stimulation of TNFalpha on PGE2 output by the bovine endometrium does not show the same cyclical changes as that shown on PGF2alpha output. The stimulation of TNFalpha resulted in a decrease in the PGE2/PGF2alpha ratio only in the late luteal stage. Furthermore, TNFalpha stimulated PGE2 secretion in stromal, but not epithelial cells. The overall results suggest that TNFalpha is a potent regulator of endometrial PGE2 secretion as well as PGF2alpha secretion during the entire period of estrous cycle, and that TNFalpha plays different roles in the regulation of secretory function of bovine endometrium at different phases of the estrous cycle.

Interferon-τ Blocks the Stimulatory Effect of Tumor Necrosis Factor-α on Prostaglandin F2α Synthesis by Bovine Endometrial Stromal Cells

Abstract Tumor necrosis factor-α (TNFα) has been shown to be a potent stimulator of prostaglandin (PG) F2α synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-τ (IFNτ) on TNFα-stimulated PGF2α synthesis and the intracellular mechanisms of TNFα and IFNτ action in the stromal cells. When cultured bovine stromal cells were exposed to TNFα (0.006–0.6 nM) for 24 h, the production of PGF2α and cyclooxygenase (COX)-2 gene expression were stimulated by TNFα (0.06–0.6 nM, P < 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNFα on PGF2α production (P < 0.05). Although IFNτ (0.03–30 ng/ml) did not stimulate basal PGF2α production in the stromal cells, it suppressed TNFα action in PGF2α production dose dependently (P < 0.05). Moreover, the stimulatory effect of TNFα (0.6 nM) on COX-2 gene expression was completely blocked by IFNτ (30 ng/ml; P < 0.05), although the gene expression of COX-2 was not influenced by IFNτ. The overall results indicate that the stimulatory effect of TNFα on PGF2α production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFNτ on luteolysis is the inhibition of TNFα action in PGF2α production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNFα.

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.

Tumor necrosis factor-α and its receptor in the corpus luteum of pregnant cows.

The objective of this study was to investigate the presence of tumor necrosis factor (TNF)‐α mRNA and TNF‐α receptors in the bovine corpus luteum (CL) during the gestation period. TNF‐α mRNA and TNF‐α receptors were determined on bovine CL from pregnant cows at three stages: trimester I (fetal crown‐rump length; 6–20 cm), trimester II (25–45 cm) and trimester III (50–80 cm). TNF‐α mRNA was detected by an RT‐PCR analysis in the CL of all stages of gestation. A Scatchard analysis revealed the presence of a high‐affinity binding site (Kd; 5.1–6.9 nM) in the CL membranes collected at each stage of gestation. Furthermore, the concentrations of TNF‐α receptors in the CL of trimesters I (24.0 ± 1.95 pmol/mg protein) and III (21.6 ± 2.39 pmol/mg protein) of gestation were significantly higher than the concentration in trimester II (14.9 ± 2.07 pmol/mg protein) (P < 0.05). These results indicate that TNF‐α is locally produced and that TNF‐α receptors are present in bovine CL during the gestation period, and suggest that TNF‐α plays one or more roles as a paracrine factor in regulating bovine CL function during the entire gestation period. Mol. Reprod. Dev. 55:406–411, 2000.

Tumor necrosis factor-α stimulates prostaglandin F2α secretion by bovine luteal cells via activation of mitogen-activated protein kinase and phospholipase A2 pathways

It has been well demonstrated that tumor necrosis factor‐α (TNFα) stimulates prostaglandin (PG) F2α secretion by bovine corpus luteum (CL) in vitro. The objective of the present study was to clarify the intracellular signaling pathway of TNFα to stimulate PGF2α production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid‐ (days 8–12 after ovulation) CL were incubated with TNFα (0.6 nM) and/or various compounds as follows: U‐73122 (an inhibitor of phospholipase [PL] C), ACA (an inhibitor of PL‐A2), H‐89 (an inhibitor of protein kinase [PK] A), calphostin C (an inhibitor of PK‐C), L‐NAME/L‐NORG (inhibitors of nitric oxide synthase), and PD98059 (an inhibitor of mitogen‐activated protein kinase [MAPK] kinase). Although U‐73122 (0.1–10 μM), H‐89 (0.1–10 μM), calphostin C (0.01–1 μM) and L‐NAME/L‐NORG (1–100 μM) did not affect TNFα‐induced PGF2α secretion by the cultured cells, ACA (1–100 μM) and PD98059 (0.1–100 μM) inhibited TNFα‐stimulated PGF2α secretion by the cells in a dose‐dependent fashion (P < 0.05 or lower). These findings suggest that TNFα activates the MAPK and PL‐A2 pathways in bovine luteal cells to stimulate PGF2α secretion. Mol. Reprod. Dev. 56:387–391, 2000.