Conjugated linoleic acid decreases prostaglandin synthesis in bovine luteal cells in vitro

Feeding conjugated linoleic acids (CLA) improves reproductive performance in dairy cows; however, the molecular mechanisms by which CLA improves reproduction are not understood. The effect of the CLA isomers, trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA on synthesis of progesterone, PGE₂, and PGF_2α, in bovine luteal cells was determined in this study. Luteal cells from three cows were cultured in medium containing 0 or 0.1 µM of trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA in varying ratios in the presence and absence of 1 µM of forskolin. Prostaglandin and progesterone concentrations were not altered by CLA isomer and ratio. Luteal cells cultured in the presence of CLA had lower PGF_2α concentrations (62.6 ± 13.4 pg/ml vs. 55.7 ± 12.2 pg/ml; P = 0.005) and, in the absence of forskolin, lower PGE₂ concentrations (65.3 ± 15.1 pg/ml vs. 32.4 ± 14.1 pg/ml; P = 0.002) in culture media, while progesterone concentrations were not altered (P = 0.63). Relative steady‐state mRNA amounts of COX‐2 (1.7‐fold decrease; P = 0.002), PGE synthase (1.5‐fold decrease; P = 0.03) and 3β‐hydroxysteroid dehydrogenase (1.6‐fold decrease; P = 0.0003) were lower in CLA‐treated cultures, but CLA did not significantly alter mRNA amounts of PGE₂ 9‐keto‐reductase, StAR, and cytochrome P450 side chain cleavage enzyme. In conclusion, a potential mechanism exists by which trans‐10, cis‐12 CLA and cis‐9, trans‐11 CLA may improve reproductive performance in dairy cows, by suppressing PGF_2α synthesis in luteal cells via attenuation of COX‐2 gene expression. Mol. Reprod. Dev. 78:328–336, 2011. © 2011 Wiley‐Liss, Inc.     

Endothelin-1 is a potent stimulator of alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells

Endothelin-1 (ET) is known to stimulate mesangial cell (MC) proliferation, extracellular matrix (ECM) synthesis, and thereby contribute to the progression of glomerulonephritis (GN). To clarify the molecular and cellular mechanisms of how ET is involved in the development of glomerular sclerosis, we investigated the influence of ET on the MC-alpha1beta1 integrin-mediated collagen matrix reorganization using a collagen gel contraction assay. ET enhanced MC-alpha1beta1 integrin-mediated gel contraction in a dose-dependent manner. Addition of the endothelin A (ETA) receptor antagonist, BQ123, into collagen gels abolished ET-induced gel contraction by MC. Cell behavior involved in ET-induced gel contraction was investigated in combination with function-blocking anti-alpha1-integrin antibody. Migration and adhesion assays revealed that ET stimulated alpha1beta1 integrin-mediated MC migration but did not influence cell adhesion to type I collagen (collagen I). Integrin-function blocking studies using anti-alpha1 integrin antibody indicated that MC-alpha1beta1 integrin is required not only for collagen-dependent migration, but also for gel contraction. Zymography showed that ET increased MC matrix metalloproteinase-2 (MMP-2) activity in a dose-dependent manner during MC-induced gel contraction process. Finally, flow cytometry analysis indicated that ET did not affect the cell surface expression of the MC-alpha1beta1 integrin within the collagen gel. These data suggested that ET promotes collagen matrix reorganization through the enhancement of MC-alpha1beta1 integrin-dependent migration and MMP-2 activity. We therefore conclude that ET is a potential molecule inducing pathological collagen matrix remodeling observed in progressive GN.     

Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture

Interleukin-1 beta (IL-1 beta) strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin and epidermal growth factor (EGF). Its effect was dose-dependent and was maximal at 2 ng/ml. IL-1 beta had no cytotoxic effect but changed the cells from a flat to a spindle shape as shown by phase-contrast microscopy. The inhibition of DNA synthesis by IL-1 beta was closely correlated with a decrease in the labeling index. This inhibitory effect was observed only when IL-1 beta was added for 10 h to cultured hepatocytes in the G1 phase within 12 h after addition of insulin and EGF: it was not observed in the S phase, which starts about 24 h after addition of the mitogens. Exposure of the hepatocytes to IL-1 beta for two 1-h periods, one at an early stage (0-6 h) and one at a late stage (6-12 h) of the G1 phase, resulted in the same marked inhibition of DNA synthesis as exposure to IL-1 beta for 10 h in the G1 phase. This requirement of IL-1 beta at two stages in the G1 phase for inhibition of DNA synthesis of hepatocytes is different from that with transforming growth factor-beta, which is required for only 1 h in the early G1 phase for a similar inhibition. These findings suggest that IL-1 beta acts at two distinct stages in the G1 phase and that its cooperative actions are necessary to inhibit growth of adult rat hepatocytes in primary culture. Other cytokines, such as IL-6/B-cell stimulating factor-2, were less potent, but caused significant inhibition of DNA synthesis of adult rat hepatocytes at 2 ng/ml, whereas IL-2 and tumor necrosis factor did not affect hepatocyte growth. From these results it is suggested that Kupffer cells in liver lobules and macrophages in the blood may play important roles, mainly via IL-1, in repair of liver damage and regeneration.     

Progesterone is a suppressor of apoptosis in bovine luteal cells

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.     

Gossypolone suppresses progesterone synthesis in bovine luteal cells

Gossypolone, a proposed major metabolite of gossypol, was synthesized and investigated for its effect on progesterone synthesis in cultured bovine luteal cells. Gossypolone inhibited human chorionic gonadotropin(hCG)-stimulated progesterone secretion, reduced substrate-enhanced conversions of 25-hydroxycholesterol to pregnenolone and of pregnenolone to progesterone in a dose-dependent fashion. These findings indicate that gossypolone inhibits not only 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, as gossypol does, but also side-chain cleavage enzyme complex (cytochrome P450scc activity. However, the two compounds appear to have a similar potency in inhibiting progesterone secretion. Both gossypolone and gossypol (8.5 μM) induced morphological changes in cellular organelles.     

Interleukin-1 beta is more potent than interleukin-1 alpha in suppressing follicle-stimulating hormone-induced differentiation of ovarian granulosa cells

Most studies have shown that the immune and inflammatory actions of interleukin-1 alpha and beta exhibit the identical biological spectrums of activity with similar dose-response curves. We have previously demonstrated that interleukin-1 beta suppresses follicle-stimulating hormone-induced differentiation of ovarian granulosa cells. In these experiments, we show that although the human recombinant preparations of interleukin-1 alpha and beta exhibit a similar directional inhibition of ovarian granulosa cell differentiation, there is a significant difference in the dose-response relationships between the two forms. Interleukin-1 beta was 31 times and 18 times more potent than interleukin-1 alpha in suppressing follicle-stimulating hormone-induced luteinizing hormone receptor development and progesterone secretion, respectively, from rat granulosa cells. However, there was no difference in the dose-dependent activities of interleukin-1 alpha and beta in stimulating murine thymocyte proliferation. These results suggest that interleukin-1 beta is more effective in influencing ovarian granulosa cell function than interleukin-1 alpha.     

Regulation of steroidogenesis and cholesterol synthesis by prostaglandin F-2 alpha and lipoproteins in bovine luteal cells

Bovine luteal cells can utilize low density lipoprotein (LDL) or high density lipoprotein (HDL) as a source of cholesterol for steroidogenesis, and administration of PGF-2 alpha in vitro suppresses lipoprotein utilization. The objective of this study was to examine the mechanism by which PGF-2 alpha exerts this effect. Cultured bovine luteal cells received 0.25 microCi[14C]acetate/ml, to assess rates of de-novo sterol and steroid synthesis, with or without lipoproteins. Both LDL and HDL enhanced progesterone production (P less than 0.01), but caused a significant reduction in the amount of radioactivity in the cholesterol fraction. PGF-2 alpha treatment inhibited the increase in lipoprotein-induced progesterone synthesis (P less than 0.01), but did not prevent the reduction in de-novo cholesterol synthesis brought about by LDL or HDL. PGF-2 alpha alone reduced cholesterol synthesis (P less than 0.01), but it was not as effective as either LDL or HDL. Both lipoproteins and PGF-2 alpha also decreased the amount of radioactivity in the progesterone fraction (P less than 0.01), and the effect of PGF-2 alpha was similar to that of the lipoproteins. It is concluded that lipoproteins can enhance progesterone production and also suppress de-novo cholesterol synthesis in bovine luteal cells, but only the former effect of lipoproteins is inhibited by PGF-2 alpha. Therefore, it is suggested that PGF-2 alpha allows entry of lipoprotein cholesterol into the cell, but prevents utilization for steroidogenesis. In addition, PGF-2 alpha alone can suppress cholesterol synthesis, as well as decrease conversion of cholesterol to progesterone.     

Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth.

Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 ng/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-beta 1. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes.     

Localization of prostaglandin F2 alpha inhibition of lipoprotein use by bovine luteal cells.

Prostaglandin F2 alpha (PGF2 alpha) inhibits lipoprotein-stimulated progesterone production by bovine luteal cells in vitro and the objective of this study was to localize the site of action of PGF2 alpha. Cultured bovine luteal cells were treated with PGF2 alpha for seven days, and then with either lipoproteins or 25-hydroxycholesterol in the presence of aminoglutethimide (which inhibits cholesterol side-chain cleavage) for the final 48 h. The effects of PGF2 alpha on progesterone production, cellular cholesterol content, mitochondrial cholesterol content and cholesterol side-chain cleavage activity were determined. As expected, PGF2 alpha inhibited (P less than 0.05) lipoprotein-stimulated progesterone production. However, PGF2 alpha did not inhibit low-density lipoprotein-stimulated, or high density lipoprotein-stimulated, increases in cellular cholesterol (P less than 0.05) or inhibit lipoprotein-induced increases in mitochondrial cholesterol content (P less than 0.05). Additionally, cholesterol content of mitochondria increased (P less than 0.05) in the presence of PGF2 alpha alone. To determine if the PGF2 alpha-induced inhibition of steroidogenesis occurred at, or after, the side-chain cleavage reaction, we treated cells with the readily diffusable sterol, 25-hydroxycholesterol. Prostaglandin F2 alpha did not inhibit 25-hydroxycholesterol-stimulated progesterone production (P less than 0.05). Prostaglandin F2 alpha may therefore exert its luteolytic effect at a site after cholesterol transport to the mitochondria but before cholesterol side-chain cleavage.     

Interleukin-1 beta stimulates decidual stromal cell cyclo-oxygenase enzyme and prostaglandin production.

The cytokine interleukin-1 (IL-1 beta) increased prostaglandin production by decidual stromal cells in culture in a time and dose dependent manner. Optimum conditions for stimulation were found to be for 24 hours at a concentration of 100 pg IL-1 beta/ml. An apparent increase in cyclo-oxygenase enzyme synthesis accompanied the increase in prostaglandin production, and both changes were inhibited by the protein synthesis inhibitor cycloheximide. This implicates protein synthesis in the stimulatory effects of IL-1 beta, which may be mediated through the increase in cyclo-oxygenase enzyme. A pre-incubation period of 72 hours was found to be necessary to observe the stimulatory effect of IL-1 beta on prostaglandin production, but this did not seem to be due to any change in the sensitivity of the cells to IL-1 beta; the increase in the number of cyclo-oxygenase positive cells was the same if IL-1 beta was added on day 1, day 2 or day 3 of culture, even though prostaglandin production was not stimulated on day 1 or day 2. Cycloheximide increased prostaglandin production on the first two days of culture and had no effect on the third day of culture. This was interpreted as indicating that a factor inhibiting cyclo-oxygenase activity was synthesised during the initial period of culture, which prevented any increase in prostaglandin production following the increase in enzyme synthesis.     

Ethanol is a potent stimulator of phosphatidylcholine breakdown in cultured rat hepatocytes.

Addition of ethanol (17 to 340 mM) to cultured rat hepatocytes stimulated the breakdown of phosphatidylcholine phospholipases D and C as measured by an increase in the rate of release of choline and phosphocholine into the medium. The effects of ethanol were mimicked by propanol, dimethylsulfoxide and to a lesser extent methanol. The magnitude of the stimulation seen with ethanol was equivalent to and additive to that produced by glucagon vasopressin, norepinephrine, A23187 or PMA. In contrast, ethanol (340 mM) stimulated PI-specific phospholipase C activity by less than 20%. An equivalent stimulation of PC-specific phospholipase D and C was seen with as little as 20 mM ethanol and a 100% increase was seen with 340 mM ethanol. Ethanol did not significantly affect the ability of vasopressin, norepinephrine, ATP or A23187 to stimulate PI-specific phospholipase C. It is concluded that while ethanol is only a weak stimulator of PI-specific phospholipase C, it is a potent stimulator of phosphatidylcholine breakdown in rat hepatocytes.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

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 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) 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.     

Interleukin-1beta induces chronic activation and de novo synthesis of neutral ceramidase in renal mesangial cells.

The lipid signaling molecule ceramide is formed by the action of acid and neutral sphingomyelinases and degraded by acid and neutral ceramidases. Short-term stimulation of mesangial cells with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) leads to a rapid and transient increase in neutral sphingomyelinase activity (Kaszkin, M., Huwiler, A., Scholz, K., van den Bosch, H., and Pfeilschifter, J. (1998) FEBS Lett. 440, 163-166). In this study, we report on a second delayed peak of activation occurring after hours of IL-1beta treatment. This second phase of activation was first detectable after 2 h of treatment and steadily increased over the next 2 h, reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity was observed, accounting for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity was due to expressional up-regulation, as detected by an increase in mRNA levels and enhanced de novo protein synthesis. The increase in neutral ceramidase protein levels and activity could be blocked dose- dependently by the p38 MAPK inhibitor SB 202190, whereas the classical MAPK pathway inhibitor U0126 and the protein kinase C inhibitor Ro 318220 were ineffective. Moreover, cotreatment of cells for 24 h with IL-1beta and SB 202190 led to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation was not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase-2, which is a downstream substrate of p38 MAPK, thus suggesting that the p38 MAPK-mediated induction of neutral ceramidase occurs independently of the MAPK-activated protein kinase-2 pathway. In summary, our results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens

We investigated the roles of estrogens and androgens in the progesterone biosynthesis of bovine luteal cells. The responsiveness of primary luteal cells to the stimulation of tropic agents was observed in a dose-dependent manner. Estrogens and androgens significantly inhibited tropic agent-induced progesterone secretions, but glucocorticoids did not, which indicated the inhibitions were specific. The failure of exogenous 8-Br-cAMP to prevent these inhibitions suggested that took place at the post-cAMP steps. The immunoblot showed that testosterone remarkably decreased the amount of induced P450scc protein after 6-hour treatment, yet 17beta-estradiol did not. The 3beta-HSD activity assays demonstrated that both 17beta-estradiol and testosterone efficiently blocked induced 3beta-HSD activities. Both inhibitory effects of E2 and T on progesterone synthesis were observed one hour after treatment and accompanied with suppressed 3beta-HSD activities. This study presents that estrogens and androgens specifically inhibit bovine luteal function through different mechanisms.     

3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Fas-mediated apoptosis is suppressed by calf serum in cultured bovine luteal cells

Calf serum (CS) is a common supplement used in cell culture. It has been suggested that CS contains substances protecting cells against apoptosis. To examine whether a culture system including CS is appropriate for studying apoptosis in bovine luteal cells, we examined the influence of CS on the expression of Fas, bcl-2 and bax gene. Since progesterone (P(4)) is known to be an anti-apoptotic factor in bovine luteal cells, the present study was carried out to examine the P(4) effect on apoptosis. Bovine mid-luteal cells were exposed to Fas ligand (Fas L) in the presence or in the absence of P(4) antagonist (onapristone, OP) in a basal medium (BM) containing 5% CS (BM-CS) or BM containing 0.1% BSA (BM-BSA). Although Fas L alone, OP alone or Fas L plus OP did not show any cytotoxic effect on the cells cultured in BM-CS, administration of OP or OP in combination with Fas L resulted in the killing of 30% and 55% of the cells cultured in BM-BSA medium, respectively (p<0.05). Concomitantly, CS inhibited bax mRNA expression and stimulated bcl-2 expression in the cells (p<0.05). Moreover, in the cells cultured with BM-CS, Fas mRNA expression was smaller than that of cells incubated in BM-BSA medium (p<0.05). The overall results suggest that CS suppressed Fas-mediated cell death in cultured bovine luteal cells by promoting the ratio of bcl-2 to bax expression and by inhibiting Fas expression. Therefore, it may be suggested that CS contains such anti-apoptotic substances (growth factors) amplifying the cell survival pathways in the bovine corpus luteum (CL) in vitro.