Barbara Jana

The concentration of GnRH in hypothalamus, LH and FSH in pituitary, LH, PRL and sex steroids in peripheral and ovarian venous plasma of hypo- and hyperthyroid, cysts-bearing gilts

The aim of this work was to investigate the hormonal pattern in hypo- and hyperthyroid gilts with experimentally induced cystic ovarian disease (COD). A total of 70 adult, nulliparous gilts divided into six groups were used for the experiment. Group I was euthyroid and control. Group II was made hypothyroid by oral administration of methylthiouracyl for 24 days. Group III represented euthyroid gilts injected with gonadotropins (PMSG and hCG). Group IV consisted of hypothyroid gilts injected with gonadotropins. Group V was treated with L-thyroxine for 24 days and Group VI with thyroxine and with gonadotropins for the last 10 days of the test. The treatment of all groups was terminated on the 4th-5th day of the next estrous cycle. The peripheral blood of the gilts was collected on Day 0, and on Day 24. On the 25th day the gilts were laparotomized and cannulas were inserted into utero-ovarian veins of each ovary for blood collection. Simultaneously, peripheral blood samples were collected during 1 to 3 consecutive days. The animals were then slaughtered and the hypothalamus, pituitary and ovaries were frozen and preserved for further analyses. In hypothalamic tissue the content of GnRH: in the pituitary the concentration of LH and FSH; and in peripheral and ovarian blood plasma the level of LH, PRL, E1, P4, A4, T and cortisol (Cl) were estimated by RIA procedure. The level of GnRH in the hypothalamus, and LH and FSH in the pituitary showed a tendency to parallel with thyroid function which may indicate a role of this gland in their production or secretion. In hypothyroid animals an increase of LH and PRL and a slight decrease of secretory function of the ovaries were noted. Injections of gonadotropins in euthyroid or hypo- and hyperthyroid gilts intensified the function of the ovaries, which was manifested by numerous follicular cysts and corpora lutea. The hormonal milieu of gilts from these groups showed a low level of LH, PRL and an increased content of sex steroids in peripheral and ovarian blood. The ovarian steroidogenesis of cyst-bearing gilts was disturbed, which was indicated by an increased level of E1, P4, A4, T, and Cl, but a low level of E2. These disturbances in steroidogenesis in cystic gilts may be caused by a deficiency in LH secretion as the consequence of the pituitary gonadotropin suppression by the used gonadotropins. The steroid hormone pattern of cyst-bearing gilts strongly resembles the endocrine profile noted in polycystic ovarian disease in women.

Long-term estradiol-17β administration reduces population of neurons in the sympathetic chain ganglia supplying the ovary in adult gilts

Elevated levels of endogenous estrogens occurring in the course of pathological states of ovaries (follicular cysts, tumors) as well as xenoestrogens may result in hyperestrogenism. In rat, a close relationship between estrogens and sympathetic and sensory neurons supplying the genito-urinary system was reported. Recently, we have shown that long-term estradiol-17β (E(2)) administration affected morphological and immunochemical organization of the sympathetic ovarian neurons in the caudal mesenteric ganglion of adult gilts. In this study, the influence of E(2) overdose on the number and distribution of neurons in the sympathetic chain ganglia (SChG) projecting to the ovary of adult pigs was investigated. The numbers of ovarian dopamine-β-hydroxylase (DβH-), neuropeptide Y (NPY-), somatostatin (SOM-), galanin (GAL-) and estrogen receptors (ERs-) immunoreactive perikarya as well as the density of the intraganglionic nerve fibers containing DβH and/or NPY, SOM, GAL were also determined. 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 day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with E(2), while the control gilts were receiving oil. After the last E(2)/oil injection, the SChG Th16-S2 were collected and processed for double-labeling immunofluorescence. Injections of E(2): (1) increased the E(2) level in the peripheral blood ~4-5 fold, (2) reduced the total number of Fast Blue-positive postganglionic neurons in the ganglia under investigation, (3) decreased the number of perikarya in the L2-L4 ganglia, (4) reduced the number of perikarya in the ventral, dorsal and central regions of the SChG, (5) decreased the numbers of DβH(+)/NPY(+) and DβH(+)/GAL(+) perikarya and the numbers of DβH(+) but NPY(-), SOM(-) and GAL(-) perikarya in the SChG, (6) decreased the number of perikarya expressing ERs subtype α and β, and (7) decreased the total number of the intraganglionic nerve fibers containing DβH and/or NPY. These results show that long-term E(2) treatment of adult gilts down-regulates the population of both noradrenergic and ERs expressing the SChG ovary supplying neurons. Our findings suggest also that elevated E(2) levels that occur during pathological states may regulate gonadal function(s) by affecting ovary supplying neurons.

The effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 on chorioamnion secretion of prostaglandins (PG)F2α and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.

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.

Long term estradiol-17β administration changes population of the dorsal root ganglia neurons innervating the ovary in the sexually mature gilts.

The influence of estradiol-17β (E₂) overdose on the number and distribution of neurons in the dorsal root ganglia (DRGs) supplying the ovary of adult pigs was investigated. The numbers of ovarian substance P (SP)-, calcitonin gene-related peptide (CGRP)-, galanin (GAL)-, pituitary adenylate cyclase-activating polypeptide (PACAP)-, neuronal isoform of nitric oxide synthase (nNOS)- and estrogen receptors (ERs)-immunoreactive perikarya were also determined. On day 3 of the estrous cycle, the ovaries of both the control and experimental gilts were injected with retrograde tracer Fast Blue. From day 4 of the estrous cycle to the expected day 20 of the second studied cycle, the experimental gilts were injected with E₂, while the control gilts received oil. The DRGs Th16-L5 were then collected and processed for double-labelling immunofluorescence. Injections of E₂ increased the E₂ level in the peripheral blood ∼4-5-fold and reduced the following in the DRGs: the total number of Fast Blue-positive perikarya, the number of large perikarya, the population of perikarya in the L2 and L3 ganglia, the numbers of SP- and/or CGRP-, PACAP-, nNOS-immunoreactive perikarya and the number of large perikarya expressing ERs subtype α and β. These results show that long-term E₂ treatment of adult gilts affects both the spatial and neurochemical organization pattern of ovary sensory innervation. Our findings suggest that elevated E₂ levels occurring during pathological states may regulate the transmission of sensory modalities from the ovary to the spinal cord.

Expression of steroidogenic enzymes in porcine polycystic ovaries.

In the present study the expression pattern of the cholesterol side-chain cleavage cytochrome (P450(scc)), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and aromatase (P450(arom)) was analyzed in the health and polycystic ovaries of gilts by means of the Western blot and immunohistochemistry. The polycystic status of ovaries was induced by i.m. dexamethasone (DXM) injections on days 7-21 of the estrous cycle. Macroscopic observation of ovaries of DXM-treated gilts revealed the presence of cysts (1-2 cm in diameter, with a mean number of 7.0+/-1.2 per ovary), a decrease (P<0.05) in number of small follicles (1-3 mm in diameter), as well as the lack of medium-sized follicles (4-6 mm in diameter) and corpora lutea, as compared to the control animals. The expression of P450(scc) (P<0.01), 3beta-HSD (P<0.05) and P450(arom) (P<0.001) proteins in the cysts was higher than in the medium-sized follicles of the control gilts. Moreover, DXM injections resulted also in an enhancement (P<0.05) in the level of P450(scc) protein in the walls of small follicles as compared to the control gilts. Following DXM administration the immunoreactivity (IR) of P450(scc) in the primordial follicles was lower than in the control group. Comparing to the control gilts, the reaction for this enzyme in DXM-treated animals was observed in secondary follicles, while for 3beta-HSD, in primordial, primary, as well as secondary follicles. The immunostaining for P450(scc) (theca cells) and P450(arom) (granulosa cells) in the small follicles of the DXM-treated gilts were more prominent than those found in the gonads of control animals. However, IR for P450(scc) was not found in the granulosa cells of small follicles in the gilts receiving DXM. The intensity of P450(scc) and P450(arom) labelling was distinctly enhanced in the cysts as compared to the medium follicles of the control animals. Furthermore, in contrary to the medium follicles of the control animals, faint IR for 3beta-HSD was found in the granulosa cell layer of cysts. Our data revealed that both the expression of P450(scc), 3beta-HSD and P450(arom) and localization of these enzymes in polycystic ovaries were different from those, found under physiological conditions. These results suggested that above-mentioned enzymes may, by influencing the ovarian steroid synthesis, play an essential role in the creation and/or course of cystic ovarian disease.

Localization of substance P, calcitonin gene related peptide and galanin in the nerve fibers of porcine cystic ovaries.

In a previous study, we showed that both the noradrenergic and cholinergic component of ovarian innervation is markedly changed in porcine cystic ovaries. The present study is aimed at elucidating the distribution pattern of substance P- (SP), calcitonin gene related peptide CGRP- and/or galanin (GAL)-containing nerve fibers within porcine cystic ovaries. The status polycysticus was induced by dexamethasone phosphate disodium salt i.m. injections performed from the 7(th) until the 21(st) day of the first studied estrous cycle. During the same period of time, gilts of the control group received saline. All animals were slaughtered on the expected 11(th) day of the second studied estrous cycle, and their ovaries were collected. When compared to control gonad, a distinct difference in the distribution pattern and the density of SP-, CGRP- and/or GAL-immunoreactive (GAL-IR) nerve fibers was observed. Thus, unlike in the control gonad, SP- and/or CGRP-IR perivascular nerve fibers were found to supply medullar blood vessels of polycystic ovary. Furthermore, the number of GAL-IR nerve fibers contributing to the ground plexus in polycystic ovaries was higher than that observed in the control gonads. Thus, as may be judged from the profound changes in the distribution pattern of differently chemically coded afferent terminals within polycystic gonads, it appears possible that neuropeptides released from these terminals may take part in the etiopathogenesis of this disorder.

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.

Exogenous long-term treatment with 17β-oestradiol alters the innervation pattern in pig ovary.

The aim of the present study was to determine the effect of long-term 17β-oestradiol (E2) exposure, a simulation of pathological states that occur with oestrogen overproduction, on the innervation patterns of ovaries in adult gilts. The intraovarian distribution and density of nerve fibres immunoreactive (IR) to protein gene product (PGP) 9.5 and containing dopamine-β-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM) and galanin (GAL) were determined. From Day 4 of the first oestrous cycle to Day 20 of the second cycle studied, experimental gilts were injected with E2 (1000μg every 12h) whereas control gilts were injected with corn oil. After E2 administration, there was an increase in the number of PGP9.5-, DBH-, NPY- and GAL-IR fibres. Numerous PGP9.5-IR terminals were observed within the ground plexus around secondary follicles and small or medium tertiary follicles. Long-term E2 treatment increased the density of DBH- and NPY-IR fibres in the cortical part of the ground plexus, DBH- and GAL-IR fibres in the medullary part of the ground plexus, DBH-IR fibres near small and medium tertiary follicles and NPY-IR fibres around medullary arteries. The data indicate that long-term exposure of gilts to E2 increases the total number of intraovarian fibres, including sympathetic fibres. These results suggest that elevated E2 levels that occur during pathological states may affect the innervation patterns of ovaries and their function(s).