Marta J. Siemieniuch

Actions of a nitric oxide donor on prostaglandin production and angiogenic activity in the equine endometrium

Nitric oxide (NO) plays an important role in prostaglandin secretion and angiogenesis in the reproductive system. In the present study, the roles of the NO donor spermine NONOate and tumour necrosis factor-alpha (TNF; as a positive control) in prostaglandin production and angiogenic activity of equine endometria during the oestrous cycle were evaluated. In addition, the correlation between NO production and the expression of key prostaglandin synthase proteins was determined. The protein expression of prostaglandin F synthase (PGFS) increased in early and mid-luteal stages, whereas that of prostaglandin E synthase (PGES) was increased in the early luteal stage. The in vitro release of NO was highest after ovulation. There was a high correlation between NO production and PGES expression, as well as NO release and PGFS expression. There were no differences detected in prostaglandin H synthase 2 (PTGS-2) throughout the oestrous cycle and there was no correlation between PTGS-2 expression and NO. In TNF- or spermine-treated endometria, the expression of prostaglandin (PG) E(2) increased in the early and mid-luteal phases, whereas that of PGF(2alpha) increased in the follicular and late luteal phases. Bovine aortic endothelial cell (BAEC) proliferation was stimulated in TNF-treated follicular-phase endometria. However, in spermine-treated endometria, NO delivered from its donor had no effect, or even an inhibitory effect, on BAEC proliferation. In conclusion, despite no change in PTGS-2 expression throughout the oestrous cycle in equine endometrial tissue, there were changes observed in the expression of PGES and PGFS, as well as in the production of PGE(2) and PGF(2alpha). In the mare, NO is involved in the secretory function of the endometrium, modulating PGE(2) and PGF(2alpha) production. Even though TNF caused an increase in the production of angiogenic factors and prostaglandins, its complex action in mare uterus should be elucidated.

Effects of Tumor Necrosis Factor-α and Nitric Oxide on Prostaglandins Secretion by the Bovine Oviduct Differ in the Isthmus and Ampulla and Depend on the Phase of the Estrous Cycle:

To determine the possible roles of tumor necrosis factor-α (TNFα) and nitric oxide (NO) in the bovine oviduct, ampulla and isthmus collected during the estrous cycle were exposed for 18 h to TNFα, NO donor (NONOate), NO synthase inhibitors (L-NOARG, L-NAME and AMT) and oxytocin (OT) as a positive control. Prostaglandins (PGs) and NO2/NO3 in conditioned media were measured. TNFα stimulated PGF2α secretion on Day 0 (onset of estrus = Day 0) and Days 2–3, in both the ampulla and isthmus, but on Days 18–20 only in ampulla. TNFα increased PGE2 secretion in both fragments in each phase. NONOate did not affect PGF2α secretion on Days 18–20, whereas this NO donor stimulated PGF2α secretion in both fragments on Day 0 and Days 2–3. TNFα increased NO2/NO3 production in every examined phase in the ampulla and on Days 2–3 in the isthmus. L-NAME lowered NO2/NO3 production regardless of phase or fragment. L-NOARG and AMT lowered NO2/NO3 production in both fragments on Day 0 and Days 2–3. The possible role of TNFα, NO or PGs on the oviductal contractility during the early-luteal phase was also examined. Neither TNFα nor NONOate influenced contractility in either fragment. Although PGF2α stimulated the contraction in both fragments, PGE2 decreased it. When taken together, TNFα seems to play some role as a modulator of PGF2α and PGE2 production and for transferring the embryo from the oviduct to the uterus by stimulating NO production in the bovine oviduct.

Steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy in domestic cats

BackgroundUntil recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy.MethodsThe mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA.ResultsLuteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age.ConclusionsThese results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.

Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells

The aims of this study were to determine the effects of lipopolysaccharides (LPS), tumor necrosis factor (TNF), interleukin 1 alpha (IL-1α), nitric oxide donor (NONOate), or the combination of TNF + IL-1α + NONOate on the following: (i) secretion of prostaglandin (PG)-F(2α), PGE(2), leukotriene (LT)-B(4), and LTC(4) by epithelial cells of the teat cavity and lactiferous sinus of bovine mammary gland; (ii) messenger RNA (mRNA) transcription of enzymes responsible for arachidonic acid (AA) metabolism (prostaglandin-endoperoxide synthase 2 [PTGS2], prostaglandin E synthase [PTGES], prostaglandin F synthase [PGFS], and arachidonate 5-lipooxygenase [ALOX5]); and (iii) proliferation of the cells. The cells were stimulated for 24 h. Prostaglandins and LT were measured by enzyme immunoassay, mRNA transcription of enzymes was determined by real-time reverse transcription polymerase chain reaction, and the cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. All factors increased PG secretion, but the highest stimulation was observed after TNF and IL-1α (P < 0.001). Tumor necrosis factor, NONOate, and TNF + IL-1α + NONOate increased LTB(4) production (P < 0.01), whereas LTC(4) was increased by LPS, TNF, and IL-1α (P < 0.01). Lipopolysaccharides, TNF, IL-1α, and the reagents combination increased PTGS2, PTGES, and PGFS mRNA transcription (P < 0.01), whereas ALOX5 mRNA transcription was increased only by TNF (P < 0.001). Lipopolysaccharides, TNF, IL-1α, NONOate, and the combination of reagents increased the cell number (P < 0.001). Mediators of acute-clinical Escherichia coli mastitis locally modulate PG and LT secretion by the epithelial cells of the teat cavity and lactiferous sinus, which might be a useful first line of defense for the bovine mammary gland. Moreover, the modulation of PG and LT secretion and the changing ratio of luteotropic (PGE(2), LTB(4)) to luteolytic (PGF(2α), LTC(4)) metabolites may contribute to disorders in reproductive functions.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells.

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO₂/NO₃) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17β-estradiol (E₂; 10⁻⁹ M) and/or progesterone (P₄; 10⁻⁷ M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻⁵ M). Prostaglandin F(2α) and PGE₂ secretion was measured in medium by ELISA. The pretreatment of cells with P₄ (progesterone), E₂ (17 β-estradiol), or E₂/P₄ augmented TNF-α-induced PGF(2α) and PGE₂ secretion (P < 0.01). The pretreatment of cells with E₂ or E₂/P₄ increased NONOate-induced PGF(2α) and PGE₂ secretion (P < 0.01). TNF-α induced NO₂/NO₃ production by BOECs. The pretreatment of cells with E₂ augmented only TNF-α-induced NO₂/NO₃ production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻⁵ M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

mRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F(2α) synthase, PGFS; and prostaglandin E(2) synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenneys Category II) and severe phases of fibrosis (Kenneys Category III; endometrosis), compared with healthy endometrium (Kenneys Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE(2) and PGF(2α) in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF(2α) concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E(2) concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.

Are glucocorticoids auto– and/or paracrine factors in early bovine embryo development and implantation?

We determined the transcript content of three genes involved in the metabolism of glucocorticoids (GC) in bovine in vitro fertilized embryos (2-blastomere stage until hatched blastocyst), trophoblast as well as the oviduct (Day 2-4 of the estrous cycle) and endometrium (Day 16 of the cycle and pregnancy). Since mRNA expression of the glucocorticoid receptor and two enzymes responsible for GC production (11β-HSD1 and 2) was demonstrated in the embryos in all pre-implantation stages as well as in the endometrium and oviduct, it is suggested that GC may serve as auto-/paracrine factors in the development of bovine pre-implantation embryos.

Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical endometritis in mares

BackgroundEndometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice.MethodsA total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis.ResultsPositive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively.ConclusionMicrobiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare’s uterus in clinical practice.

Ovarian steroids regulate prostaglandin secretion in the feline endometrium.

Sex steroids, i.e. progesterone (P(4)) and 17beta-estradiol (E(2)), fluctuate during the feline estrous cycle and their alterations correspond to many events in cat reproduction. In order to investigate possible effects of sex steroids on prostaglandin (PG) secretion in the cultured endometrial cells, mRNA expressions coding for PG-endoperoxide synthase (PTGS2) in the epithelial and stromal cells harvested with sex steroids were studied by RT-PCR. The effects of ovarian steroids on PG secretion in the epithelial and stromal cells were also investigated. E(2) at a dose 10(-7)M significantly increased prostaglandin F2alpha (PGF(2alpha)) secretion in the epithelial cells (P<0.01). PGF(2alpha) production was intensified by the treatment in combination with both steroids (P<0.001). P(4) at any dose alone had no effect on PGF(2alpha) secretion in the epithelial cells, whereas at a dose 10(-5)M enhanced prostaglandin E2 (PGE(2)) production (P<0.05). The ovarian steroids stimulated both PGF(2alpha) and PGE(2) in the epithelial cells of the feline endometrium via an E(2) receptor (ESR1)- and P(4) receptor (PGR)-dependent genomic-pathway. In contrast to the epithelial cells, neither P(4) nor E(2) affected PG secretion in the stromal cells. PTGS2 mRNA expression was not affected by ovarian steroids in either cell types. The overall results suggest that PG secretion is regulated by P(4) and E(2) and this effect is not due to changes in PTGS2 mRNA expression.

Prostaglandin endoperoxide synthase 2 (PTGS2) and prostaglandins F2α and E2 synthases (PGFS and PGES) expression and prostaglandin F2α and E2 secretion following oestrogen and/or progesterone stimulation of the feline endometrium.

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF(2α) synthase (PGFS) and PGE(2) synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E(2) and/or P(4) on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E(2) and/or P(4) treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E(2) plus P(4) at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P(4) at late pseudopregnancy (p < 0.05). Simultaneous incubation with E(2) and P(4) up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E(2) significantly increased PGE(2) secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E(2) and/or P(4) affected neither PGF(2α) secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE(2) secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E(2) and P(4) at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE(2) are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.