RESUMO
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.
Assuntos
Interleucina-13 , Transcriptoma , Gravidez , Animais , Feminino , Cavalos , Interleucina-17 , Citocinas/genética , Endométrio , Inflamação/genética , FibroseRESUMO
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 (nâ¯=â¯6 for each stages of endometrosis) and the follicular phase of the estrous cycle (nâ¯=â¯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 (Pâ¯<â¯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 (Pâ¯<â¯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 (nâ¯=â¯6, Kenney and Doig category I) were stimulated with vehicle or TGF-ß1 (1, 5, 10â¯ng/ml) for 24, 48 or 72â¯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 (Pâ¯<â¯0.05). Additionally, TGF-ß1 at a dose of 10â¯ng/ml increased α-SMA protein expression and COL1, COL3, FN production after 72â¯h of stimulation (Pâ¯<â¯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.
Assuntos
Actinas/metabolismo , Colágeno/metabolismo , Endométrio/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Cavalos , Fator de Crescimento Transformador beta1/farmacologia , Actinas/genética , Animais , Células Cultivadas , Colágeno/genética , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/metabolismoRESUMO
This article describes the application of a simplified Bayesian method for estimation of doses from a mixed field using cytogenetic biological dosimetry, taking as an example neutron and gamma radiation emitted from the MARIA nuclear research reactor in Poland. The Bayesian approach is a good alternative to the commonly used iterative method, which allows separate dose estimation. In the present paper, a computer program, which uses the iterative and simplified Bayesian methods to calculate mixed radiation doses, is introduced.
Assuntos
Biometria/métodos , Análise Citogenética , Raios gama , Teorema de Bayes , Humanos , RadiometriaRESUMO
The aim of the study was to determine the effects of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF), interleukin-1-alpha (IL-1α), and nitric oxide donor (NONOate) on both in vivo and in vitro secretion of prostaglandin (PG)E2, PGF2α, leukotriene (LT)B4, and LTC4 by the bovine mammary gland. In the first experiment, tissues isolated from the teat cavity and lactiferous sinus were treated in vitro with LPS (10 ng/mL), TNF (10 ng/mL), IL-1α (10 ng/mL), NONOate (10(-4) M), and the combination of TNF + IL-1α + NONOate for 4 or 8 h. PGE2 or PGF2α secretion was stimulated by all treatments (P < 0.05) excepting NONOate alone, which did not stimulate PGF2α secretion. Moreover, all factors increased LTB4 and LTC4 secretion (P < 0.05). In the second experiment, mastitis was experimentally mimicked in vivo by repeated (12 h apart) intramammary infusions (5 mL) of (1) sterile saline; (2) 250-µg LPS; (3) 1-µg/mL TNF; (4) 1-µg/mL IL-1α; (5) 12.8-µg/mL NONOate; and (6) TNF + IL-1α + NONOate into 2 udder quarters. All infused factors changed PGE2, 13,14-dihydro,15-keto-PGF2α, and LT concentrations in blood plasma collected from the caudal vena cava, the caudal superficial epigastric (milk) vein, the jugular vein, and the abdominal aorta (P < 0.05). In summary, LPS and other inflammatory mastitis mediators modulate PG and LT secretion by bovine mammary gland in both in vivo and in vitro studies.
Assuntos
Citocinas/farmacologia , Leucotrienos/metabolismo , Lipopolissacarídeos/farmacologia , Glândulas Mamárias Animais/metabolismo , Óxido Nítrico/farmacologia , Prostaglandinas/metabolismo , Animais , Bovinos , Dinoprosta/análogos & derivados , Dinoprosta/sangue , Dinoprosta/metabolismo , Dinoprostona/sangue , Dinoprostona/metabolismo , Feminino , Interleucina-1alfa/farmacologia , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Leucotrienos/sangue , Glândulas Mamárias Animais/efeitos dos fármacos , Mastite Bovina/fisiopatologia , Doadores de Óxido Nítrico/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although prostaglandin (PG) F2α analogues are routinely used for oestrus synchronisation in cattle, their effects on the function of the bovine corpus luteum (CL), and on ovarian arterial contractility, may not reflect the physiological effects of endogenous PGF2α. In the first of two related experiments, the effects of different analogues of PGF2α (aPGF2α) on the secretory function and apoptosis of cultured bovine cells of the CL were assessed. Enzymatically-isolated bovine luteal cells (from between days 8 and 12 of the oestrous cycle), were stimulated for 24h with naturally-occurring PGF2α or aPGF2α (dinoprost, cloprostenol or luprostiol). Secretion of progesterone (P4) was determined and cellular [Ca(2+)]i mobilisation, as well as cell viability and apoptosis were measured. Naturally-occurring PGF2α and dinoprost stimulated P4 secretion (P<0.05), whereas cloprostenol and luprostiol did not influence P4 synthesis. The greatest cytotoxic and pro-apoptotic effects were observed in the luprostiol-treated cells, at 37.3% and 202%, respectively (P<0.001). The greatest effect on [Ca(2+)]i mobilisation in luteal cells was observed post-luprostiol treatment (200%; P<0.001). In a second experiment, the influence of naturally-occurring PGF2α and aPGF2α on ovarian arterial contraction in vitro, were examined. No differences in the effects of dinoprost or naturally-occurring PGF2α were found across the studied parameters. The effects of cloprostenol and luprostiol on luteal cell death, in addition to their effects on ovarian arterial contractility, were much greater than those produced by treatment with naturally-occurring PGF2α.
Assuntos
Artérias/efeitos dos fármacos , Bovinos , Dinoprosta/análogos & derivados , Células Lúteas/efeitos dos fármacos , Ovário/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Animais , Cloprostenol/farmacologia , Dinoprosta/farmacologia , Feminino , Células Lúteas/fisiologia , Prostaglandinas F Sintéticas/farmacologiaRESUMO
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.
Assuntos
Bovinos , Corpo Lúteo/crescimento & desenvolvimento , Luteólise , Animais , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/fisiologia , Citocinas/fisiologia , Dinoprosta/fisiologia , Endotelina-2/fisiologia , Feminino , Hormônios Esteroides Gonadais/fisiologia , Leucotrienos/fisiologia , Hormônio Luteinizante/fisiologia , Lisofosfolipídeos/fisiologia , Neovascularização Fisiológica , Prostaglandinas/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Information on the prevalence of subclinical endometritis and its mechanism in repeat breeding cows is very limited. The aims of this study were: a) to evaluate the incidence of this disorder with cytobrush cytology b) to analyze mRNA expression of tumor necrosis factor alpha (TNFalpha) and inducible nitric oxide synthase (iNOS) in endometrial biopsy samples collected from repeat breeding cows with and without subclinical endometritis. Two experiments were carried out. In experiment 1,112 (12.4%) repeat breeding cows (inseminated at least 3 times and not pregnant) were selected out of 902 cows from 8 dairy herds. Cytobrush cytology was performed on these cows, using the threshold of 10% PMNs in uterine smears. The results showed that 45 out of the 112 cows (40.2%) were diagnosed as having subclinical endometritis. In experiment 2, uterine biopsy samples were taken from repeat breeding cows with subclinical endometritis (n = 10) and without this disorder (n = 10). Using reverse transcription-PCR, the mRNA expression of TNFalpha and iNOS was determined. A statistically significant increase in expression of both substances was measured in the group of cows with subclinical endometritis (p <0.05). These results provide evidence for a high prevalence of subclinical endometritis in repeat breeding cows as well as the involvement of TNFalpha and iNOS pathways in the regulation of this pathological condition.
Assuntos
Endometrite/veterinária , Endométrio/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/patologia , Endometrite/patologia , Feminino , Regulação da Expressão Gênica/fisiologia , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
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 (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's 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.
Assuntos
Endométrio/metabolismo , Endométrio/patologia , Cavalos/genética , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/análise , Dinoprostona/sangue , Dinoprostona/metabolismo , Endométrio/enzimologia , Feminino , Fibrose/enzimologia , Fibrose/genética , Fibrose/metabolismo , Cavalos/sangue , Cavalos/metabolismo , Concentração Osmolar , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/fisiologiaRESUMO
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.
Assuntos
Bovinos/fisiologia , Cortisona/metabolismo , Cortisona/farmacologia , Dinoprosta/metabolismo , Hidrocortisona/metabolismo , Fase Luteal/fisiologia , Animais , Cortisona/administração & dosagem , Cortisona/sangue , Dinoprosta/análogos & derivados , Dinoprosta/sangue , Dinoprosta/genética , Endométrio/metabolismo , Feminino , Hidrocortisona/sangueRESUMO
Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10(-7)m) for 24 h. The concentrations of PGE(2) and PGF(2α) in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE(2) and PGF(2α) release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.
Assuntos
Técnicas de Cultura de Células/veterinária , Endométrio/citologia , Cavalos , Ocitocina/farmacologia , Prostaglandinas/metabolismo , Animais , Criopreservação/veterinária , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Tumor necrosis factor-α (TNF-α) is involved in the tissue remodeling that occurs in the corpus luteum (CL) during its development and regression. This cytokine is also implicated in the regulation of reproduction by its actions on ovarian steroidogenic cells. The aim of this study was to examine the influence of TNF-α on (1) progesterone (P(4)) output by the bovine CL and on (2) the responsiveness of the CL to LH or prostaglandin E(2) (PGE(2)) in vitro. In experiment 1, CL (days 8 to 10 of the estrous cycle) were perfused by using an in vitro microdialysis system with TNF-α (0.1, 0.5, or 1 µg/mL) alone or with TNF-α (1 µg/mL) followed by LH (1000 ng/mL) or PGE(2) (2 × 10(-5) M). Basal P(4) release (P < 0.05) was increased by TNF-α (0.5 or 1 µg/mL). Moreover, TNF-α (1 µg/mL) inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). In experiment 2, 4 h after intrauterine infusion of TNF-α (0.01 µg/mL or 1 µg/mL), CL (days 8 to 10 of the estrous cycle) were collected by colpotomy, cultured, and stimulated with LH (10 ng/mL) or PGE(2) (10(-6) M). Intrauterine infusion of TNF-α at a concentration of 1 µg/mL increased basal P(4) output by CL (P < 0.05). Moreover, the intrauterine infusion of TNF-α at a concentration of 0.01 µg/mL inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). These results indicate that TNF-α (1) does not have an effect on the autonomous, pulsatile release of P(4); (2) increases P(4) secretion by bovine CL with increasing doses, and (3) reduces in a dose-dependent manner the responsiveness of CL to luteotropic factors both directly (after infusion to CL) and indirectly (after intrauterine infusion).
Assuntos
Bovinos/fisiologia , Corpo Lúteo/metabolismo , Hormônio Luteinizante/farmacologia , Progesterona/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Corpo Lúteo/efeitos dos fármacos , Dinoprostona/farmacologia , Antagonistas de Estrogênios/farmacologia , Ciclo Estral , Feminino , Antagonistas de Hormônios/farmacologia , Técnicas In VitroRESUMO
Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.
Assuntos
Corpo Lúteo/fisiologia , Dinoprostona/metabolismo , Cavalos/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/farmacologia , Progesterona/metabolismo , Animais , Bovinos , Divisão Celular , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/enzimologia , Dinoprostona/biossíntese , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Feminino , Células Lúteas/enzimologia , Fase Luteal/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , Progesterona/biossínteseRESUMO
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.