Joanna Czarzasta

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.

Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli

Leukotrienes (LTs) are lipid mediators that play a significant role in the inflammatory process. Their production in inflamed uteri is not fully understood. The present experiment aimed to determine LTB4 and LTC4 amounts, 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA levels and protein expression in inflamed porcine uteri. On Day 3 of the oestrous cycle (Day 0 of the study), either Escherichia coli suspension or saline were infused into uterine horns. Collection of uterine tissues and washings took place eight or sixteen days later. In gilts suffering from endometritis increased LTB4 and LTC4 levels in the endometrium and washings and 5-LO mRNA levels in the myometrium on Days 8 and 16, 5-LO protein levels in the endometrium and myometrium on Day 8, LTAH mRNA and protein levels in the endometrium and myometrium on Days 8 and 16, respectively. Although LTCS mRNA and protein expression in the myometrium and LTCS protein expression in the endometrium were enhanced on Day 16 after Escherichia coli inoculation, LTCS mRNA levels decreased on Day 8 in both tissues. Our study shows the upregulation of LT production in inflamed porcine uteri, which suggests the importance of these factors to the process 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 17β‐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‐β‐hydroxylase (DβH) and/or neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL) small and large perikarya; the numbers of small perikarya containing DβH (but not NPY, SOM, GAL); and the density of DβH 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.

Long-Term Estradiol-17β Administration Changes the Population of Paracervical Ganglion Neurons Supplying the Ovary in Adult Gilts

The aim of this study was to determine the influence of estradiol-17β (E2) overdose on the number and distribution of ovarian parasympathetic neurons in the paracervical ganglion (PCG) in adult pigs. To identify the neurons innervating gonads 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 E2, while control gilts received oil. The PCG were then collected and processed for double-labeling immunofluorescence. Injections of E2 increased the E2 level in the peripheral blood approximately four- to fivefold 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 vesicular acetylcholine transporter (VAChT)+/somatostatin+, VAChT+/vasoactive intestinal polypeptide (VIP)+, VAChT+/neuronal isoform of nitric oxide synthase+, VAChT+/VIP−, VAChT+/dopamine β-hydroxylase (DβH)−, VAChT−/VIP−, and VAChT−/DβH− perikarya; and the total number of perikarya expressing estrogen receptors (ERs) subtype α and/or β. In summary, long-term E2 treatment of adult gilts downregulates the population of both cholinergic and ERs expressing the PCG ovary-projecting neurons. Our results suggest that elevated E2 levels occurring during pathological states may regulate gonadal function(s) by affecting ovary-supplying neurons.

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.

Morphological and neurochemical characterization of the ovarian sympathetic chain ganglia perikarya in testosterone-treated sexually matured pigs.

We studied the effect of testosterone overdose on the number, distribution and chemical coding of ovarian neurons in the sympathetic chain ganglia (SChGs) in pigs. On day 3 of the estrous cycle the ovaries of both the control and experimental gilts were injected with retrograde neuronal tracer Fast Blue to identify the neurons innervating gonads. From the following day to the expected day 20 of the second studied cycle the experimental pigs were injected with testosterone, while the control pigs received oil, and subsequently the SChG Th16-S2 were collected. Testosterone injections increased testosterone (∼3.5 fold) and estradiol-17β (∼1.6 fold) levels in the peripheral blood, and reduced the following in the SChGs: the total number of Fast Blue-positive neurons, the numbers of perikarya in the L3-L5 ganglia, the numbers of perikarya in the ventral, dorsal and central regions of the SChGs, and the numbers of DβH(+)/NPY(+), DβH(+)/GAL(+), DβH(+)/NPY(-), DβH(+)/SOM(-) and DβH(+)/GAL(-) perikarya. In the testosterone-affected SChGs, the perikarya DβH(-)/SOM(+), DβH(-)/GAL(+) and DβH(-)/NPY(-) were absent. In these ganglia, the population of androgen receptor-positive perikarya was increased, while the population of estrogen receptor-expressing perikarya was lowered. Our data indicate that in the pig SChGs elevated androgen levels occurring during pathological states may affect the morphology and chemical coding of ovarian neurons.

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-17β (∼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(+)/DβH(-), VAChT(-)/SOM(-), VAChT(-)/VIP(-), VAChT(-)/nNOS(-) and VAChT(-)/DβH(-) perikarya, In the T-affected PCG, the populations of ovarian perikarya coded VAChT(-)/SOM(+), VAChT(-)/VIP(+) and VAChT(-)/DβH(+), and expressing androgen receptor were increased. After T treatment within the PCG dropped the density of nerve fibers expressing VAChT and/or SOM, VIP, DβH. Obtained data suggest that elevated androgen levels occurring during pathological processes may regulate ovary function(s) by affecting the PCG gonad-supplying neurons.

Effect of lipopolysaccharide and cytokines on synthesis and secretion of leukotrienes from endometrial epithelial cells of pigs

In the present study, effects were studied of lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-4 and IL-10 on the mRNA and protein expression of 5-lipooxygenase (5-LO), leukotriene (LT)A4 hydrolase (LTAH) and LTC4 synthase (LTCS), and secretion of LTB4 and LTC4 from endometrial epithelial cells of pigs, as well as on viability of these cells. Cells were incubated for 24h with LPS (10 or 100ng/ml of medium), TNF-α, IL-1β, IL-4 or IL-10 (each cytokine: 1 or 10ng/ml of medium). Larger doses of TNF-α and IL-10 and both doses of IL-1β increased the relative abundance of mRNA/protein of 5-LO in the cells. A similar effect was exerted by the smaller dose of LPS on 5-LO mRNA content. Smaller doses of LPS and IL-4, and the larger dose of IL-10 increased the relative abundance of mRNA/protein LTAH, while both doses of TNF-α and the larger dose of IL-1β increased the protein content of this enzyme. Relative abundance of the mRNA/protein of LTCS was greater with the smaller dose of LPS, both doses of TNF-α and greater doses of IL-1β and IL-10, while relative abundance of LTCS mRNA was greater in response to the larger dose of LPS and both doses of IL-4. The LTB4 and LTC4 release was increased by the smaller dose of LPS, both doses of TNF-α and larger doses of IL-1β and IL-10. The IL-4 at the smaller dose exerted a stimulatory effect on LTB4 release. Larger doses of TNF-α and IL-4 enhanced cell viability. Interactions with LPS and cytokines revealed in this study may represent mechanisms important for the regulation of endometrium functions of pigs under physiological or pathological conditions.

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.