Lipopolysaccharide and cytokines modulate leukotriene (LT)B4 and LTC4 production by porcine endometrial endothelial cells.

Uterine inflammatory response is mediated by inflammatory mediators including eicosanoids and cytokines produced by immune and endometrial cells. Interactions between lipopolysaccharide (LPS) and cytokines, and leukotrienes (LTs) in endothelium, important for the host defence during the inflammation, are unknown. We studied the effect of LPS, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4 and IL-10 on 5-lipooxygenase (5-LO), LTA4  hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA and protein expression, LTB4 and LTC4 release from porcine endometrial endothelial cells, and cell viability. For 24 hr, cells were exposed to LPS (10 or 100 ng/ml of medium) and cytokines (each 1 or 10 ng/ml). 5-LO mRNA/protein expression augmented after incubation with larger doses of LPS, TNF-α, IL-4 and IL-10 and smaller dose of IL-1ß. Larger dose of TNF-α, smaller doses of LPS and IL-1ß and both doses of IL-10 increased LTAH mRNA/protein expression. LTAH protein content was up-regulated by larger dose of LPS, but it was reduced in response to both doses of IL-4. LTCS mRNA expression was elevated by larger doses of LPS, IL-4 and IL-10 or both doses of TNF-α and IL-1ß. LTCS protein level increased after treatment with both doses of IL-1ß, IL-4 and IL-10, smaller dose of LPS and larger dose of TNF-α. Both doses of LPS and larger doses of TNF-α and IL-10 increased LTB4 release. LPS, IL-1ß and IL-10 at smaller doses, or TNF-α and IL-4 at larger doses stimulated LTC4 release. Smaller doses of TNF-α and IL-1ß or both doses of IL-4 enhanced the cell viability. This work provides new insight on the participation of LPS, TNF-α, IL-1ß, IL-4 and IL-10 in LTB4 and LTC4 production/release from porcine endometrial endothelial cells, and the effect of above factors on these cells viability. The used cellular model gives the possibility to further establish the interactions between inflammatory mediators.

The influence of leukotrienes C4 and D4 on the contractility of an inflamed porcine uterus.

The effect of leukotriene (LT) C4 (LTC4) and LTD4 on the contractility of an inflamed porcine uterus was investigated. On Day 3 of the estrous cycle (Day 0 of the study), either saline or Escherichia coli suspension was injected into each uterine horn. Although acute endometritis developed in all bacteria-inoculated gilts, a severe acute endometritis was noted more often on Day 8 than on Day 16. Myometrial and endometrial/myometrial strips were incubated with LTC4 or LTD4 alone, or together with a cysteinyl-LT receptor antagonist (BAY-u9773). Leukotriene C4 increased the contraction intensity in the saline- and bacteria-treated uteri on Day 8; however, its effect was lower in the myometrium of inflamed uteri. Contraction frequency was found to decrease in the saline-treated uteri as opposed to inflamed ones, in which it was elevated. On Day 16, contraction intensity increased in response to LTC4 in the saline-treated uteri but was reduced in the inflamed organs. The value of this parameter was lower in the inflamed uteri than that in the saline-treated ones. Leukotriene D4 (Days 8 and 16) augmented contractility in the saline-treated uteri, but despite increasing its intensity in the inflamed organs, it decreased contraction frequency. Leukotriene C4 or LTD4, added to BAY-u9773-pretreated saline- and bacteria-treated uteri on both days, decreased the contraction intensity. On Day 16 after treatment with BAY-u9773 and LTC4, contraction intensity in the endometrium/myometrium of the inflamed uteri was lower than that in the saline-treated organs. Data show that both LTC4 and LTD4 affect the contractility of inflamed porcine uteri, though LTC4 exerts a weaker contractile effect.

Pro- and anti-inflammatory mediators change leukotriene B4 and leukotriene C4 synthesis and secretion in an inflamed porcine endometrium.

We studied the effect of lipopolysaccharide (LPS), proinflammatory cytokines (tumor necrosis factor α [TNF-α] and interleukin [IL]-1ß), and anti-inflammatory cytokines (IL-4 and IL-10) on leukotriene (LT) A4 hydrolase and LTC4 synthase (LTCS) protein expression in, and LTB4 and LTC4 secretion from, an inflamed porcine endometrium. On day 3 of the estrous cycle (day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) CFU/mL) was injected into each uterine horn of gilts (n = 12 per group). Endometrial explants, obtained 8 and 16 days later, were incubated for 24 h with LPS (10 or 100 ng/mL of medium), TNF-α, IL-1ß, IL-4, and IL-10 (each cytokine: 1 or 10 ng/mL of medium). Although acute endometritis developed in all bacteria-inoculated gilts, a severe form of acute endometritis was diagnosed more often on day 8 of the study than on day 16. The amount of the LTA4 hydrolase (LTAH) protein in the inflamed endometrium on day 8 was greater after applying the lower dose of TNF-α (P < 0.001) and both doses of IL-1ß (P < 0.001) and IL-4 (1 ng, P < 0.01 and 10 ng, P < 0.001) than in the saline-treated uteri. A similar situation was observed in the case of the inflamed tissue on day 16 in response to LPS (100 ng, P < 0.01), TNF-α (10 ng, P < 0.05), and IL-4 (1 ng, P < 0.001). The content of LTC4 synthase in the inflamed endometrium on day 8 was reduced by LPS (100 ng, P < 0.05), IL-1ß (10 ng, P < 0.05), IL-4 (1 and 10 ng, P < 0.05), and IL-10 (1 ng, P < 0.01) but increased after the application of LPS (100 ng, P < 0.05) and TNF-α (1 and 10 ng, P < 0.001), IL-1ß, and IL-4 (1 ng, P < 0.05 and 10 ng, P < 0.001) on day 16. On day 8, endometrial secretion of LTB4 from the saline-injected and E coli-injected organs was similar in response to all of the used mediators. On the other hand, the contents of LTB4 in the medium decreased after incubating the inflamed tissues from day 16 with TNF-α (1 ng, P < 0.05 and 10 ng, P < 0.01), IL-1ß (1 ng, P < 0.01), and IL-10 (10 ng, P < 0.05) compared with the saline-treated ones. Secretion of LTC4 from the inflamed uteri on day 8 was elevated by the lower doses of TNF-α (P < 0.01) and IL-10 (P < 0.05), whereas on day 16, such an effect occurred in response to the higher doses of IL-4 (P < 0.01) and IL-10 (P < 0.05). The obtained results show that pro- and anti-inflammatory mediators participate in the synthesis/secretion of LTs from an inflamed porcine endometrium. Our data suggest that inflammatory mediators may indirectly affect the processes regulated by LTs by influencing LT production.

Long-term testosterone administration affects the number of paracervical ganglion ovary-projecting neurons in sexually mature gilts.

The influence of testosterone (T) overdose on the number and distribution of ovarian neurons in the paracervical ganglion (PCG) in pigs was examined. To identify the ovarian neurons, on day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue. From next day to the expected day 20 of the second studied cycle, experimental gilts were injected with T, while control gilts received oil. The PCG was then collected and processed for double-labeling immunofluorescence. T injections increased the T (∼3.5-fold) and estradiol-17ß (∼1.6-fold) levels in the peripheral blood, and reduced the following in the PCG: the total number of Fast Blue-positive neurons, the number of perikarya in the lateral part of the PCG, the numbers of VAChT(+)/SOM(+), VAChT(+)/VIP(+), VAChT(+)/nNOS(+), VAChT(+)/VIP(-), VAChT(+)/DßH(-), VAChT(-)/SOM(-), VAChT(-)/VIP(-), VAChT(-)/nNOS(-) and VAChT(-)/DßH(-) perikarya, In the T-affected PCG, the populations of ovarian perikarya coded VAChT(-)/SOM(+), VAChT(-)/VIP(+) and VAChT(-)/DßH(+), and expressing androgen receptor were increased. After T treatment within the PCG dropped the density of nerve fibers expressing VAChT and/or SOM, VIP, DßH. Obtained data suggest that elevated androgen levels occurring during pathological processes may regulate ovary function(s) by affecting the PCG gonad-supplying neurons.

Inflammation changes the expression of leukotriene receptors in porcine uteri.

Leukotrienes (LTs) are important as pro-inflammatory mediators to the innate immune response, inflammation, and allergy. Although it is known that inflammation of the uterus in gilts leads to an increase in LTB4 and LTC4 synthesis, researchers lack knowledge regarding the expression of LT receptors in the inflamed uterus. The aim of this experiment was to determine the levels of cysteinyl LT receptors type 1 (CysLT(1)R) and type 2 (CysLT(2)R), as well as of LTB4 receptor type 1 (LTB4R1) mRNA and protein expression, and the cellular localization of these receptors in the inflamed porcine uterus. On Day 3 of the estrous cycle (Day 0 of the study), 50 ml of either saline or Escherichia coli suspension (109 colony-forming units/ml) was injected into each uterine horn. Injections of bacteria increased CysLT(1)R mRNA levels in the endometrium on Days 8 and 16 of the study and in the myometrium on Day 16, as well as the CysLT(1)R protein level in the endometrium on Day 8. In the inflamed uteri, CysLT(2)R mRNA levels in the myometrium were increased on Days 8 and 16; however, the protein levels of this receptor decreased in the endometrium (Day 16) and myometrium (Day 8). After bacterial treatment, LTB4R1 mRNA and protein levels in the endometrium on Day 8, and LTB4R1 mRNA levels in the myometrium on Days 8 and 16 were found to have increased. Data obtained show that inflammation of porcine uteri changes the expression of LT receptors, suggesting their importance in the course/consequences of uterine inflammation.

Reduction of the number of neurones in the caudal mesenteric ganglion innervating the ovary in sexually mature gilts following testosterone administration.

The effect of testosterone on the morphological and chemical plasticity of the porcine caudal mesenteric ganglion (CaMG) ovary-projecting neurones was investigated. To identify the neurones on day 3 of the oestrous cycle, the ovaries of both the control and experimental gilts were injected with Fast Blue retrograde neuronal tracer. From next day until day 20 of the anticipated second studied cycle, experimental gilts were injected with testosterone, whereas control gilts received oil. Testosterone injections increased testosterone (by approximately 3.5-fold) and 17ß-oestradiol (by approximately 1.6-fold) levels in the peripheral blood and decreased the following in the CaMG: the total number of Fast Blue-positive perikarya (including small ones); the population of small perikarya in the caudal, ventral and dorsal ganglional regions; the numbers of dopamine-ß-hydroxylase (DßH) and/or neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) small and large perikarya; the numbers of small perikarya containing DßH (but not NPY, SOM, GAL); and the density of DßH and/or NPY, SOM nerve fibres. A disappearance of small and large non-noradrenergic perikarya and an increase in the total number of androgen receptor-immunoreactive perikarya was noted. Our results suggest that elevated androgen levels occurring during pathological states may regulate ovary function(s) by affecting the CaMG gonad-supplying neurones.

Synthesis of prostacyclin and its effect on the contractile activity of the inflamed porcine uterus.

The goal of the study was to estimate the content of prostacyclin (PGI(2)), the levels of PGI synthase (PTGIS) and receptor (PTGIR) protein expression, and the cellular localization of these factors in the inflammatory-changed porcine uterus. The effect of PGI(2) on the contractility of the inflamed uteri was also determined. On Day 3 of the estrous cycle (Day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) colony-forming units/mL) were injected into each uterine horn. Acute endometritis developed in all bacteria-inoculated gilts, however on Day 8 of the study a severe form of acute endometritis was noted more often than on Day 16. Bacteria injections increased the contents of 6-keto-prostaglandin F(1α) in endometrium, myometrium, washings, and the level of PTGIS in endometrium on Days 8 and 16, and the content of PTGIR in endometrium on Day 16. In the inflamed uteri on both study days, stronger immunoreactivity for PTGIS was observed in part of the luminal and glandular epithelial cells and in a portion of the endometrial arteries, and for PTGIR in part of the luminal epithelium and endothelial cells in a portion of the endometrial arteries. On Day 8, PGI(2) decreased contraction intensity in endometrium/myometrium and myometrium of the saline-treated uteri and increased the contraction intensity in both types of strips from the inflamed organs. Our study reveals that inflammation of the porcine uterus upregulates PGI(2) synthesis and that PGI(2) increases contractility, which suggests that PGI(2) might be essential for the course of uterine inflammation.

Isolation and characterization of zinc-binding proteins of canine seminal plasma.

Zinc-binding proteins from seminal plasma (ZnBPs) originate in the secretions of different accessory sex glands and are implicated in key events associated with sperm-egg fertilization processes. This study describes the isolation and characterization of the ZnBPs of canine seminal plasma. Ejaculates were collected from three crossbred dogs for a 2-week period. The ZnBPs as well as non zinc-binding proteins (nZnBPs) were isolated by zinc-dependent affinity chromatography. The isolated fractions were subjected to native gel electrophoresis (one-dimensional polyacryamide gel electrophoresis, PAGE) and sodium dodecyl sulphate polyacryamide gel electrophoresis (SDS-PAGE), using denaturing and reducing conditions. Zinc-elution profile using affinity chromatography displayed two protein fractions represented by the nZnBPs and ZnBPs, respectively. Using native gel electrophoresis, it was found that both the nZnBPs and ZnBPs occurred in their native state as aggregates, ranging from 140 to 669 kDa. The nZnBPs were disaggregated into 8 protein bands, with molecular weights ranging from 10.7 to 79.7 kDa, following SDS-PAGE analysis. By contrast, SDS-PAGE analysis of the ZnBPs revealed 13 protein bands, with molecular weights ranging from 11.6 to 152.3 kDa. Densitometric analysis showed that 46-48% of nZnBPs could be accounted by protein fractions with molecular weights of 10.7 and 14.2 kDa. Also, 2 protein fractions with molecular weights of 11.6 and 14.3 kDa, were predominant in ZnBPs, accounting for approximately 28-30% of the total proteins. These results demonstrate the zinc-binding capacity of proteins secreted by the canine prostate. The findings of this study indicate that ZnBPs of canine seminal plasma comprise several protein fractions, which might be implicated in the reproductive processes in the dog.