Cultured Porcine Luteal Cells Research Articles

Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) mediates the LH-induced stimulation of progesterone synthesis in the porcine corpus luteum.

Progesterone (P4) synthesized by the corpus luteum (CL) plays a key role in the establishment and maintenance of pregnancy. The LH signal is important for luteinisation and P4 synthesis in pigs. In a previous study, we demonstrated that mitogen-activated protein kinase kinase kinase 8 (MAP3K8) regulates P4 synthesis in mouse CL, but whether the function and mechanism of MAP3K8 in the pig is similar to that in the mouse is not known. Thus, in the present study we investigated the effects of MAP3K8 on porcine CL. Abundant expression of MAP3K8 was detected in porcine CL, and, in pigs, MAP3K8 expression was higher in mature CLs (or those of the mid-luteal phase) than in regressing CLs (late luteal phase). Further functional studies in cultured porcine luteal cells showed that P4 synthesis and the expression of genes encoding the key enzymes in P4 synthesis are significantly reduced when MAP3K8 is inhibited with the MAP3K8 inhibitor Tpl2 kinase inhibitor (MAP3K8i, 10μM). After 12-24h treatment of luteal cells with 100ngmL-1 LH, MAP3K8 expression and P4 secretion were significantly upregulated. In addition, the 10μM MAP3K8 inhibitor blocked the stimulatory effect of LH on P4 synthesis and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in porcine luteal cells. The LH-induced increases in MAP3K8 phosphorylation and expression, ERK1/2 phosphorylation and P4 synthesis were all blocked when protein kinase A was inhibited by its inhibitor H89 (20 μM) in porcine luteal cells. In conclusion, MAP3K8 mediates the LH-induced stimulation of P4 synthesis through the PKA/mitogen-activated protein kinase signalling pathway in porcine CL.

The influence of IL-1β, TNFα and IL-6 on induction of apoptosis in cultured porcine luteal cells

The influence of IL-1β, TNFα and IL-6 on induction of apoptosis in cultured porcine luteal cells

Effect of prostaglandin E2 and tumor necrosis factor α on the VEGF‐Receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.

Tumor Necrosis Factor-.ALPHA. Induces Apoptosis in Cultured Porcine Luteal Cells

Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.

Effect of growth hormone and insulin-like growth factor-I on spontaneous apoptosis in cultured luteal cells collected from early, mature, and regressing porcine corpora lutea

The aim of the present study was to test the hypothesis that growth hormone (GH) and insulin-like growth factor-I (IGF-I) act at a local level to inhibit luteal cell apoptosis. Luteal cells collected from the corpora lutea at different stages of the luteal phase were cultured for 24 h in M 199 medium supplemented with 5% of calf serum to cause attachment cells to the plastic. After 24 h, the media were changed and various concentrations of GH (10, 100 or 200 ng/ml) or IGF-I (30, 50 or 100 ng/ml) were added to the culture medium. Twenty-four hours later, cells were fixed for morphological assessment of apoptotic cells utilising a Hoechst staining technique. To support morphological observations, measurements of caspase-3 activity in cultured porcine luteal cells were performed. Increased incidence of apoptotic bodies and caspase-3 activity accompanied luteal regression and was associated with a decreased progesterone (P4) secretion by luteal cells. GH stimulated P4 secretion by luteal cells collected from developing (ELP) and mature (MLP) corpora lutea but had no effect on its secretion by cells collected from regressing corpora lutea (LLP). Moreover, it had no effect on the incidence of apoptotic bodies in all types of corpora lutea. However, suppression of caspase-3 activity was observed with 100 and 200 ng of GH/ml in all types of corpora lutea. IGF-I had a stimulatory effect on P4 secretion by ELP and MLP, decreased the incidence of apoptotic bodies and suppressed caspase-3 activity in cultures treated with all doses used. In conclusion, our results indicate that both GH and IGF-1 trigger anti-apoptotic effects either indirectly, by increasing progesterone secretion, or directly, through the inhibition of caspase-3 activity and subsequent prevention of apoptotic body formation.

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay.

The role of the basic fibroblast growth factor (basic FGF) in the control of secretion of the ovarian protein hormone relaxin (RLX) by porcine large luteal cells (LLCs) was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled sheep erythrocytes. In the presence of complement and porcine relaxin antiserum, a zone of hemolysis (a plaque) developed around a RLX-releasing LLC. The rate of plaque development in time-course experiments was used as an index of the rate of RLX secretion. Monolayers were bathed in medium containing graded concentrations of basic FGF in the presence or absence of a stimulatory secretagogue [0.01 microM prostaglandin E2 (PGE2)]. Exposure of luteal cell-containing monolayers to basic FGF resulted in a significant reduction (P less than 0.05) in the rate of RLX-induced plaque formation, evidence of an inhibitory effect of basic FGF on the rate of basal RLX secretion. This suppressive effect was variable in onset (1-3 h of incubation) and dose related. Minimally and maximally effective doses were about 10 and 30 ng/ml basic FGF, respectively. Higher doses of basic FGF (20 and 30 ng/ml) entirely suppressed RLX from a substantial subset of LLCs (10-20% of all LLCs), an indication of a differentially sensitive subpopulation. Addition of basic FGF (30 ng/ml) to monolayes also treated with PGE2 resulted in a significant (P less than 0.05) attenuation of the stimulatory effect of PGE2 on RLX secretion, evidence that these agents functionally interact in the modulation of RLX. We conclude that these results taken in association with the prior demonstration of the presence of basic FGF in luteal tissue suggest that basic FGF serves as a local inhibitory mechanism that regulates RLX secretion. Furthermore, the ability of basic FGF to counteract the effect of PGE2 implies that intraluteal stimulatory/inhibitory agents may act in concert to achieve fine control of RLX secretion. The observation of a preferentially responsive subpopulation is consistent with the possibility that basic FGF is implicated in heterogenous RLX secretion. Nevertheless, the physiological role(s) of basic FGF in the control of RLX secretion and the interrelationships of basic FGF with other local and systemic secretagogues remain to be clearly defined.

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay

Basic fibroblast growth factor inhibits basal and stimulated relaxin secretion by cultured porcine luteal cells: analysis by reverse hemolytic plaque assay

Analysis of Relaxin Release by Cultured Porcine Luteal Cells Using a Reverse Hemolytic Plaque Assay: Effects of Arachidonic Acid, Cyclo- and Lipooxygenase Blockers, Phospholipase A2, and Melittin*

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 microM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55-65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 microM, respectively. In the presence of 10 microM AA, maximal plaque formation occurred significantly faster (1-2 h) than in controls (4-8 h; P less than 0.05). The percentage of plaque-forming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 microM AA-treated monolayers than in controls during the first 3-4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A2 and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 microM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 microM); but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 microM). However, the same dose of ibuprofen (20 microM) failed to modulate the stimulatory effect of prostaglandin E2 (1 microM) or phorbol diester (4 beta-phorbol 12 beta-myristate 13 alpha-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.

Inhibitory effect of oxytocin and vasopressin on steroid release by cultured porcine luteal cells.

Previously, we have demonstrated the presence of substances reacting like arginine vasopressin (AVP) and oxytocin (OXT) in acid extracts of corpora lutea (CL) of pigs by RIA. The present study examined purified extracts of CL by using HPLC. The results of these experiments show that CL of nonpregnant sows contain AVP and OXT. Little is known about possible auto- and paracrine effects of AVP and OXT in the ovary. Therefore, we investigated the influence of AVP and OXT on progesterone, estradiol, and androstenedione secretion in porcine luteal cell cultures from nonpregnant sows. Progesterone and androstenedione secretion increased significantly (P less than 0.05) in the presence of ovine LH (oLH), whereas no change in basal estradiol levels could be observed under the same conditions. When AVP or OXT was added to the culture system a dose-dependent inhibition of basal as well as oLH-stimulated progesterone secretion was measured. Under basal conditions, a dose of 1 pg AVP/ml decreased progesterone secretion significantly (P less than 0.05), but to reach the same effect in the presence of OXT a dose of 100 ng/ml was necessary. In the presence of oLH the addition of as little as 0.01 pg AVP/ml inhibited progesterone secretion significantly (P less than 0.05). On the other hand, 10 ng OXT/ml or higher doses were needed to decrease oLH-stimulated progesterone release. In the presence of specific peptide antagonists the inhibitory effect on progesterone release was abolished. These results suggest that AVP and OXT effects are mediated through specific receptors. OXT and AVP also inhibited androstenedione secretion, but had no effect on estradiol secretion. Calculation of the ED50 data from dose-response curves of both peptides show that AVP is about 10(4)-fold more active than OXT in inhibiting in vitro progesterone and androstenedione secretion. This suggests that AVP as well as OXT may play an important role in the regulation of ovarian function.

Prostacyclin stimulates relaxin release from cultured porcine luteal cells.

The release of relaxin from cultured porcine luteal cells (derived from pregnant sows) was detected by a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. Treatment with a prostacyclin analogue (carba-prostacyclin; 10 and 100 ng/ml) significantly accelerated the rate of plaque formation in a dose-dependent manner. In contrast, the culture of luteal cells in the presence of cyclooxygenase inhibitors (indomethacin, ibuprofen, and diclofenac sodium) resulted in no change in the rate of basal relaxin release. These results add weight to the view that prostacyclin may contribute alone, or perhaps in combination with other prostanoids, to the overall regulation of ovarian endocrine function and relaxin release during pregnancy in the pig. However, the data are inconsistent with the possibility that prostacyclin arises from relaxin-releasing luteal cells themselves.

Detection of relaxin release by porcine luteal cells using a reverse hemolytic plaque assay: effect of prostaglandins E2 and F2 alpha, human chorionic gonadotropin, and oxytocin.

The release of relaxin from cultured porcine luteal cells derived from pregnant sows was detected by a reverse hemolytic plaque assay. In this assay, luteal cells are cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis--a plaque--develops around relaxin-releasing luteal cells. Treatment with prostaglandin E2 (10(-8) and 10(-6) M) significantly accelerated the rate of plaque formation; in contrast, human chorionic gonadotropin (10-1,000 IU/ml) inhibited the rate of plaque formation. Oxytocin (10(-8) to 10(-4) M) had no detectable effect on relaxin release. However, none of these treatments or long-term preexposure to prostaglandin F2 alpha increased the total proportion of large luteal cells that released relaxin, which remained at about 50%. These results are consistent with the idea that prostaglandins of uterine and/or luteal origin and pituitary luteinizing hormone may contribute, alone or perhaps in combination, to the overall regulation of ovarian relaxin release during pregnancy in the sow. In addition, the results indicate that the effects of prostaglandins are restricted to a subpopulation of large luteal cells that release detectable amounts of relaxin in culture.

Analysis of relaxin release from cultured porcine luteal cells by reverse hemolytic plaque assay: influence of gestational age and prostaglandin F2 alpha.

Relaxin release from dispersed luteal cells was detected by a reverse hemolytic plaque assay to determine the influence of gestational age on basal relaxin secretion. Monodispersed luteal cells were derived by collagenase treatment of corpora lutea obtained from pigs in early (days 19-26), mid-(days 47-62), and late (days 80-99) gestation. The rate of plaque development under nonstimulated conditions progressively accelerated as gestation advanced, as did the rate of increase in plaque size. These results unequivocally demonstrate that basal relaxin release increases with advancing gestation. However, only about 50% of large luteal cells released relaxin at all stages of pregnancy examined up to day 100. These data indicated not only that basal relaxin release increases during pregnancy, but also that considerable heterogeneity exists with respect to relaxin output by individual cells. In contrast, both the basal rate of relaxin release and the percentage of cells committed to relaxin release declined significantly when luteal cells derived from preparturient sows (days 107-112 of gestation) were examined. Exposure of cultured luteal cells to prostaglandin F2 alpha (10(-8) and 10(-6) M) resulted in a rapid stimulation of relaxin secretion, but this agent did not recruit additional cells into the secretory pool. These data are consistent with the idea that autonomous changes and the action of secretagogues may combine at different times to achieve overall regulation of relaxin release by the corpus luteum. The significance of nonrelaxin-releasing luteal cells remains to be determined.