Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos / Caracterization of caveolin -1 and -2 proteins in cloned and transgenic placenta of cattle

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Caveolinas y modulación del sistema del óxido nítrico en el hipotiroidismo según avanza la edad / Age-related Influence of Hypothyroidism on Caveolins and Nitric Oxide Modulation

Introducción El hipotiroidismo y la edad impactan sobre la producción de óxido nítrico (NO) cardíaco y renal. Las caveolinas, moduladores negativos de la actividad enzimática de la NO sintetasa (NOS), se afectan con ambos factores. Objetivos Evaluar la implicación de las caveolinas (cav) en la modulación de la actividad de la NOS cardíaca y renal en animales hipotiroideos adultos. Material y métodos Se utilizaron ratas macho Sprague-Dawley eutiroideas e hipotiroideas [metimazol 0,02% (v/v) en el agua de bebida durante 28 días]. Los animales fueron sacrificados para extraer el corazón y los riñones. Resultados La actividad de la NOS en la aurícula derecha disminuyó con la edad y el hipotiroidismo. La expresión de cav-1 aumentó con la edad y el hipotiroidismo. La actividad de la NOS en el ventrículo izquierdo aumentó con el avance de la edad y el hipotiroidismo. La expresión de ambas caveolinas disminuyó en los grupos adulto e hipotiroideo. En la médula renal, el hipotiroidismo disminuyó la actividad de la NOS en jóvenes y la aumentó en adultos. La expresión de cav-1 disminuyó con la edad y en jóvenes hipotiroideos. Los niveles proteicos de cav-3 disminuyeron en animales adultos hipotiroideos. Conclusiones El hipotiroidismo impacta sobre la actividad de la NOS y de sus moduladores, las caveolinas, en el sistema cardiovascular y renal. El hipotiroidismo intensifica los efectos del avance de la edad en ambos sistemas.

Introduction Hypothyroidism and age impact on cardiac and renal nitric oxide (NO) production. Caveolins, which are negative modulators of NO synthase (NOS) activity, are affected by both factors. Objectives The aim of this study was to evaluate caveolin (CAV) participation in the modulation of renal and cardiac NOS activity in adult hypothyroid animals. Methods Euthyroid and hypothyroid [methimazole 0.02% (v/v) in the drinking water during 28 days] male Sprague-Dawley rats were used. Animals were sacrificed to remove the heart and kidneys. Results Right atrial NOS activity decreased with age and hypothyroi-dism. Caveolin-1 expression increased with age and hypothyroidism. Conversely, left ventricular NOS activity increased with aging and hypothyroidism and the expression of both CAV isoforms decreased in adult and hypothyroid groups. In the renal medulla, hypothyroidism reduced NOS activity in young and raised it in adult animals and CAV-1 expression decreased with age and in hypothyroid young animals. Caveolin-3 protein levels decreased in adult hypothyroid animals. Conclusions Hypothyroidism impacts on NOS activity and on that of its modulators, caveolins, in the cardiovascular and renal systems. Hypothyroidism enhances the effects of aging in both systems.