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1.
Front Vet Sci ; 7: 582211, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33195599

RESUMO

Although proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; ß-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2, and MMP9, as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h (P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment.

2.
Zookeys ; 992: 105-138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33223907

RESUMO

Seven new species of the subfamily Neanurinae from north-western Iran are described and illustrated in detail. Endonura agnieskae sp. nov. differs from the most similar congener, E. reticulata (Axelson, 1905), in chaetotaxic details and the arrangement of tubercles on the dorsal side of the body. Endonura annae sp. nov. can be easily recognised by its wide labrum, the absence of chaetae C on the head and the presence of a toothed claw. Endonura schwendingeri sp. nov. is especially distinctive due to the absence of chaetae A and Ocp on the head and the presence of the male ventral organ. Deutonura breviseta sp. nov. is related and most similar to D. persica Smolis, Shayanmehr & Yoosefi-Lafooraki, 2018, described recently and known from Mazandran Province in Iran. The new species can be easily distinguished by the following set of features: dark pigmented body, presence of chaetae C and Dl3 on the head, absence of microchaetae on the furca rudimentary, presence of thickened macrochaetae on dorsal side of body and absence of cryptopygy. The main characteristics of Deutonura sengleti sp. nov. include a white body with dark pigmented eyes, the fusion of tubercles Di and De on the first thoracic segment and the presence of the male ventral organ. Deutonura iranica sp. nov. is superficially similar to D. gibbosa Porco, Bedos & Deharveng, 2010, a species known from the Alps and Jura in Europe, but it differs in the body colour and the number of labial chaetae and chaetae (L+So) on the head. Paravietnura rostrata sp. nov., the first member of this enigmatic and intriguing genus known from Iran, is characterised by an unusually elongate ogival labrum and extreme reduction of dorsal chaetotaxy. Furthermore, new records of several other species of the subfamily: Cryptonura maxima Smolis, Falahati & Skarzynski, 2012; C. persica Smolis, Falahati & Skarzynski, 2012; Deutonura persica; Endonura longirostris Smolis, Shayanmehr, Kuznetsova & Yoosefi-Lafooraki, 2017; E. paracentaurea Smolis, Shayanmehr, Kuznetsova & Yoosefi-Lafooraki, 2017; Neanura deharvengi Smolis, Shayanmehr & Yoosefi-Lafooraki, 2018; N. muscorum (Templeton, 1835) and Protanura papillata Cassagnau & Delamare Deboutteville, 1955 are given. The present study is based on the rich material collected by Antoine Senglet and loaned by Peter J. Schwendinger.

3.
BMC Vet Res ; 16(1): 343, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943074

RESUMO

BACKGROUND: Equine endometrosis is a chronic degenerative condition, described as endometrial fibrosis that forms in the stroma, under the basement membrane and around the endometrial glands. The role of lysophosphatidic acid (LPA) in the development of tissue fibrosis varies depending on the organ, and its profibrotic role in mare endometrosis remains unclear. The study aimed to establish the endometrial presence of LPA and its receptors (LPAR1-4), together with its effects on connective tissue growth factor (CTGF) and prostaglandins (PG) secretion from equine endometrium under physiological (estrous cycle), or pathological conditions (endometrosis). Mare endometria in the mid-luteal phase (n = 5 for each category I, IIA, IIB, III of Kenney and Doig) and in the follicular phase (n = 5 for each category I, IIA, III and n = 4 for IIB) were used. In experiment 1, the levels of LPA, LPAR1-4 mRNA level and protein abundance were investigated in endometria at different stages of endometrosis. In experiment 2, the in vitro effect of LPA (10- 9 M) on the secretion of CTGF and PGs from endometrial tissue explants at different stages of endometrosis were determined. RESULTS: Endometrial LPA concentration was higher in the mid-luteal phase compared to the follicular phase in category I endometrium (P < 0.01). There was an alteration in endometrial concentrations of LPA and LPAR1-4 protein abundance in the follicular phase at different stages of endometrosis (P < 0.05). Additionally, LPA increased the secretion of PGE2 from category I endometrium in both phases of the estrous cycle (P < 0.05). The effect of LPA on the secretion of CTGF and PGF2α from endometrial tissue was altered depending on different stages of endometrosis (P < 0.05). CONCLUSION: Our data indicate that endometrosis disturbs proper endometrial function and is associated with altered endometrial LPA concentration, its receptor expression and protein abundance, PGE2/PGF2α ratio, and CTGF secretion in response to LPA. These changes could influence several physiological events occurring in endometrium in mare during estrous cycle and early pregnancy.

4.
Animals (Basel) ; 10(5)2020 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-32429399

RESUMO

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory effect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.

5.
Theriogenology ; 153: 74-84, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32442743

RESUMO

An increasing number of studies have shown that prostaglandins (PGs) exert multiple regulatory actions in the processes associated to tissue remodeling and fibrosis. Extracellular matrix (ECM) turnover is mediated by matrix metallopeptidases (MMPs). The knowledge about the regulation of their expression in mare endometrium is still limited. Thus, the aim of this study was to investigate whether: (i) profibrotic transforming growth factor (TGF)-ß1 modulates PG production in equine endometrium; and (ii) PGE2 and PGF2α modulate MMPs, their tissue inhibitors (TIMPs), and collagen 1 (COL1) expression. In experiment 1, the effect of TGF-ß1 (5 ng/mL) on PG secretion and PG synthases mRNA transcription, after 24 and 48 h treatment of mare endometrial fibroblast and epithelial cells was investigated using ELISA and qPCR. In experiment 2, the effects of PGE2 and PGF2α in doses 10-7M and 10-8M on secretion and MMP1, 2, 9, 13, TIMP1, 2, and COL1A1 mRNA transcription in mare endometrial fibroblasts were assessed. Transforming growth factor-ß1 treatment decreased secretion of PGF2α by endometrial fibroblasts (P < 0.05) and PGF2α and PGE2 by endometrial epithelial cells (P < 0.05). Prostaglandin E2 increased MMP-2 and MMP-9, and decreased MMP-13 secretion by endometrial fibroblasts (P < 0.05). Additionally, PGF2α treatment increased MMP-2, MMP-13 and COL1, but decreased MMP-1 secretion by endometrial fibroblasts (P < 0.05). Prostaglandins may be involved in the processes associated to pathological endometrial remodeling by their effect on MMP expression. The effect of PGF2α on COL1 secretion from fibroblasts suggests its profibrotic role in pathological endometrial remodeling.

6.
Theriogenology ; 150: 150-157, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31973963

RESUMO

The innate and adaptive immune mechanisms are key components of regulation of reproductive physiological function and uterine disorders in equine uterus. The predominant immunological response in equine endometrium, characterized by an innate immune response, occurs under estrogens influence, in the follicular phase. Although, the increase in immune-related genes in equine endometrium during estrus has been suggested to play a role in uterine clearance after mating, immune cells and their product, i.e. cytokines play also mandatory role in the luteal development and maintenance, regression of equine corpus luteum, as well as in early pregnancy. Innate immune response is nonspecific and acts as the first line of defense against pathogens, foreign stimuli that include constituents of seminal fluid and local infections (endometritis). It has been recently established that a phagocytosis-independent mechanism to restrain bacteria, by means of neutrophil extracellular traps (NETs) formation, is involved in pathogenesis of in mare endometrial fibrosis (endometrosis). Moreover, persistent macrophages and mast cell activation could also have pro-fibrotic roles by secreting great amounts of pro-fibrotic factors and lead to fibrosis. This review will highlight the involvement of immune key components of the innate and adaptive immune system and their products in equine uterus and their contribution to reproductive physiological function and uterine disorders.

7.
Sci Rep ; 10(1): 1119, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980722

RESUMO

Equine endometrial fibrosis (endometrosis) is described as a degenerative chronic condition in the uterus. Its characteristic feature is excessive deposition of extracellular matrix (ECM) components around the endometrial glands and stroma. Although matrix metallopeptidases (MMPs) that mediate ECM turnover are important factors in the process of fibrosis, knowledge of their expression and regulation in endometrosis is limited. In other species, one of the important regulators of MMPs and tissue inhibitors of MMPs (TIMPs) is transforming growth factor (TGF)-ß1. The goal of this study was to determine (i) endometrial expression of MMPs and TIMPs during endometrosis and (ii) the effect of TGF-ß1 on expression of MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells. In the follicular phase of the estrous cycle, MMP-1, -2, -9, and TIMP concentrations were higher during endometrosis than in healthy endometrium (P < 0.05). In the midluteal phase, MMP-3 concentration was lower in severe endometrosis compared to healthy endometrium (P < 0.05). In fibroblasts, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP1, but downregulated MMP-3 secretion (P < 0.05). In epithelial cells, TGF-ß1 upregulated MMP-1, -9, -13, and TIMP secretion (P < 0.05). Endometrial expression of MMPs and TIMPs is altered during endometrosis. TGF-ß1 is a regulator of endometrial ECM remodeling via its effect on MMPs and TIMPs in equine endometrial fibroblasts and epithelial cells.


Assuntos
Endometriose/veterinária , Regulação Enzimológica da Expressão Gênica , Doenças dos Cavalos/fisiopatologia , Metaloproteinases da Matriz/biossíntese , Fator de Crescimento Transformador beta1/fisiologia , Animais , Células Cultivadas , Endometriose/enzimologia , Endometriose/fisiopatologia , Endométrio/metabolismo , Endométrio/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ciclo Estral , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Doenças dos Cavalos/enzimologia , Cavalos , Metaloproteinases da Matriz/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Inibidores Teciduais de Metaloproteinases/biossíntese , Inibidores Teciduais de Metaloproteinases/genética , Fator de Crescimento Transformador beta1/farmacologia
8.
Theriogenology ; 142: 196-206, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606658

RESUMO

The maternal endometrium undergoes transformations during early pregnancy period to regulate the paracellular permeability across the epithelium and to enable adhesion between the trophoblast and endometrial epithelial cells. These transformations, under the influence of ovarian hormones, are associated with a partial loss in polarity of epithelial cell that is regulated by tight junctions (TJ), adherens junctions (AJ) and associated polarity protein complexes. This study examined the change in expression and distribution of proteins associated with TJs, AJs and apical partition defective (PAR) complex in porcine endometrium on Days 10, 13 and 16 of estrous cycle and pregnancy. Moreover, effect of hormones, progesterone (P4) and 17-ß estradiol (E2) on polar phenotype of endometrial epithelial cells was also investigated in vitro. There was pregnancy induced increase in gene and protein expression of TJ associated claudin-1 (CLDN1) on Day 13 of pregnancy as compared to corresponding day of estrous cycle and a decrease in TJ protein, zona occludens-1 (ZO-1) and PAR complex associated PAR6 expression levels on Day 16 of pregnancy (P < 0.05). Immunofluorescence studies revealed that on Days 10 and 13, TJ proteins occludin (OCLN) and ZO-1were primarily present in the apical region of lateral epithelial membrane. On Day 16 of pregnancy, whereas, OCLN redistributed into cytoplasm, ZO-1 decreased apically but was found to localize in the basal epithelium. The AJ proteins cadherin and ß-catenin were located at the apical epithelium on Day 10 of estrous cycle and pregnancy and Day 13 of estrous cycle. On Days 13 and 16 of pregnancy both proteins were expressed in the lateral membrane and co-localization between these proteins was observed on Day 16. On Day 10, PAR complex proteins PAR3, cell division control protein 42 (CDC42) and atypical protein kinase C (aPKC) ζ were observed in apical epithelium and in lateral membrane and CDC42 was also present in the cytoplasm of epithelium. Pregnancy induced redistribution of aPKCζ to cytoplasm and CDC42 to apical surface of luminal epithelium was observed on Days 13 and 16. The in vitro P4 and E2 treatment of epithelial cells mimicked in vivo results. These results indicate that P4 and E2 regulate alterations in epithelium that may facilitate embryo implantation and given the role of cadherin, catenin and CDC42 in embryo invasion, change in distribution of these proteins may limit the invasiveness of porcine conceptuses into the stroma.


Assuntos
Polaridade Celular/genética , Endométrio/metabolismo , Moléculas de Adesão Juncional/genética , Moléculas de Adesão Juncional/metabolismo , Prenhez , Suínos , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Células Cultivadas , Implantação do Embrião/genética , Feminino , Expressão Gênica , Idade Gestacional , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Gravidez , Prenhez/genética , Prenhez/metabolismo , Suínos/embriologia , Suínos/genética , Suínos/metabolismo , Junções Íntimas/genética , Junções Íntimas/metabolismo , Distribuição Tecidual
9.
Artigo em Inglês | MEDLINE | ID: mdl-31798533

RESUMO

The luteinization of the follicular cells, following a LH surge, causes extensive molecular and structural changes in preovulatory follicles (POF) that lead to ovulation and ultimate formation of the corpus luteum (CL). The objective of this study was to identify proteins expressed in porcine POF before the LH surge and a new CL formed, 2-3 days after ovulation, and evaluate proteome changes associated with formation of the CL from a follicle. We used 2D-gel electrophoresis-based proteomics and tandem mass spectrometry followed by a functional analysis using Ingenuity Pathway analysis (IPA) to evaluate functional pathways associated with the luteinization process. Protein lysates were prepared from isolated POFs and from the newly formed CL. A total of 422 protein spots were identified in both structures. A total of 15 and 48 proteins or their proteoforms were detected only in the POFs and CL, respectively. An IPA analysis of a POF proteome showed that most of the follicular proteins were involved in cellular infiltration, endoplasmic stress responses, and the protein ubiquitination pathway. Most of the early luteal proteins were associated with steroid metabolism, cell death and survival, free radical scavenging, and the protein ubiquitination pathway. A comparison of a follicular proteome with that of an early luteal proteome revealed that 167 identified proteins or their proteoforms were differentially regulated between POFs and the newly formed CL (p < 0.05 and a fold change of >1.8). Proteins that were significantly more abundant in follicles included cAMP-dependent protein kinase, histone binding protein RBBP4, reticulocalbin, vimentin, and calumenin; more abundant luteal proteins included albumin, farnesyl diphosphate synthase, serine protease inhibitors, elongation factor-1, glutaredoxin, and selenium-binding protein. Proteins that were significantly altered with luteal formation were found to be associated with cholesterol biosynthesis, cell death and survival, and acute phase response. Moreover, upstream regulators of differentially abundant proteins in CL were identified that included insulin growth factor-1, sterol regulatory element-binding transcription factor-1, and nuclear factor erythroid-derived 2. We have identified novel proteins that advance our understanding of (1) processes associated with differentiation of POFs into the CL, (2) possible mechanisms of luteal cell survival, and (3) pathways regulating steroidogenesis in the newly formed CL.

10.
Artigo em Inglês | MEDLINE | ID: mdl-31803139

RESUMO

The corpus luteum (CL) is an important tissue of the female reproductive process which is established through ovulation of the mature follicle. Pulsatile release of prostaglandin F2α from the uterus leads to the regression of luteal cells and restarts the estrous cycle in most non-primate species. The rapid functional regression of the CL, which coincides with decrease of progesterone production, is followed by its structural regression. Although we now have a better understanding of how the CL is triggered to undergo programmed cell death, the precise mechanisms governing CL protein degradation in a very short period of luteolysis remains unknown. In this context, activation of ubiquitin-proteasome pathway (UPP), unfolded protein response (UPR) and autophagy are potential subcellular mechanisms involved. The ubiquitin-proteasome pathway (UPP) maintains tissue homeostasis in the face of both internal and external stressors. The UPP also controls physiological processes in many gonadal cells. Emerging evidence suggests that UPP dysfunction is involved in male and female reproductive tract dysfunction. Autophagy is activated when cells are exposed to different types of stressors such as hypoxia, starvation, and oxidative stress. While emerging evidence points to an important role for the UPP and autophagy in the CL, the key underlying transcriptional mechanisms have not been well-documented. In this review, we propose how CL regression may be governed by the ubiquitin-proteasome and autophagy pathways. We will further consider potential transcription factors which may regulate these events in the CL.

11.
BMC Vet Res ; 15(1): 416, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31752870

RESUMO

BACKGROUND: Prostaglandin F2α (PGF2α) may differentially affect viability of luteal cells by inducing either proliferation or cell death (via apoptosis or necroptosis). The diverse effects of PGF2α may depend on its local vs. systemic actions. In our study, we determined changes in expression of genes related to: (i) apoptosis: caspase (CASP) 3, CASP8, BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and (ii) necroptosis: receptor-interacting protein kinase (RIPK) 1, RIPK3, cylindromatosis (CYLD), and mixed lineage kinase domain-like (MLKL) in the early and mid-stage corpus luteum (CL) that accompany local (intra-CL) vs. systemic (i.m.) analogue of PGF2α (aPGF2α) actions. Cows at day 4 (n = 24) or day 10 (n = 24) of the estrous cycle were treated by injections as follows: (1) systemic saline, (2) systemic aPGF2α (25 mg; Dinoprost), (3) local saline, (4) local aPGF2α (2.5 mg; Dinoprost). After 4 h, CLs were collected by ovariectomy. Expression levels of mRNA and protein were investigated by RT-q PCR, Western blotting and immunohistochemistry, respectively. RESULTS: We found that local and systemic administration of aPGF2α in the early-stage CL resulted in decreased expression of CASP3 (P < 0.01), but CASP8 mRNA expression was up-regulated (P < 0.05). However, the expression of CASP3 was up-regulated after local aPGF2α treatment in the middle-stage CL, whereas systemic aPGF2α administration increased both CASP3 and CASP8 expression (P < 0.01). Moreover, we observed that both local and systemic aPGF2α injections increased RIPK1, RIPK3 and MLKL expression in the middle-stage CL (P < 0.05) while CYLD expression was markedly higher after i.m. aPGF2α injections (P < 0.001). Moreover, we investigated the localization of necroptotic factors (RIPK1, RIPK3, CYLD and MLKL) in bovine CL tissue after local and systemic aPGF2α injections in the bovine CL. CONCLUSION: Our results demonstrated for the first time that genes related to cell death pathways exhibit stage-specific responses to PGF2α administration depending on its local or systemic actions. Locally-acting PGF2α plays a luteoprotective role by inhibiting apoptosis and necroptosis in the early CL. Necroptosis is a potent mechanism responsible for structural CL regression during PGF2α-induced luteolysis in cattle.


Assuntos
Bovinos , Morte Celular/efeitos dos fármacos , Corpo Lúteo/efeitos dos fármacos , Dinoprosta/farmacologia , Ocitócicos/farmacologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Corpo Lúteo/citologia , Corpo Lúteo/fisiologia , Dinoprosta/administração & dosagem , Esquema de Medicação , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Progesterona/sangue , Progesterona/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Zootaxa ; 4608(3): zootaxa.4608.3.11, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31717140

RESUMO

A new species of the genus Xenylla Tullberg, 1869 is described. The new species, Xenylla weinerae sp. nov., from China, resembles X. acauda Gisin, 1947, X. brevicauda Tullberg, 1869, X. caudata Jordana, 1993, X. lesnei Denis, 1935, X. nirae Gama Oliveira, 1994, X. wandae Queiroz Mendonça, 2016 and X. vanharteni Weiner et al., 2012 due to unique feature--six thickened cylindrical sensilla on antennal segment IV. X. weinerae sp. nov. having moderately modified chaetotaxy (b h1 h2 q t, head with dorsal setae l1 and l3 subequal, dorsal setae a3 on Abd. IV present) and well-developed furca (mucro separated from the dens, bearing 2 setae) is also similar to some populations of X. obscura Imms, 1912 sensu Thibaud et al. (2004). An identification key to Asian Xenylla species is provided.


Assuntos
Artrópodes , Animais , China , Cabeça , Urodelos
13.
Artigo em Inglês | MEDLINE | ID: mdl-31632347

RESUMO

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

14.
Reprod Domest Anim ; 54 Suppl 3: 46-52, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31512314

RESUMO

Inflammation and fibroproliferative diseases may be modulated by epigenetic changes. Therefore, we suggest that epigenetic mechanisms could be involved in equine endometrosis pathogenesis. DNA methylation is one of the methods to evaluate epigenetics, through the transcription of methyltransferases (DNMT1, DNMT3A, DNMT3B). The correlation between DNMTs and collagen (COL) transcripts was assessed for the different Kenney and Doig's (Current Therapy in Theriogenology. Philadelphia: WB Saunders; 1986) endometrium categories. Endometrial biopsies were randomly collected from cyclic mares. Histological classification (category I, n = 13; II A, n = 17; II B, n = 12; and III, n = 7) and evaluation of COL1A2, COL3A1 and DNMTs transcripts by qPCR, were performed. Data were analysed by one-way analysis of variance (ANOVA), Kruskal-Wallis test and Pearson correlation. As mares aged, there was an increase in endometrium fibrosis (p < .01), and in DNMT1 mRNA (p < .001). Considering DNMT3B transcripts for each category, there was an increase with fibrosis (p < .05). No changes were observed for DNMT1 and DNMT3A transcripts. However, DNMT3A mRNA levels were the highest in all categories (p < .01). In category I endometrium, a positive correlation was observed for transcripts of all DNMTs in both COLs (p < .01). In category IIA, this correlation was also maintained for all DNMTs transcripts in COL1A2 (p < .05), but only for DNMT3B in COL3A1 (p < .05). In category IIB, there was a positive correlation between DNMT3B and COL3A1 (p < .05). In category III, a positive correlation was only observed between DNMT3B and COL3A1 (p < .05). Our results suggest that there is a disturbance in COLs and DNMTs correlation during fibrosis.


Assuntos
Colágeno/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Endometrite/veterinária , Doenças dos Cavalos/metabolismo , Envelhecimento/fisiologia , Animais , Colágeno/genética , Metilação de DNA , Endometrite/genética , Endometrite/metabolismo , Endométrio/patologia , Feminino , Fibrose/fisiopatologia , Doenças dos Cavalos/genética , Cavalos , RNA Mensageiro
15.
Vet Immunol Immunopathol ; 209: 37-44, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30885304

RESUMO

Bacterial infections of the genital tract are the major cause of reproductive failure in the mares. MiRNAs are important regulators of gene expression, mostly through transcriptional and translational regression. We hypothesized that LPS induced aberrant expression of miRNAs and their targets, which are involved in regulation of uterine homeostasis. Three groups of primary endometrial epithelial and stromal cells, and endometrial tissue explants were cultured. The 1st group was kept as control, while the 2nd and 3rd groups were challenged with low (0.5 µg/mL) or high (3.0 µg/mL) doses of Lipopolysaccharides (LPS). Cell pellets and tissue explants were collected after 24 and 48 h, for total RNA isolation and qRT-PCR of the selected miRNAs and their targets. Culture media and cell lysates were collected after 24 and 48 h, for cytokines (IL6 and TNFα) and prostaglandins (PGE2 & PGFα2) measurement. Both endometrial cells expressed TLR4 and its accessory molecules (MyD88 & CD14) that are required for triggering inflammatory immune response after LPS, via up-regulation of TRAF6, TNFα, IL6 and IL8, compared to the respective control. After both doses of LPS challenge, miR-155, miR-223 and miR-17 were significantly increased; miR-181b, miR-21 and let-7a were significantly decreased compared to respective controls. Interestingly, miR-24 and miR-532-5p were clearly up-regulated after only the low LPS dose. TNFα, IL6 and PGs in culture media and from cell lysates revealed dose- and time-dependent patterns, after LPS. Results indicated that both epithelial and stromal cells have a primary role in innate immune response after LPS challenge, while this recognition occurred via TLR4 and its accessory molecules. Dysregulation of miRNAs and their targets expression after LPS might affect normal uterine function through perturbation of PG and cytokine secretion.


Assuntos
Endometrite/veterinária , Endométrio/imunologia , Doenças dos Cavalos/imunologia , Inflamação/genética , Inflamação/imunologia , MicroRNAs/fisiologia , Animais , Sobrevivência Celular , Endometrite/genética , Endometrite/imunologia , Endométrio/citologia , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doenças dos Cavalos/genética , Cavalos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , MicroRNAs/biossíntese , MicroRNAs/genética , Células Estromais/metabolismo , Receptor 4 Toll-Like/genética
16.
Theriogenology ; 128: 193-200, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776689

RESUMO

Necroptosis is an alternative form of programmed cell death regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent. In the present study, to clarify if necroptosis in luteal endothelial cells (LECs) participates and contributes for bovine luteolysis, we investigated RIPK1 and RIPK3 localization in luteal tissue and their expression in cultured LECs after treatment with selected immune factors - mediators of luteolytic action of prostaglandin F2α (PGF). In addition, effects of tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM), and/or nitric oxide donor - NONOate (100 µM) on viability and CASP3 activity in the cultured LECs were investigated. Furthermore, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 µM) on RIPKs and CASPs expression, were evaluated. Localization of RIPK1 and RIPK3 protein in the cultured LECs were determined. In cultured LECs, expression of RIPKs mRNA were up-regulated by TNF + IFNG at 12 h, and by PGF (1 µM) or NONOate at 24 h, respectively (P < 0.05). Although NONOate decreased cell viability, it prevented TNF + IFNG-stimulated CASP3 activity in cultured LECs. Nec-1 prevented TNF + IFNG-induced RIPK1 and CASP3 mRNA expression at 12 h and prevented RIPK3 mRNA expression. These findings suggest that RIPKs-dependent necroptosis which are induced by TNF + IFNG, PGF or NO could be potent mechanism responsible for LECs cell death and disappearance of luteal capillaries in regressing bovine CL.


Assuntos
Bovinos/fisiologia , Morte Celular/fisiologia , Células Endoteliais/citologia , Luteólise/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Corpo Lúteo/metabolismo , Feminino , Imuno-Histoquímica
17.
Domest Anim Endocrinol ; 67: 1-10, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30522057

RESUMO

Prostaglandin E2 (PGE2) has contradictory effects in many organs. It may have proinflammatory, anti-inflammatory, or anti-fibrotic roles, depending on the type of receptors to which it binds. By signaling through its receptors EP2 and EP4, PGE2 mediates anti-inflammatory and anti-fibrotic actions. In spite of chronic endometrial fibrosis (endometrosis) being a major cause of mare infertility, its pathogenesis is not fully understood. We have shown that contact of mare endometrium in vitro with neutrophil extracellular traps (NETs) proteases favors endometrial collagen type I production. Therefore, we investigated the involvement of the PGE2 pathway in collagen deposition in mare endometrium, challenged in vitro with proteases present in NETs. Mare endometria (Kenney and Doig categories I/IIA and IIB/III), obtained in the follicular phase (FLP) and mid-luteal phase (MLP), were incubated for 24 h with components found in NETs (elastase, cathepsin-G, and myeloperoxidase). Secretion of PGE2 and transcripts for specific PGE synthase (PGES) and PGE2 receptors (EP2 and EP4) were evaluated. Impaired PGE2 production and low EP2 transcript abundance depended on the endometrial category and estrous cycle phase. Impairment of PGE2 and/or EP2 might play a role in FLP (category IIB/III) and MLP (I/IIA) endometrial fibrogenesis because of the reduction in its antifibrotic capacity. In conclusion, priming of the endometrium with endogenous ovarian steroids might inhibit the antifibrotic PGE2 pathway either in healthy or pathologic tissues with collagen formation after NETs proteases action.


Assuntos
Dinoprostona/fisiologia , Endometriose/veterinária , Endométrio/patologia , Armadilhas Extracelulares/fisiologia , Doenças dos Cavalos/etiologia , Animais , Colágeno/metabolismo , Dinoprostona/biossíntese , Endometriose/etiologia , Endometriose/metabolismo , Endométrio/química , Endométrio/metabolismo , Ciclo Estral/fisiologia , Armadilhas Extracelulares/enzimologia , Feminino , Fibrose , Doenças dos Cavalos/patologia , Cavalos , Infertilidade Feminina/etiologia , Infertilidade Feminina/veterinária , Peptídeo Hidrolases/metabolismo , Prostaglandina-E Sintases/genética , RNA Mensageiro/análise , Receptores de Prostaglandina E/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos
18.
Reprod Domest Anim ; 53 Suppl 2: 66-69, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30238664

RESUMO

We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n = 8) and mid luteal-MLP (n = 7) phases explants were cultured for 24-48 hr with medium alone (Control), ELA (0.5 µg/ml,1 µg/ml), sivelestat - ELA inhibitor (INH,10 µg/ml), or ELA (0.5 µg/ml,1 µg/ml) + INH (10 µg/ml). COL1 gene transcription was done by qRT-PCR and PGE2 and PGF2 α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE2 production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF2 α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE2 (p < 0.05). PGF2 α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up-regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF2 α production (p < 0.05), but PGF2 α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE2 production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE2 and inhibiting pro-fibrotic PGF2 α.


Assuntos
Dinoprosta/metabolismo , Dinoprostona/metabolismo , Endométrio/efeitos dos fármacos , Cavalos/fisiologia , Elastase Pancreática/farmacologia , Animais , Colágeno/genética , Colágeno/metabolismo , Ciclo Estral , Feminino
19.
Theriogenology ; 116: 17-27, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29763784

RESUMO

During early pregnancy, uterine epithelial cells undergo major transformations in their cytoskeleton that make the endometrium receptive for conceptus attachment. Actin binding proteins (ABPs) such as cofilin, gelsolin, and vinculin are involved in regulating actin polymerization, severing or crosslinking actin to integrins. However, whether ABPs are involved in epithelial remodeling or embryo adhesion in pigs is unknown. Therefore, the expression and distribution of these proteins were investigated in porcine endometrium on Days 10 and 13 (pre-implantation period), and 16 (attachment phase) of the estrous cycle or pregnancy. While day and pregnancy status had no effect on ABP gene expression, the protein abundance of vinculin was significantly higher on Day 13 than on Day 10 (p < 0.05) of the estrous cycle, and its abundance was highest on Day 16 in the pregnant endometrium. Immunofluorescent staining showed alterations in the distribution of these proteins depending on the day of the estrous cycle or early pregnancy examined. Double immunofluorescent staining for the ABPs and actin revealed that while cofilin co-localized with actin in the apical epithelium on Days 13 and 16 of the estrous cycle, in pregnant animals, it was strongly associated with actin in the sub-epithelial stroma of the endometrium. Gelsolin was also co-localized with actin in the apical epithelium on Days 13 and 16 of the estrous cycle, but this association was absent in the pregnant endometrium. Vinculin co-localized with actin in the sub-epithelial stroma on Days 13 and 16 irrespective of the reproductive status, but was additionally associated with actin in the apical epithelium on Day 16 of pregnancy. Vinculin interacted with phosphorylated focal adhesion kinase in the endometrial epithelium, and the interaction was dependent on estradiol-17ß, a conceptus-secreted pregnancy-recognition factor in pigs. Furthermore, silencing vinculin in the endometrial epithelial cells negatively affected trophoblast adhesion to them. In conclusion, the influence of stage and reproductive status on the specific localization of actin and its binding proteins in the porcine endometrium suggests that they play a role in regulating the endometrial cytoskeleton. Moreover, vinculin may facilitate conceptus attachment to the epithelium by interacting with focal adhesion kinase.


Assuntos
Actinas/metabolismo , Implantação do Embrião , Proteínas dos Microfilamentos/metabolismo , Prenhez/metabolismo , Suínos , Actinas/fisiologia , Animais , Citoesqueleto , Endométrio/metabolismo , Epitélio/metabolismo , Feminino , Proteínas dos Microfilamentos/fisiologia , Gravidez , Útero/metabolismo
20.
Cytokine ; 110: 316-327, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29627157

RESUMO

In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p < 0.05), and downregulated mRNA and protein of StAR, CYP11A1, cPGES and mPGES1 (p < 0.05). Conversely, TGF augmented PGF2a concentration in culture media, through PTGS2 and PGFS gene expression activation (p < 0.05). When cells were incubated with PGF2a, both TGF and ALK5 were upregulated (p < 0.05). Additionally, treatment with the pharmacological inhibitor of ALK5, ALK4 and ALK7 - SB431542 (SB) attenuated PGF2a functional and structural luteolytic actions. Indeed, SB blocked: (i) PGF2a inhibitory effect on StAR, CYP11A1, 3BHSD and mPGES1; (ii) PGF2a auto-amplification signal via PTGS2 and PGFS expression (p < 0.05); (iii) the PGF2a-induced BAX and FASL expression (p < 0.05). Finally, TGF decreased cell viability (p < 0.05) and promoted caspase 3 activity (p = 0.08) and the expression of pro-apoptotic FASL and BAX (p < 0.05). Our results suggest that TGF supports functional regression and structural luteolysis, and also confirm the importance of ALK5, ALK4 and ALK7 activation during PGF2a mediated luteolysis in mares.


Assuntos
Sobrevivência Celular/fisiologia , Células Lúteas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Corpo Lúteo/metabolismo , Dinoprostona/metabolismo , Regulação para Baixo/fisiologia , Feminino , Expressão Gênica/fisiologia , Cavalos , Luteólise/metabolismo , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
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