Effect of proinflammatory cytokines on endometrial collagen and metallopeptidase expression during the course of equine endometrosis.

Equine endometrosis (endometrial fibrosis) is a degenerative chronic process that occurs in the uterus of the mare and disturbs proper endometrial function. Fibrosis is attributed to excessive deposition of extracellular matrix (ECM) components. The turnover of ECM is mediated by matrix metallopeptidases (MMP). Previously, it was shown that cytokines modulate MMP expression in other tissues and may regulate fibrosis indirectly by attracting inflammatory cells to the site of inflammation and directly on various tissues. However, the regulation of MMP expression in equine endometrosis is still relatively unknown. Thus, our aim was to determine if interleukin (IL)-1ß and IL-6 regulate ECM, MMPs, or their inhibitors (TIMPs) and whether this regulation differs during endometrosis in the mare. Endometrial fibrosis was divided into four categories according to severity: I (no degenerative changes), IIA (mild degenerative changes), IIB (moderate degenerative changes) and III (severe degenerative changes) according to Kenney and Doig classification. Endometrial explants (n = 5 for category I, IIA, IIB and III according to Kenney and Doig) were incubated with IL-1ß (10 ng/ml) or IL-6 (10 ng/ml) for 24 h. Secretion and mRNA transcription of collagen type 1 (Col1a1) and type 3 (Col3a1), fibronectin (Fn1), Mmp-1, -2, -3, -9, -13, Timp-1, -2 were analyzed by real-time PCR and ELISA, respectively. IL-1ß treatment up-regulated secretion of COL1, MMP-2, TIMP1, and TIMP2 in category I endometrial fibrosis tissues (P < 0.05). IL-6 treatment up-regulated secretion of ECM, MMP-2, and MMP-3 and down-regulated secretion of MMP-9 in category I tissues (P < 0.05). In category IIA tissues, IL-1ß and IL-6 treatment up-regulated secretion of COL3 (P < 0.05; P < 0.05), and IL-6 treatment also down-regulated secretion of MMP-9 (P < 0.05). In category IIB tissues, IL-1ß treatment down-regulated secretion of COL3 (P < 0.05) and up-regulated secretion of MMP-3 (P < 0.01), while IL-6 treatment up-regulated secretion of MMP-3, MMP-9, and MMP-13 (P < 0.05). In category III tissues, IL-1ß treatment up-regulated secretion of COL1, MMP-1, MMP-9 and TIMP-2 (P < 0.05), and IL-6 up-regulated secretion of all investigated ECM components, MMPs and TIMPs. These results reveal that the effect of IL-1ß and IL-6 on equine endometrium differs depending on the severity of endometrial fibrosis. Our findings indicate an association between inflammation and development of endometrosis through the effect of IL-1ß and IL-6 on expression of ECM components, MMPs, and TIMPs in the mare.

Effects of prostaglandin F2α on angiogenic and steroidogenic pathways in the bovine corpus luteum may depend on its route of administration.

Prostaglandin (PG) F2α and its analogs (aPGF2α) are used to induce regression of the corpus luteum (CL); their administration during the middle stage of the estrous cycle causes luteolysis in cattle. However, the bovine CL is resistant to the luteolytic actions of aPGF2α in the early stage of the estrous cycle. The mechanisms underlying this differential luteal sensitivity, as well as acquisition of luteolytic sensitivity by the CL, are still not fully understood. Therefore, to characterize possible differences in response to aPGF2α administration, we aimed to determine changes in expression of genes related to (1) angiogenesis-fibroblast growth factor 2 (FGF2), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2); and (2) steroidogenesis-steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (P450scc), and hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 1 (HSD3B) in early- and middle-stage CL that accompany local (intra-CL) versus systemic (i.m.) aPGF2α injection. Cows at d 4 (early stage) or d 10 (middle stage) of the estrous cycle were treated as follows: (1) systemic saline injection, (2) systemic aPGF2α injection (25 mg), (3) local saline injection, and (4) local aPGF2α injection (2.5 mg). Progesterone (P4) concentration was measured in jugular vein blood samples during the entire set of experiments. After 4 h of treatment, CL were collected by ovariectomy, and mRNA and protein expression levels were determined by reverse transcription quantitative-PCR and Western blotting, respectively. Local and systemic aPGF2α injections upregulated FGF2 expression but decreased expression of VEGFA in both CL stages. Both aPGF2α injections increased the expression of STAR in early-stage CL, but downregulated it in middle-stage CL. In the early-stage CL, local administration of aPGF2α upregulated HSD3B, whereas systemic injection decreased its mRNA expression in early- and middle-stage CL. Moreover, we observed a decrease in the P4 level earlier after local aPGF2α injection than after systemic administration. These results indicate that aPGF2α acting locally may play a luteotrophic role in early-stage CL. The systemic effect of aPGF2α on the mRNA expression of genes participating in steroidogenesis seems to be more substantial than its local effect in middle-stage CL.

Bisphenol A alters ß-hCG and MIF release by human placenta: an in vitro study to understand the role of endometrial cells.

A proper fetomaternal immune-endocrine cross-talk in pregnancy is fundamental for reproductive success. This might be unbalanced by exposure to environmental chemicals, such as bisphenol A (BPA). As fetoplacental contamination with BPA originates from the maternal compartment, this study investigated the role of the endometrium in BPA effects on the placenta. To this end, in vitro decidualized stromal cells were exposed to BPA 1 nM, and their conditioned medium (diluted 1 : 2) was used on chorionic villous explants from human placenta. Parallel cultures of placental explants were directly exposed to 0.5 nM BPA while, control cultures were exposed to the vehicle (EtOH 0.1%). After 24-48 h, culture medium from BPA-treated and control cultures was assayed for concentration of hormone human Chorionic Gonadotropin ( ß -hCG) and cytokine Macrophage Migration Inhibitory Factor (MIF). The results showed that direct exposure to BPA stimulated the release of both MIF and ß -hCG. These effects were abolished/diminished in placental cultures exposed to endometrial cell-conditioned medium. GM-MS analysis revealed that endometrial cells retain BPA, thus reducing the availability of this chemical for the placenta. The data obtained highlight the importance of in vitro models including the maternal component in reproducing the effects of environmental chemicals on human fetus/placenta.

Physiopathologic mechanisms involved in mare endometrosis.

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFß and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1ß, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Is adenomyosis a problem in reproduction and fertility?

Adenomyosis is defined as the presence of glandular foci beside the endometrium of uterus: in the myometrium and/or perimetrium depending on the progress of the disorder. So far, adenomyosis has been diagnosed in women and rodents, and studies conducted on cows have been rare. In this review we: (1) summarize the knowledge regarding adenomyosis, (2) compare the symptoms and aetiopathology between women and cows, (3) describe angiogenic uterine processes related to adenomyosis development and (4) outline the influence of adenomyosis on proper fertility processes in cattle (conception and fertility rates).

Effect of cytokines and ovarian steroids on equine endometrial function: an in vitro study.

Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IFNG mRNA increased in late LP. Transcription of the mRNA of both TNF receptors was highest in the mid-LP. All cytokines and receptors were expressed in surface and glandular epithelium, as well as in the stroma. Expression of TNF and its receptor TNFRSF1A increased during the follicular phase (FP) and mid-LP. IFNG was expressed in the mid-LP, whereas its receptor IFNR1 was expressed in the in mid- and late LP. The highest expression of FASLG and FAS occurred during the late LP. OXT increased the secretion of prostaglandin (PG) E2 and PGF2α in the FP and mid-LP. In the mid-LP, E2 and P4+E2 stimulated PGF2α secretion, whereas TNF and P4 increased cell viability. All treatments, with the exception of P4, increased nitric oxide and angiogenic activity in both phases. The coordinated action of cytokines and ovarian hormones may regulate secretory, angiogenic and proliferative functions in the equine endometrium.

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.

Growth and regression in bovine corpora lutea: regulation by local survival and death pathways.

The bovine corpus luteum (CL) is a transient gland with a life span of only 18 days in the cyclic cow. Mechanisms controlling CL development and secretory function may involve factors produced both within and outside this gland. Although luteinizing hormone (LH) surge is the main trigger of ovulation and granulosa cells luteinization, many locally produced agents such as arachidonic acid (AA) metabolites, growth factors and cytokines were shown to complement gonadotropins action in the process of CL development. Bovine CL is a highly vascular gland, where the very rapid angiogenesis rate (until Day 5 of the cycle) results in the development of a capillary network, endowing this gland with one of the highest blood flow rate per unit mass in the body. Angiogenesis in the developing CL is later followed by either controlled regression of the microvascular tree in the non-fertile cycle or maintenance and stabilization of the blood vessels, as seen during pregnancy. Different luteal cell types (both steroidogenic and accessory luteal cells: immune cells, endothelial cells, pericytes and fibroblasts) are involved in the pro- and/or anti-angiogenic responses. The balance between pro- and anti-angiogenic responses to the main luteolysin - prostaglandin F2α (PGF2α) could be decisive in whether or not PGF2α induces CL regression. Fibroblast growth factor-2 (FGF2) may be one of the factors that modulate the angiogenic response to PGF2α. Manipulation of local production and action of FGF2 will provide new tools for reproductive management of dairy cattle. Luteolysis is characterized by a rapid decrease in progesterone production, followed by structural regression. Factors like endothelin-1, cytokines (tumour necrosis factorα, interferons) and nitric oxide were all shown to play critical roles in functional and structural regression of the CL by inhibiting steroidogenesis and inducting apoptosis.

Transcriptomic profiling of mare endometrium at different stages of endometrosis.

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-ß1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-ß1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

Acute changes in the concentrations of prostaglandin F2α (PGF) and cortisol in uterine and ovarian venous blood during PGF-induced luteolysis in cows.

Prostaglandin F2α (PGF) is considered to be the main luteolysin in cattle. We have previously demonstrated that cortisol (Cr) suppresses PGF production in non-pregnant bovine endometrium. This study was carried out to test whether exogenous PGF increases ovarian and/or uterine PGF production and to determine the temporal relationship between PGF and Cr in ovarian and uterine circulations during PGF-induced luteolysis in cows. Catheters were inserted into the ovarian vein (OV), uterine vein (UV) and jugular vein (JV) of 10 cows on Day 9 of the oestrous cycle (Ovulation = Day 0) for frequent blood collection. On Day 10, the cows were divided randomly into two groups and treated with a luteolytic dose of a PGF analogue (cloprostenol) or saline solution. Blood samples were collected at -0.25, 0, 0.25, 0.5, 1 and 2 h and then at 2-h intervals until 12 h after treatment (0 h). The basal concentrations of PGF and Cr in OV and UV plasma were not significantly different. Injection of a PGF analogue induced more than twofold increases in the levels of PGF between 0.25 and 1 h in UV plasma, but not in OV plasma. PGF increased (p < 0.05) the concentrations of Cr in OV, UV and JV plasma between 0.5 and 1 h. The Cr levels in OV, UV and JV plasma were similar. The PGF levels in UV plasma decreased after Cr reached its highest levels. The overall results suggest that the uterus rather than the ovary increases PGF production in response to PGF injection. Based on the temporal changes of PGF and Cr in the ovarian and uterine circulations, Cr may act to reduce uterine PGF production in non-pregnant cows in vivo.

Conversion of cortisone to cortisol and prostaglandin F2α production by the reproductive tract of cows at the late luteal stage in vivo.

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine-stimulated prostaglandin F(2α) (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at -2, -1, -0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone-treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non-pregnant cows.

The effect of basic fibroblast growth factor 2 on the bovine corpus luteum depends on the stage of the estrous cycle and modulates prostaglandin F2α action.

The roles of fibroblast growth factor 2 (FGF2) in the corpus luteum (CL) function and its modulatory effect on prostaglandin (PG) F2α during the bovine estrous cycle were studied using the following design of in vivo and in vitro experiments: (1) effects of FGF2 and FGF receptor 1 inhibitor (PD173074) on bovine CL function in the early (PGF2α-resistant) and mid (PGF2α-responsive) luteal stage in vivo, (2) the modulatory effect of FGF2 on PGF2α action during the luteal phase in vivo and (3) effects of FGF2 and PD173074 on bovine CL secretory function in vitro. Cows were treated by injection into the CL with: (1) saline (control), (2) FGF2, (3) PD173074, (4) FGF2 followed by intramuscular (i.m.) PGF2α, (5) PD173074 followed by i.m. PGF2α and (6) i.m. PGF2α as a positive control. For in vitro experiments, CL explants were treated with the aforementioned factors. Progesterone (P4) concentrations of blood samples or culture media were determined by radioimmunoassay. Relative mRNA expressions of the genes involved in angiogenesis and steroidogenesis were determined by quantitative real-time PCR. Although FGF2 treatment on day 4 of the estrous cycle did not change the cycle length, FGF2 with PGF2α decreased the P4 concentrations observed during the estrous cycle compared to the control group (P < 0.001). Moreover, FGF2 treatment on day 10 prolonged CL function as indicated by a significantly greater concentration of P4 on day 21 compared to the control group. In the in vitro study, FGF2 decreased cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase (HSD3B1) mRNA expression (P < 0.01) and decreased P4 production in the early-stage CL (P < 0.001). However, FGF2 + PGF2α or PGF2α alone resulted in an elevation of steroidogenic acute regulatory protein and CYP11A1 mRNA expression and P4 secretion in the early-stage CL (P < 0.01). In the mid-luteal phase, FGF2 upregulated CYP11A1 and HSD3B1 mRNA expression (P < 0.01), while FGF2 + PGF2α increased only HSD3B1 mRNA expression (P < 0.001). In conclusion, FGF2 seems to play a modulatory role in CL development or luteolysis, differentially regulating steroidogenesis and angiogenic factors as well as PGF2α actions.

Leukotrienes are auto-/paracrine factors in the bovine corpus luteum: an in vitro study.

The aim of this study was to determine leukotrienes (LTs) functions in the bovine corpus luteum (BCL) during the oestrous cycle. In steroidogenic CL cells we examined the effect of luteotropic [LH, prostaglandin E(2) (PGE(2))] and luteolytic (PGF(2α), cytokines) factors on: the levels of LTB(4) and C(4), the expression of 5-lipoxygenase (LO), LT receptors type I (LTR-I) and LTR-II, and the effects of LTB(4) and C(4) stimulations on the levels of progesterone (P4), PGE(2), F(2α) and nitric oxide (NO) metabolites. Both luteolytic and luteotropic factors stimulated 5-LO expression on days 2-4 and 17-19 of the cycle. Leukotriene receptors type I expression increased after PGE(2) and tumour necrosis factor α with interferon γ (TNF/IFN) stimulation on days 2-4 of the cycle. Leukotriene receptor type II expression increased after PGE(2α) and TNF/IFN stimulation on days 2-4 and 17-19 of the cycle, and LTR-II expression on days 8-10 of the cycle was unchanged after cell stimulation with any factor. Leukotriene B(4) level increased after BSC incubation with luteotropic factors during all examined days of the cycle and after cytokine stimulation at early- and mid-luteal stages, whereas luteolytic factors stimulated LTC(4) secretion over the entire cycle. Leukotriene B(4) stimulated P4 secretion at the mid-luteal stage and stimulated NO secretion during all examined phases. Leukotriene B(4) stimulated PGE(2) secretion at the early- and mid-luteal stage. Leukotriene C(4) inhibited P4 secretion at the mid- and regressing-luteal stage, stimulated NO (entire cycle) and PGF(2α) at mid- and regressing-luteal phases. Leukotrienes modulate steroidogenic cells functions, depending on the stage of the cycle. Leukotriene B(4) plays a luteotropic role stimulating P4 and PGE(2) secretions; LTC(4) stimulates the secretion of luteolytic factors and enhances the luteolytic cascade within BCL.

Leukotrienes affect secretory function of ovarian cells in vitro.

The aim of this study was to determine which cells are the source of production and target for leukotriene (LTs) action within the bovine ovary. Luteal (CL, days 14-16 of the oestrous cycle), steroidogenic cells (LSC) and endothelial cells (LEC) of the bovine corpus luteum (CL), and granulosa cells (GC) were isolated enzymatically, cultured in a monolayer and incubated with LTC(4), LTB(4), Azelastine (an antagonist of LTC(4)) or Dapsone (an antagonist of LTB(4)). Then cells were collected for determination of mRNA expression for LT receptors (LTRs) and 5-lipoxygenase (5-LO) by real time RT-PCR, and media were collected for determination of prostaglandin (PG)E(2), F(2α), progesterone (P4; LSC only), endothelin-1 (ET-1; LEC only) and 17-ß oestradiol (E2; GC only). The greatest mRNA expression for LTR-II and 5-LO were found in LEC, whereas LTR-I mRNA expression did not differ among cell types. The level of PGE(2) increased after LTs treatment in each type of ovarian cell, excluding LTC(4) treatment in LEC. The secretion of PGF(2α) was also increased by LTs, but decreased after LTB(4) treatment of LSC. In GC cultures, both LTs stimulated E2 secretion; in LEC cultures, LTB(4) stimulated whereas LTC(4) inhibited P4 secretion; in LEC cultures, LTC(4) stimulated but LTB(4) inhibited ET-1 secretion. The results show that LTs are produced locally and are involved in PGs production/secretion in all examined cells (LSC, LEC and GC) of bovine ovary. Leukotriene treatment modulate secretion of E2, by GC, P4 by LSC and ET-1 by LEC, which indicates that LTs are involved in regulation of ovarian secretory functions.

Tumor necrosis factor-alpha as a possible auto-/paracrine factor affecting estrous cycle in the cat uterus.

Tumor Necrosis Factor-alpha (TNFalpha) is a pleiotrophic cytokine, affects either normal or tumor cells, and influences cellular differentiation. TNFalpha role in female reproduction has been proven to be mediated through an influence on prostaglandin (PGs) synthesis and output. To evaluate the possible role of TNFalpha in an auto-/paracrine regulation in the cat uterus, mRNA expression coding for TNFalpha and its receptors (TNFR1 and TNFR2), and TNFalpha protein content at different stages of the estrous cycle were investigated. Additionally, TNFalpha involvement in PG secretion at different stages of the estrous cycle was investigated by in vitro tissue culture. Gene expressions coding for TNFalpha and TNFR1 were the highest at diestrus (P < 0.05). TNFalpha protein expression was the lowest at interestrus (P < 0.05). Nevertheless, TNFR2 was not affected by the estrous stage. TNFalpha at a dose of 1 ng/ml significantly increased PGF2alpha secretion at estrus (P < 0.01) and PGE2 secretion at diestrus (P < 0.001) after 12h incubation. Overall findings indicate that TNFalpha locally produced in the cat's uterus, stimulates PG secretion in an estrous cycle-related manner.

Luteinizing hormone and ovarian steroids affect in vitro prostaglandin production in the equine myometrium and endometrium.

Prostaglandins (PGs) play crucial roles in the regulation of the oestrus cycle and establishment of pregnancy in animals. Luteinizing hormone (LH) and ovarian steroids are involved in regulating endometrial PG production in many species. Their effects on PG production and associated pathways in the mare myometrium and endometrium are the subjects of our interest. This study aimed to evaluate the specific effects of LH and ovarian steroids on equine myometrial and endometrial tissues on (i) PGE2 and PGF2α secretion and (ii) transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin-endoperoxide synthase (PTGS2), PGE2 synthases (PGES), PGF2α synthases (PGFS), and PGI2 synthases (PGIS), using equine myometrial and endometrial explants. Equine myometrial and endometrial tissues were collected at the mid-luteal (n = 6) and follicular (n = 6) phases of the oestrus cycle and were exposed to: (1) vehicle (control), (2) arachidonic acid (AA, 50 ng/mL, positive control), (3) LH (10 ng/mL), (4) progesterone (P4, 10-7M) and (5) 17-ß oestradiol (E2, 10-9M) for 24 h. After exposure, PGF2α and PGE2 concentrations were determined using direct enzyme immunoassays. Alterations in PG synthase mRNA expression were determined using RT-qPCR. After 24 h, LH and P4 increased PGE2 and PGF2α secretion by myometrial tissues at the mid-luteal phase (P < 0.05), whereas PG secretion was augmented by LH and E2 during the follicular phase (P < 0.01). In contrast, LH and E2 increased PGE2 and PGF2α secretion by endometrial tissues during the mid-luteal phase (P < 0.05), while E2 enhanced PGE2 secretion during the follicular phase of the oestrus cycle (P < 0.01). These results indicate that LH and ovarian steroids modulate PG production in equine myometrial and endometrial tissues and affect PG synthase expression at the mRNA level. We conclude that the equine myometrium is an alternative source of PG production and participates in the regulation of uterus function during the oestrus cycle.

Effects of exogenous tumour necrosis factor-alpha on the secretory function of the bovine reproductive tract depend on tumour necrosis factor-alpha concentrations.

The aim of study was to correlate tumour necrosis factor-alpha (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F(2alpha), PGE(2) nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic--1 microg or luteotrophic--10 microg). In the peripheral blood, 1 microg TNF concentrations achieved within the range of 30-45 pg/ml, and 10 microg TNF provoked a sharp increase in achieved concentrations at a range of 250-450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF(2alpha) and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE(2) (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis, metritis).

Influence of nonsteroidal anti-inflammatory drugs on progesterone production by cultured bovine luteal cells.

The objective of the present study was to determine the influence of nonsteroidal anti-inflammatory drugs (NSAIDs) representing different chemical groups on progesterone (P4) production by cultured bovine steroidogenic luteal cells. The cells were enzymatically isolated from corpora lutea collected on days 8-12 of the estrous cycle. After 24 h preincubation they were incubated for 24 h with medium only (control) or stimulated with bovine luteinizing hormone - LH (100 ng/ml; positive control) or increasing concentrations (10(-8) to 10(-4) M) of acetylsalicylic acid, indomethacin, ibuprofen, naproxen, piroxicam, phenylbutazone, dipyrone or nimesulide. Concentartions of P4 in the culture media were determined by enzyme immunoassay. LH significantly increased P4 secretion, while acetylsalicylic acid and indomethacin did not affect the production of this hormone. A significant increase in P4 secretion was observed after administration of dipyrone at all concentrations, piroxicam at concentrations of 10(-8), 10(-7) and 10(-5) M, phenylbutazone and naproxen at concentrations of 10(-7) and 10(-6) M and ibuprofen at concentrations of 10(-5) and 10(-4) M. Nimesulide did not affect P4 production at concentrations of 10(-8) - 10(-5) M, while at a concentration of 10(-4) M it inhibited P4 secretion. The results obtained indicate that NSAIDs may change the production of P4 in bovine luteal cells, however, these changes are dependent on the substance used.

Effect of transforming growth factor -ß1 on α-smooth muscle actin and collagen expression in equine endometrial fibroblasts.

Transforming growth factor (TGF)-ß1 not only regulates cell growth, development, and tissue remodeling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the concentration of TGF-ß1 is correlated with endometrosis (equine endometrial fibrosis). In other tissues, TGF-ß1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and an ability to deposit excessive amounts of extracellular matrix (ECM) components. The aim of the study was to determine whether TGF-ß1 plays a role in the development of equine endometrosis. In Exp. 1, endometrial expression of α-SMA in different stages of endometrosis was determined. In endometrial tissues from the mid luteal phase (n = 6 for each stages of endometrosis) and the follicular phase of the estrous cycle (n = 5 for each stages of endometrosis), mRNA transcription and protein expression of α-Sma were evaluated by Real-time PCR and Western-blot, respectively. The α-Sma mRNA transcription and protein expression levels were correlated with the severity of endometrosis (P < 0.05). In both phases of the estrous cycle, α-SMA protein expression was up-regulated in final stage of endometrosis compared to initial stage (P < 0.05). In Exp. 2, the dose- and time-dependent effects of TGF-ß1 on expression of α-SMA and ECM components were determined, as well as cell proliferation of equine fibroblasts. Equine endometrial fibroblasts (n = 6, Kenney and Doig category I) were stimulated with vehicle or TGF-ß1 (1, 5, 10 ng/ml) for 24, 48 or 72 h. Then, mRNA transcription of α-Sma, collagen type I (Col1a1), collagen type III (Col3a1) and fibronectin 1 (Fn1) were determined by Real-time PCR. The production of ECM components was determined by ELISA. Transforming growth factor-ß1 increased the mRNA transcription of α-Sma and ECM components in a dose- and time-dependent manner in cultured endometrial fibroblasts (P < 0.05). Additionally, TGF-ß1 at a dose of 10 ng/ml increased α-SMA protein expression and COL1, COL3, FN production after 72 h of stimulation (P < 0.05). The data showed a positive linkage between the presence of myofibroblasts and severity of endometrosis. We conclude that TGF-ß1 may participate in pathological fibrotic changes in equine endometrial tissue by induction of myofibroblast differentiation, increased production of ECM components and fibroblast proliferation.

Effects of follicular ablation and induced luteolysis on LH and follicular fluid factors during the periovulatory period in mares.

Haemorrhagic anovulatory follicles (HAFs) are the most common pathological anovulatory condition in the mare. To enhance understanding of the physiopathology of HAFs, the aim of the present study was to determine the effects of an induced-follicular wave on LH concentrations and follicular fluid factors relevant to the ovulatory process. Mares were allocated to treatment or control groups (n = 7/group) in a crossed over design during 14 oestrous cycles with a period of one cycle occurring when there were no treatments between the times when treatments were administered. In the treatment group, all antral follicles ≥8 mm were ablated on Day 10 after ovulation followed by administration of a luteolytic dose of PGF2α. All mares of both groups were treated with 1500 IU of hCG when a follicle ≥32 mm was detected (Hour 0), and follicular fluid was aspirated 35 h later. Blood samples were collected every 48 h from Day 10 until Hour 0 from all mares. Follicular fluid was assayed for PGE2, estradiol and progesterone. Plasma was assayed for LH concentrations. A follicular wave followed follicle ablation in the treated mares. Concentrations of LH were greater (P = 0.05) in mares ot the treatment compared with control group. Concentrations of PGE2, estradiol and progesterone in follicular fluid did not differ between groups (P > 0.05). Treatment resulted in an earlier increase in circulating LH, however, there was no effect on concentrations of intra-follicular PGE2, estradiol or progesterone in hCG-stimulated preovulatory follicles.