RESUMO
The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-ß3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-ß3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy.
Assuntos
Endométrio , Interferon gama , Animais , Feminino , Bovinos , Endométrio/metabolismo , Interferon gama/genética , Gravidez , Fertilização , Parto , Citocinas/genética , Citocinas/metabolismoRESUMO
The alteration of signaling molecules involved in the general metabolism of animals can negatively influence reproduction. In dairy cattle, the development of follicular cysts and the subsequent appearance of ovarian cystic disease (COD) often lead to decreased reproductive efficiency in the herd. The objective of this review is to summarize the contribution of relevant metabolic and nutritional sensors to the development of COD in dairy cows. In particular, we focus on the study of alterations of the insulin signaling pathway, adiponectin, and other sensors and metabolites relevant to ovarian functionality, which may be related to the development of follicular persistence and follicular formation of cysts in dairy cattle. The results of these studies support the hypothesis that systemic factors could alter the local scenario in the follicle, generating an adverse microenvironment for the resumption of ovarian activity and possibly leading to the persistence of follicles and to the development and recurrence of COD.
Assuntos
Doenças dos Bovinos , Cistos Ovarianos , Feminino , Bovinos , Animais , Cistos Ovarianos/veterinária , Cistos Ovarianos/metabolismo , Folículo Ovariano/metabolismo , Reprodução , Insulina/metabolismo , Doenças dos Bovinos/metabolismo , Microambiente TumoralRESUMO
The cytokines of the interleukin-1 (IL-1) family are closely involved in the resolution of inflammation in cows with metritis and endometritis. However, little is known about the role of these cytokines beyond uterine regression in the absence of disease, especially around conception. Thus, the aim of this study was to examine the gene and protein expression of IL-1α, IL-1ß, IL-1RI, IL-1RII and IL-1Ra in endometrial biopsies previous to conception, to evaluate the possible association of these cytokines with delayed conception in dairy cows. Gene and protein expression levels were evaluated by real-time PCR and immunohistochemistry, respectively. The gene expression levels of cytokines were not associated with the duration of the period to conception following parturition. However, high protein expression of IL-1ß and low protein expression of IL-1Ra were significantly associated with early conception. These results suggest that an imbalanced protein expression of IL-1ß and IL-1Ra in the endometrium of dairy cows could be part of the maternal immune response mechanism necessary to propitiate early conception and probably to maintain pregnancy.
Assuntos
Doenças dos Bovinos , Endometrite , Feminino , Gravidez , Bovinos , Animais , Proteína Antagonista do Receptor de Interleucina 1/genética , Endométrio , Fertilização , Endometrite/genética , Endometrite/veterinária , Biópsia/veterinária , Doenças dos Bovinos/genéticaRESUMO
Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C-C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary.
Assuntos
Hormônio Adrenocorticotrópico , Ovário , Feminino , Bovinos , Animais , Hormônio Adrenocorticotrópico/farmacologia , Interleucina-17 , Ligantes , LeucócitosRESUMO
Stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, reducing fertility by interfering with the mechanisms that regulate the timing of events within the follicular phase of the estrous cycle. In the HPA axis, melanocortin 2 receptor (MC2R) mediates responses to adrenocorticotropic hormone (ACTH) in concert with melanocortin receptor accessory protein 2 (MRAP2). The aims of the present study were: (1) to evaluate the effects of ACTH administered in cows in the preovulatory period on the expression of the MC2R/MRAP2 complex in the dominant follicle; and (2) to analyze the involvement of Extracellular signal Regulated Kinase 1 (ERK1) signaling in the activation of MC2R and the expression of key enzymes involved in the biosynthesis of glucocorticoids (GCs) in the dominant follicle. To this end, 100 IU ACTH was administered to Holstein cows from a local dairy farm during pro-estrus every 12 h for four days until ovariectomy, which was performed before ovulation. Protein immunostaining of MC2R was higher in the dominant follicles of ACTH-treated cows (p < 0.05). Also, Western blot analysis showed higher activation of the ERK1 signaling pathway in ACTH-treated cows (p < 0.05). Finally, immunohistochemistry performed in the dominant follicles of ACTH-treated cows detected higher expression of CYP17A1 and CYP21A2 (p < 0.05). These results suggest that the bovine ovary is able to respond locally to ACTH as a consequence of stress altering the expression of relevant steroidogenic enzymes. The results also confirm that the complete GC biosynthesis pathway is present in bovine dominant follicle and therefore GCs could be produced locally.
Assuntos
Hormônio Adrenocorticotrópico , Sistema Hipotálamo-Hipofisário , Hormônio Adrenocorticotrópico/metabolismo , Animais , Bovinos , Feminino , Sistema Hipotálamo-Hipofisário/metabolismo , Ovulação , Sistema Hipófise-Suprarrenal , Receptor Tipo 2 de Melanocortina/metabolismoRESUMO
The nutritional conditions and immune status of dairy cows affect reproductive performance. This study was conducted with the aim to analyze the phagocytic activity (PA) and phagocytic capacity (PC) of circulating monocytes after the period of transition from pregnancy to lactation, to evaluate possible associations with duration of time period to conception following parturition. Results indicated PA was not associated with duration of time period to conception following parturition. In contrast, cows with a lesser PC conceived earlier (98 ± 9 days in milk, DIM) than those with a greater PC (168 ± 15 DIM). Based on these results, to analyze the association of the hormonal and metabolic milieu with the PA and PC, the animals were grouped considering the days to conception following parturition. In the group with the greater number of days to conception (>168 DIM), the PA was associated with concentrations of progesterone and beta-hydroxybutyrate (BHB) at 90 DIM and glucose at 120 DIM, whereas PC was associated with the concentrations of progesterone, cortisol and glucose at 90 DIM, non-esterified fatty acids (NEFA) at 120 DIM, 17ß-estradiol at 150 DIM, and 17ß-estradiol and BHB at 180 DIM. Overall, these results represent a new perspective related to the reproductive performance of dairy cows. The modifications of cellular functions may be useful for predicting the onset of health complications in dairy cows and to manage cows in ways that result in an enhanced fertility during the subsequent lactational period.
Assuntos
Bovinos/fisiologia , Fertilização/fisiologia , Monócitos/fisiologia , Fagocitose/fisiologia , Animais , Feminino , Lactação/fisiologia , Parto , GravidezRESUMO
Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (pâ¯<â¯0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (pâ¯<â¯0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (pâ¯<â¯0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.
Assuntos
Doenças dos Bovinos/genética , Doenças dos Bovinos/fisiopatologia , Cistos Ovarianos/genética , Cistos Ovarianos/fisiopatologia , Folículo Ovariano/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Estudos de Casos e Controles , Bovinos/fisiologia , Doenças dos Bovinos/metabolismo , Feminino , Cisto Folicular/genética , Cisto Folicular/metabolismo , Cisto Folicular/fisiopatologia , Expressão Gênica , Cistos Ovarianos/metabolismo , Ovário/metabolismo , Ovário/patologia , Ovulação/genética , Ovulação/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. The main signs of this infertility are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1ß, IL-1RI, IL-1RII, IL-1RA and IL-4 in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged administration of progesterone in dairy cows. Protein expression of IL-1ß, IL-1RI, IL-1RII, IL-1RA and IL-4 was evaluated by immunohistochemistry. Additionally, IL-1ß and IL-4 concentrations in follicular fluid and serum were determined by ELISA. In granulosa cells, IL1-RII and IL-4 expression was higher in follicles with different persistence times than in the control dominant follicles. IL-1RA expression was higher in persistent follicles of the P15 group (15 days of follicular persistence) than in those of the control group. In theca cells, IL-1RII expression was higher in persistent follicles of the P0 group (expected time of ovulation) than in dominant follicles from the control group (pâ¯<â¯.05) and the other persistence groups, whereas IL-4 expression was higher in persistent follicles of groups P0 and P15 than in the dominant follicles of the control group (pâ¯<â¯.05). Differences between serum and follicular fluid within each group were detected only in P0 for IL-1ß, and in the control, P10 and P15 groups for IL-4 (pâ¯<â¯.05). These results complement previous results, evidencing that early development of COD in cows is concurrent with an altered expression of cytokines in different ovarian follicular structures and may contribute to the follicular persistence and ovulation failure found in cattle with follicular cysts.
Assuntos
Anovulação/metabolismo , Bovinos/fisiologia , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/metabolismo , Interleucina-4/metabolismo , Folículo Ovariano/fisiologia , Receptores Tipo II de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Animais , Anovulação/veterinária , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/fisiopatologia , Sobrevivência Celular , Indústria de Laticínios , FemininoRESUMO
Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. The initiation of the IGF response is a result of interactions between IGF-binding proteins (IGFBPs) and IGFBP proteases, mainly pregnancy-associated plasma protein A (PAPP-A). IGFBPs bind IGFs with high affinity and consequently regulate their access to IGF receptors (IGFRs). The aim of this research was to determine variations in components of the IGF system in the ovaries of cows with persistent follicles induced by long-term administration of progesterone. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes occur early in the development of COD. The concentrations of IGF1 and IGFBP4 in follicular fluid were similar in all groups with follicular persistence and in control antral follicles. IGFR1 and IGFBP4 expression in situ were higher in granulose cells in persistent follicles than in control follicles. No differences were found in PAPP-A concentration within follicular fluid in persistent follicles relative to control antral follicles. These data support the hypothesis that the IGF system is altered in the initial stages of development of follicular persistence and has a determinant role in ovarian function in cattle.
Assuntos
Doenças dos Bovinos/metabolismo , Cistos Ovarianos/veterinária , Somatomedinas/metabolismo , Animais , Bovinos , Doenças dos Bovinos/patologia , Feminino , Folículo Ovariano/patologiaRESUMO
In dairy cattle, cystic ovarian disease (COD) is an important cause of subfertility, and two of the main signs are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1α, IL-6, IL-8 and TNF-α in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged treatment with progesterone in dairy cows. Protein expression of IL-1α, IL-6, IL-8 and TNF-α was evaluated by immunohistochemistry. Additionally, IL-6 concentration in follicular fluid and serum was determined by ELISA. IL-1α, IL-6, IL-8 and TNF-α expression was increased in follicles with different persistence times in relation to the control dominant follicles, in granulosa cells. For IL-6, IL-8 and TNF-α, this increase was detected early (P0: expected time of ovulation and/or P5: 5 days of follicular persistence). Additionally, theca cells showed an increase in IL-6 in antral (groups P10 and P15) and persistent follicles (group P10) related to dominant follicles from the control group (p < 0.05). Serum concentration of IL-6 was higher in groups P5, P10 and P15 than in control cows (p < 0.05). The results show evidence that early development of COD in cows is concurrent with altered expression of these cytokines in different ovarian follicular structures and may contribute to the follicular persistence and endocrine changes found in cattle with follicular cysts.
Assuntos
Bovinos/fisiologia , Citocinas/metabolismo , Folículo Ovariano/fisiologia , Animais , Busserrelina/administração & dosagem , Busserrelina/farmacologia , Cloprostenol/administração & dosagem , Cloprostenol/farmacologia , Citocinas/genética , Sincronização do Estro/efeitos dos fármacos , Feminino , Fármacos para a Fertilidade Feminina/administração & dosagem , Fármacos para a Fertilidade Feminina/farmacologia , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/farmacologia , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Luteolíticos/administração & dosagem , Luteolíticos/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Glucocorticoids (GCs) such as cortisol and corticosterone are important steroid hormones with different functions in intermediate metabolism, development, cell differentiation, immune response and reproduction. In response to physiological and immunological stress, adrenocorticotropic hormone (ACTH) acts on the adrenal gland by stimulating the synthesis and secretion of GCs. However, there is increasing evidence that GCs may also be synthesized by extra-adrenal tissues. Here, we examined the gene and protein expression of the enzyme 11ß-hydroxylase P450c11 (CYP11B1), involved in the conversion of 11-deoxycortisol to cortisol, in the different components of the bovine ovary and determined the functionality of CYP11B1 in vitro CYP11B1 mRNA was expressed in granulosa and theca cells in small, medium and large antral ovarian follicles, and CYP11B1 protein was expressed in medium and large antral follicles. After stimulation by ACTH, we observed an increased secretion of cortisol by the wall of large antral follicles. We also observed a concentration-dependent decrease in the concentration of cortisol in response to metyrapone, an inhibitor of CYP11B1. This decrease was significant at 10-5 µM metyrapone. In conclusion, this study demonstrated for the first time the presence of CYP11B1 in the bovine ovary. This confirms that there could be a local synthesis of GCs in the bovine ovary and therefore a potential endocrine responder to stress through these hormones.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/enzimologia , Esteroide 11-beta-Hidroxilase/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Bovinos , Células Cultivadas , Feminino , Hormônios/farmacologia , Hidrocortisona/metabolismo , Folículo Ovariano/citologia , Esteroide 11-beta-Hidroxilase/genéticaRESUMO
The most important regulators of tissue remodelling during ovarian follicular growth, development, ovulation and atresia are gonadotropins, steroid hormones, growth factors and different proteolytic enzymes. Matrix metalloproteinases (MMPs) such as collagenase or gelatinase (i.e. MMP-1, -8, -2 and -9) and associated tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4) control connective tissue remodelling during follicular rupture. In this study, we hypothesized that an imbalance in the MMP-TIMP system may be an intra-ovarian component that contributes to the pathogenesis of cystic ovarian disease (COD) in cows. Taking into account that the control of MMP activity by TIMPs could determine their effects in both physiological and pathological conditions, MMP and TIMP mRNA and protein expression was examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively, in ovaries from control cows and cows with COD. Expression of mRNA encoding MMP-2, TIMP-1 and TIMP-2 was lower in follicular cysts than in control pre-ovulatory follicles, while the results by IHC showed this imbalance only for TIMP-2 protein expression. Additional analysis by zymography to evaluate the gelatinase activity of MMP-2 and MMP-9 demonstrated higher MMP-2 activity in follicular fluid (FF) of cysts than in FF of pre-ovulatory follicles. On the other hand, MMP-9 activity was increased in follicular cysts and absent in the FF of pre-ovulatory follicles. These findings suggest that the altered mRNA expression and activities of the MMP-TIMP system may be related to the failure in ovulation and follicular development observed in COD.
Assuntos
Doenças dos Bovinos/enzimologia , Metaloproteinases da Matriz/metabolismo , Cistos Ovarianos/veterinária , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Western Blotting , Bovinos , Feminino , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The aim of this study was to evaluate the effect of a probiotic/lactose inoculum on haematological and immunological parameters and renal and hepatic biochemical profiles before and during a Salmonella Dublin DSPV 595T challenge in young calves. Twenty eight calves, divided into a control and probiotic group were used. The probiotic group was supplemented with 100 g lactose/calf/d and 1010 cfu/calf/d of each strain of a probiotic inoculum composed of Lactobacillus casei DSPV318T, Lactobacillus salivarius DSPV315T and Pediococcus acidilactici DSPV006T throughout the experiment. The pathogen was administered on day 11 of the experiment, at an oral dose of 109 cfu/animal (LD50). Aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), urea, red blood cells, haemoglobin, haematocrit, mean cell haemoglobin (MCH), mean corpuscular volume, mean corpuscular haemoglobin concentration (MCHC), white blood cells, lymphocytes, neutrophils, band neutrophils, monocytes, eosinophils, basophils and the neutrophils/lymphocytes ratio were measured on days 1, 10, 20 and 27 of the experiment. In addition, animals were necropsied to evaluate immunoglobulin A (IgA) production in the jejunal mucosa. The most significant differences caused by the administration of the inoculum/lactose were found during the acute phase of Salmonella challenge (9 days after challenge), when a difference between groups in neutrophils/lymphocytes ratio were detected. These results suggest that the probiotic/lactose inoculum administration increases the calf's ability to respond to the disease increasing the systemic immune response specific. No differences were found in haemoglobin, haematocrit, MCH, MCHC, AST, urea, GGT, band neutrophils, eosinophils, monocytes and IgA in the jejunum between the two groups of calves under the experimental conditions of this study. Further studies must be conducted to evaluate different probiotic/pathogens doses and different sampling times, to achieve a greater understanding of the effects of this inoculum on intestinal infections in young calves and of its mechanism of action.
Assuntos
Probióticos/uso terapêutico , Salmonelose Animal/terapia , Ração Animal , Animais , Análise Química do Sangue/veterinária , Bovinos , Contagem de Eritrócitos/veterinária , Hematócrito/veterinária , Testes Hematológicos/veterinária , Hemodinâmica/efeitos dos fármacos , Hemoglobinas/metabolismo , Imunoglobulina A/análise , Jejuno/imunologia , Lacticaseibacillus casei , Ligilactobacillus salivarius , Lactose/administração & dosagem , Contagem de Leucócitos/veterinária , Pediococcus acidilactici , Salmonelose Animal/sangue , Salmonella typhimurium , Análise de SobrevidaRESUMO
Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.
Assuntos
Doenças dos Bovinos/fisiopatologia , Cistos Ovarianos/veterinária , Proteína Plasmática A Associada à Gravidez/fisiologia , Receptor IGF Tipo 1/fisiologia , Animais , Bovinos , Doenças dos Bovinos/etiologia , Feminino , Líquido Folicular/química , Expressão Gênica , Células da Granulosa/química , Imuno-Histoquímica , Cistos Ovarianos/química , Cistos Ovarianos/fisiopatologia , Folículo Ovariano/química , Gravidez , Proteína Plasmática A Associada à Gravidez/análise , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/análise , Receptor IGF Tipo 1/análise , Receptor IGF Tipo 1/genéticaRESUMO
A growing body of evidence suggests that ovulation shares many of the features of an inflammatory reaction and that cytokines play many diverse and important roles in reproductive biology. The aim of this study was to examine the expression of the pro-inflammatory cytokines interleukin (IL)-1α, IL-6 and tumour necrosis factor (TNF)-α in ovarian cells from cows with cystic ovarian disease (COD) as compared with that in ovarian structures from regularly cycling cows. Expression of genes encoding IL-1α, IL-6 and TNF-α was detected by real-time polymerase chain reaction in follicular cells from ovaries from healthy cows and cows with COD with no significant differences. However, immunohistochemistry showed increased expression of IL-1α, IL-6 and TNF-α in cystic follicles, suggesting that this expression may be related to the persistence of follicular cysts. The effect of COD was evident for IL-1α and TNF-α, and a follicular structure-disease interaction was observed in the expression of all the cytokines evaluated. Thus, altered expression of these proinflammatory cytokines may be related to ovulation failure and development of follicular cysts.
Assuntos
Doenças dos Bovinos/patologia , Citocinas/biossíntese , Cistos Ovarianos/veterinária , Folículo Ovariano/patologia , Animais , Western Blotting , Bovinos , Doenças dos Bovinos/imunologia , Citocinas/análise , Feminino , Imuno-Histoquímica , Cistos Ovarianos/imunologia , Cistos Ovarianos/patologia , Folículo Ovariano/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Com o objetivo de verificar a presença de VEGF e IGF-1 nos ovários de cadelas, foram realizadas análises imuno-histoquímicas do estroma cortical; teca e granulosa de folículos secundários, terciários e terciários pré-ovulatórios luteinizados; e ovócitos de folículos primários, secundários e terciários de ovários de cinco cadelas em anestro (Anest) e cinco em estro (Est). A identificação das fases do ciclo estral foi realizada por citologia vaginal associada a dosagem plasmática de progesterona. Os ovários foram submetidos a tratamento imuno-histoquímico para identificação de VEGF (anticorpo primário PU 360-UP, Biogenex, USA; diluição 1:30) e IGF-1 (anticorpo primário PabCa, Gro-Pep, Austrália; diluição 1:100). Determinou-se um índice de imunomarcação (IM), para cada tecido avaliado, pela razão entre a área positivamente marcada dividida pela área total analisada. Para os ovócitos, verificou-se imunomarcação positiva ou negativa. As comparações de IM entre tecidos foram realizadas pelo teste de Wilcoxon (diferentes tecidos em mesmo grupo) ou Mann-Whitney (mesmo tecido entre diferentes grupos), todas no nível de 5% de significância. VEGF e IGF-1 foram identificados, de forma semelhante (P>0,05), em todas as estruturas avaliadas em ambos os grupos experimentais. Conclui-se que esses fatores de crescimento estão presentes em cadelas no anestro e estro, no estroma cortical ovariano, folículos em diferentes estádios de desenvolvimento e ovócitos.(AU)
In order to verify the presence of VEGF and IGF-1 in the ovaries of bitches, immunohistochemical analyzes of the cortical stroma; theca and granulosa of secondary, tertiary and tertiary luteinized preovulatory follicles; and oocytes of primary, secondary and tertiary follicles of ovaries from five bitches in anestrous (Anest) and five in estrus (Est) was performed. The identification of the phases of the estrous cycle was performed by vaginal cytology associated with the measurement of plasma progesterone. The ovaries were treated for immunohistochemical identification of VEGF (PU 360 primary antibody-UP, Biogenex, USA, dilution 1:30) and IGF-1 (primary antibody PabCa, Gro-Pep, Australia; 1:100 dilution). The immunostaining index (MI) was determined for each tissue by the ratio of positively marked area divided by total analyzed area. For oocytes immunostaining was determined as positive or negative. Comparisons of IM between tissues were performed with the Wilcoxon test (deferent tissues in the same group) or Mann-Whitney test (same tissue between different groups), all at 5% significance level. VEGF and IGF-1 have been similarly identified (P>0.05) in all structures evaluated in both groups. It is concluded that in bitches in estrus and anestrous these growth factors are present in ovary cortical stroma, follicles at different stages of development and oocytes.(AU)
Assuntos
Animais , Cães , Imuno-Histoquímica/veterinária , Ovário/anatomia & histologia , Anestro , Oócitos , Ciclo Estral , Células Estromais , Folículo Ovariano/crescimento & desenvolvimento , Fatores de Crescimento do Endotélio VascularRESUMO
Com o objetivo de verificar a presença de VEGF e IGF-1 nos ovários de cadelas, foram realizadas análises imuno-histoquímicas do estroma cortical; teca e granulosa de folículos secundários, terciários e terciários pré-ovulatórios luteinizados; e ovócitos de folículos primários, secundários e terciários de ovários de cinco cadelas em anestro (Anest) e cinco em estro (Est). A identificação das fases do ciclo estral foi realizada por citologia vaginal associada a dosagem plasmática de progesterona. Os ovários foram submetidos a tratamento imuno-histoquímico para identificação de VEGF (anticorpo primário PU 360-UP, Biogenex, USA; diluição 1:30) e IGF-1 (anticorpo primário PabCa, Gro-Pep, Austrália; diluição 1:100). Determinou-se um índice de imunomarcação (IM), para cada tecido avaliado, pela razão entre a área positivamente marcada dividida pela área total analisada. Para os ovócitos, verificou-se imunomarcação positiva ou negativa. As comparações de IM entre tecidos foram realizadas pelo teste de Wilcoxon (diferentes tecidos em mesmo grupo) ou Mann-Whitney (mesmo tecido entre diferentes grupos), todas no nível de 5% de significância. VEGF e IGF-1 foram identificados, de forma semelhante (P>0,05), em todas as estruturas avaliadas em ambos os grupos experimentais. Conclui-se que esses fatores de crescimento estão presentes em cadelas no anestro e estro, no estroma cortical ovariano, folículos em diferentes estádios de desenvolvimento e ovócitos.(AU)
In order to verify the presence of VEGF and IGF-1 in the ovaries of bitches, immunohistochemical analyzes of the cortical stroma; theca and granulosa of secondary, tertiary and tertiary luteinized preovulatory follicles; and oocytes of primary, secondary and tertiary follicles of ovaries from five bitches in anestrous (Anest) and five in estrus (Est) was performed. The identification of the phases of the estrous cycle was performed by vaginal cytology associated with the measurement of plasma progesterone. The ovaries were treated for immunohistochemical identification of VEGF (PU 360 primary antibody-UP, Biogenex, USA, dilution 1:30) and IGF-1 (primary antibody PabCa, Gro-Pep, Australia; 1:100 dilution). The immunostaining index (MI) was determined for each tissue by the ratio of positively marked area divided by total analyzed area. For oocytes immunostaining was determined as positive or negative. Comparisons of IM between tissues were performed with the Wilcoxon test (deferent tissues in the same group) or Mann-Whitney test (same tissue between different groups), all at 5% significance level. VEGF and IGF-1 have been similarly identified (P>0.05) in all structures evaluated in both groups. It is concluded that in bitches in estrus and anestrous these growth factors are present in ovary cortical stroma, follicles at different stages of development and oocytes.(AU)
Assuntos
Animais , Feminino , Cães , Oócitos , Ovário , Anestro , Estro , Fator de Crescimento Insulin-Like I/isolamento & purificação , Imuno-Histoquímica/veterinária , Fator A de Crescimento do Endotélio Vascular/isolamento & purificaçãoRESUMO
Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence.
Assuntos
Doenças dos Bovinos/metabolismo , Insulina/fisiologia , Cistos Ovarianos/veterinária , Folículo Ovariano/metabolismo , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/metabolismo , Animais , Bovinos , Feminino , Regulação da Expressão Gênica/fisiologia , Insulina/sangue , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Cistos Ovarianos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Insulina/sangue , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors may contribute to follicular persistence. Transforming growth factor-beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)-induced COD. In the oestrous-synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous-synchronized control group with that in ACTH-induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH-induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.
Assuntos
Doenças dos Bovinos/metabolismo , Regulação da Expressão Gênica/fisiologia , Cistos Ovarianos/veterinária , Fator de Crescimento Transformador beta/metabolismo , Hormônio Adrenocorticotrópico/toxicidade , Animais , Bovinos , Feminino , Cistos Ovarianos/induzido quimicamente , Cistos Ovarianos/metabolismo , Ovariectomia/veterinária , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Cystic ovarian disease (COD) is an important cause of infertility in cattle, and ACTH has been involved in regulatory mechanisms related to ovarian function associated with ovulation, steroidogenesis, and luteal function. Here, we examined the localization of 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) and 11ßHSD2 proteins in the ovary of healthy cows and animals with spontaneous and ACTH-induced COD and the in vitro response of the follicular wall exposed to ACTH. After stimulation by ACTH, we documented changes in 11ßHSD expression and cortisol secretion by the follicular wall of large antral and follicular cysts. Follicular cysts showed a higher constitutive expression of both enzymes, whereas ACTH induced an increase in 11ßHSD1 in tertiary follicles and follicular cysts and a decrease in 11ßHSD2 in follicular cysts. Moderate expression of 11ßHSD1 was observed by immunohistochemistry in granulosa of control animals, with an increase (P < 0.05) from primary to secondary, tertiary, and atretic follicles. The level of immunostaining in theca interna was lower than that in granulosa. The expression of 11ßHSD2 was lower in the granulosa of primary follicles than in that of secondary, tertiary, and atretic follicles and was lower in the theca interna than in the granulosa. In ACTH-induced and spontaneously occurring follicular cysts, differences from controls were observed only in the expression of 11ßHSD1 in the granulosa, being higher (P < 0.05) than in tertiary follicles. These findings indicate that follicular cysts may be exposed to high local concentrations of active glucocorticoids and indicate a local role for cortisol in COD pathogenesis and in regulatory mechanisms of ovarian function.