RESUMEN
The aim of these studies was to investigate the involvement of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.
Asunto(s)
AMP Cíclico , Aparato Lagrimal , Ratones , Animales , AMP Cíclico/metabolismo , Adenilil Ciclasas/metabolismo , Oxitocina/farmacología , Oxitocina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Aparato Lagrimal/metabolismo , Proteínas de Unión al GTP/metabolismo , Músculo Liso , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismoRESUMEN
Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Methods: Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. Results: The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1ß or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. Conclusions: SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.
Asunto(s)
Inflamación , Aparato Lagrimal , Osteonectina , Animales , Ratones , Ratones Endogámicos NOD , Microscopía Fluorescente , Osteonectina/genética , Factores de EdadRESUMEN
In the lacrimal gland, myoepithelial cells (MEC) express muscle contractile proteins such as alpha smooth muscle actin (SMA) and calponin and therefore can contract to help expel lacrimal fluid. In a previous study, we demonstrated that lacrimal gland MEC express the oxytocin receptor (OXTR) and they contract under oxytocin (OXT) stimulation. Using NOD and MRL/lpr mice (animal models of Sjogren's syndrome), we reported a decrease in SMA and calponin protein levels plus a decline in acini contraction after stimulation with OXT. It is known that proinflammatory cytokines, such as interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ), can affect OXTR expression and signaling capacity and inhibit MEC contraction. The aim of the current study was to investigate if proinflammatory cytokines are implicated in the loss of MEC contractile ability. Thus, lacrimal gland MEC from a SMA-GFP transgenic mouse were treated with IL-1ß (10 ng/ml) for a total of 7 days. At days 0, 2, 4 and 7, GFP intensity, cell size/area, contractile proteins amounts and MEC contraction were assessed. At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing after day 4 (25%; p=0.007) and 7 (30%; p=0.0001). Mean cell area was also reduced at day 2 (34%; p=0.0005), and after 4 (51%; p<0.0001) and 7 days (30%; p=0.0015). The contraction assay at day 2 showed a 70% decrease of contraction in treated MEC (p<0.0001), 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 when compared to control. Levels of contractile proteins were measured on day 7 showing a decrease in SMA and calponin amount in treated MEC compared with the control group (around 30%; p=0.0016 and p=0.0206; respectively). Similar results were observed when TNF-α and IFN-γ were added along with IL-1ß. Taken together the present data and those from our previous studies with Sjogren's syndrome mouse models, they strongly suggest that proinflammatory cytokines affect lacrimal gland MEC contractile ability that may account for the reduced tear secretion associated with Sjogren's syndrome dry eye disease.
RESUMEN
Purpose: We reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP3) which increases intracellular calcium concentration ([Ca2+]i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC. Methods: Tear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca2+]i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. Results: OXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10-7 M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca2+]i with a maximum response at 10-6 M. Furthermore, the activation of the IP3 receptor to increase [Ca2+]i is crucial for OXT-induced MEC contraction since blocking the IP3 receptor with 2-APB completely abrogated this response. Conclusions: We conclude that OXT uses the PLC/Ca2+ pathway to stimulate MEC contraction and increase lacrimal gland secretion.
Asunto(s)
Calcio/metabolismo , Aparato Lagrimal/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Oxitócicos/farmacología , Oxitocina/farmacología , Fosfolipasas de Tipo C/fisiología , Actinas/metabolismo , Animales , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/metabolismo , Aparato Lagrimal/diagnóstico por imagen , Aparato Lagrimal/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía de Fluorescencia por Excitación Multifotónica , Reacción en Cadena en Tiempo Real de la Polimerasa , Lágrimas/fisiologíaRESUMEN
The present study focuses on the effects of E2 and EE2 environmental concentrations on different components of the reproductive axis of pejerrey (Odontesthes bonariensis), a native fish species from Pampas lakes of Argentina. The results obtained demonstrated that E2 and EE2 separate or mixed, could disrupt key pathways of the pejerrey Brain-Pituitary-Gonadal axis. First, it was observed that at the brain level, gnrh-III and cyp19a1b mRNA expression increased significantly in the exposed fish. Secondly, in the pituitary fshb and lhb mRNA expression levels, the study did not show any differences between treated and control groups. Thirdly, fshr and lhcgr transcript levels showed a significant decrease at testicular level. Nevertheless, testosterone plasmatic levels remained unchanged in exposed fish. In addition, in a histological analysis, it was possible to find pyknotic nuclei in estrogen only on treated fish testis linked to a reduction in the GSI index and a decrease in the length of spermatogenic lobules. All these findings highlighted the fact that environmental concentrations of E2, EE2 and their mixture disrupted the endocrine-reproductive axis of pejerrey, being the testis the main direct target.
Asunto(s)
Disruptores Endocrinos/toxicidad , Estradiol/toxicidad , Etinilestradiol/toxicidad , Hipófisis/efectos de los fármacos , Smegmamorpha/metabolismo , Testículo/efectos de los fármacos , Animales , Argentina , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Expresión Génica/efectos de los fármacos , Hormonas Esteroides Gonadales/genética , Masculino , Hipófisis/metabolismo , Smegmamorpha/genética , Espermatogénesis/efectos de los fármacos , Testículo/metabolismoRESUMEN
17ß-Estradiol (E2) and synthetic 17α-Ethinylestradiol (EE2) are estrogenic compounds present in surface waters as a consequence of municipal sewage discharges. The aim of this study was to evaluate the effects of E2, EE2 and its mixtures on different reproductive parameters and embryo-larval survival in pejerrey fish (Odontesthes bonariensis). In order to analyze the effect of these compounds on sperm quality, fertilization%, embryo-larval survival (%), and the point of no return (PNR), different assays were performed using concentrations 175, 350, 700 and 1400 ng/L of E2; 22.5, 45, 90 and 180 ng/L of EE2 and mixtures M1 (175 E2+22.5 EE2, ng/L), M2 (350 E2+45 EE2, ng/L), M3 (700 E2+90 EE2, ng/L) and M4 (1400 E2+180 EE2 ng/L). No significant differences in motility parameters were observed between E2 and EE2 treatments and the control group. However, a significant decrease in motility% was recorded for all mixtures tested compared with the control samples. For fertilization%, only sperm activated with M4 showed a significant decrease compared with the control group. In the case of embryo survival, there was only a significant decrease in the highest concentration of EE2 compared with the control group. For the mixtures, M3 is the one that had the most adverse effect on embryo survival. In larval survival, there was a significant decrease in concentration 175 and 700 ng/L of E2 compared with the control group. In EE2 treatments, the ones with a significant reduction in larval survival were concentration 45 and 90 ng/L. And for the mixture treatments, M1, M3 and M4 had a significantly lower larval survival than the control group. In comparison to other treatments, M1 demonstrated a significant difference in PNR when compared with the control group. The results obtained demonstrated that the exposure to mixtures of E2 and EE2 affected fish sperm motility, fertilization% and, embryo and larval survival even at relevant environmental concentrations highlighting the necessity of considering the effects of pollutants mixtures in ecotoxicological studies.
Asunto(s)
Embrión no Mamífero/efectos de los fármacos , Estradiol/toxicidad , Etinilestradiol/toxicidad , Fertilización/efectos de los fármacos , Smegmamorpha/fisiología , Espermatozoides/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Masculino , Reproducción/efectos de los fármacos , Aguas del Alcantarillado/química , Motilidad Espermática/efectos de los fármacosRESUMEN
Juveniles of pejerrey, Odontesthes bonariensis, were exposed to 0.1% Alizarin Red S (ARS) alone or with a previous immersion in 2.2% saline solution (Osmotic Induction, OI) to enhance the ARS marking method. Fish were marked in the field and immediately released in 1 m3 cages in "La Salada de Monasterio" lagoon, Chascomús, Buenos Aires , Argentina. After 73 days, clear marks were observed in the otoliths, caudal fin rays and scales with both treatments, being the intensity of the signal in the scales of OI+ARS treated fish higher. On the other hand, no marks were observed in the control group on the same structures. Approximately one year post-treatment (385 days), only marks in caudal fin rays were found clearly in OI+ARS treated fish. After this period, no significant differences in total length or weight between marked or control fish were observed and the mortality ranged between 30-40 % in all cages. These results provide strong evidence for the potential applicability of this cost-effective marking technique in differentiation of wild and hatchery-produced pejerrey. The success in the caudal fin rays marking is also important because it is easy to do and does not require the sacrifice of fish.
Juvenis de pejerrey, Odontesthes bonariensis, foram expostos a Vermelho de Alizarina S (ARS) 0,1% de duas formas, sozinho ou com uma imersão anterior em 2,2% de solução salina (Indução osmótica, IO) para melhorar o método de marcação ARS. Os procedimentos foram realizados no campo e os peixes foram liberados em gaiolas (tanques-rede) de 1 m3 na lagoa "La Salada de Monasterio", Chascomús, Buenos Aires, Argentina. Após 73 dias, marcas claras foram observadas nos otólitos, raios da nadadeira caudal e escamas em ambos os tratamentos, sendo que a intensidade do sinal nas escalas de IO + ARS de peixe tratado foi superior. Por outro lado, não foram observados marcas no grupo controle sobre as mesmas estruturas. Aproximadamente um ano pós-tratamento (385 dias), apenas marcas nos raios da nadadeira caudal foram encontrados claramente nos peixes tratados com IO+ARS. Entre os peixes observados, após este período, não houve diferenças significativas no comprimento total ou peso entre o grupo controle e marcados, ademais, a mortalidade variou entre 30-40% em todas as gaiolas. Estes resultados fornecem fortes evidências e um grande potencial para aplicação desta técnica rentável de marcação que diferencia o pejerrey selvagem e o produzido em cativeiro. O sucesso na marcação dos raios da nadadeira caudal também é de grande importância, pois sua verificação é fácil e não requer o sacrifício de peixes.
Asunto(s)
Animales , Colorantes/administración & dosificación , Peces/crecimiento & desarrollo , Sistemas de Identificación Animal , Aletas de Animales , Explotaciones PesquerasRESUMEN
In the present study it was showed for the first time the ultrastructural morphology of O. bonariensis sperm using electron microscopy techniques. Different kinds of abnormalities were described in fresh and post thawed sperm caused by crogenic protocols. Pejerrey spermatozoon is uniflagellated and is differentiated into three parts: a small roundish head (~1.80µm in length and 1.67µm in width), a midpiece or transitional region (~1.11µm in length and 1.56µm in width), and a long tail or flagellum (~29.08µm). Samples of fresh and post thawed sperm showed evidence of morphological anomalies affecting various intracellular compartments. Spermatozoa with swollen, ruptured, or absent membranes in the head showing excess of cytoplasm, and with alteration of the spatial orientation of the mitochondria were observed. A swollen flagellum was observed containing cytoplasmic vesicles, distributed along the whole length or concentrated in a restricted part of the tail. It was also found a high level of abnormalities (60%) in frozen sperm when compared with normal sperm (18%) reflecting the damage provoked by cryopreservation procedures.
No presente estudo mostrou-se pela primeira vez a morfologia estrutural dos espermatozoides de O. bonariensis utilizando técnicas de microscopia eletrônica. Diferentes tipos de anormalidades foram descritas para sêmen fresco e descongelado. O espermatozoide de Pejerrey é uniflagelado e dividido em três partes: uma cabeça pequena e arredondada (~1.80µm de comprimento e 1.67µm de largura), uma parte intermediária ou região de transição (~1.11µm de comprimento e 1.56µm de largura) e uma cauda longa ou flagelo (~29.08µm). Amostras de sêmen fresco e descongelado mostraram evidências de anormalidades morfológicas afetando vários compartimentos intracelulares. Na cabeça haviam espermatozoides com membranas dilatadas, rompidas ou ausentes, mostrando excesso de citoplasma e alteração na orientação espacial das mitocôndrias. Um flagelo dilatado foi observado contendo vesículas citoplasmáticas, as quais estavam distribuídas ao longo de todo o seu comprimento ou concentradas em uma parte restrita da cauda. Também foi encontrado um alto nível de anormalidades (60%) em sêmen congelado em comparação com o sêmen normal (18%), refletindo os danos provocados pelos procedimentos de criopreservação.