Análise histopatológica, morfométrica e imunohistoquímica de cotilédones fetais ovinos oriundos de embriões transferidos coletados no momento do parto
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Data
2016-09-30
Autores
Lourenção, Josiane Adelaide Camargo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Devido a sua facilidade de manejo, a espécie ovina é um modelo experimental para estudos nas áreas de cirurgia, obstetrícia e reprodução animal. A prática da transferência de embrião vem sendo aperfeiçoada visando a seleção de animais geneticamente superiores. A estrutura placentária permite trocas gasosas, metabólicas e transporte de nutrientes, tornando-se responsável pelo desenvolvimento fetal. O presente estudo teve como objetivo relacionar a técnica de TE às alterações morfológicas e analisar receptores de estrógeno alfa (ER-α), beta (ER-β) e progesterona (PR). Foram utilizados 20 animais, os quais foram divididos em dois grupos, 10 (TE) e 10 (controle). Foram colhidos 4 cotilédones de cada animal e o material foi fixado em formalina tamponada com pH 7,4 para inclusão em Paraplast® e glutaraldeído para historesina. As amostras foram seccionadas a 4µm e coradas em HE, que após análise prévia foram encaminhadas para coloração específica, PAS, tricômico de masson, von kossa e Perl. Para a análise morfométrica utilizou-se três amostras de cada animal de ambos os grupos, analisados da base ao ápice da microviloside no aumento de 400X em 10 campos. Para o estudo de receptores, seis cotilédones de seis animais de ambos os grupos foram selecionados, analisados e contados por três observadores em 10 campos por amostra. No presente estudo observou-se trombo arterial e venoso, esclerose, aderência de fibrina, espessamento endotelial, hialinização de vasos e infiltrados inflamatórios, tais alterações foram evidentes nas árvores vilosas primárias do grupo TE. Na região do epitélio cotiledonário, encontrou-se acúmulo de hemossiderina, células trofoblásticas binucleadas (CTBs), micronúcleo, condensação citoplasmática e nuclear nas CTBs, e projeções citoplasmáticas apicais. Ainda no epitélio observou-se presença de fosfato de cálcio, os quais também foram identificados na região do cório, também no espaço interviloso foi observado sangue extravasado. Essas características foram encontradas em ambos os grupos. Entretanto, na análise morfométrica houve diferença estatística entre as medidas M2 e M3 (p<0,05), porém entre os grupos não houve diferença estatística. Para os receptores estudados, apenas o ER-α mostrou-se ausente em todo epitélio trofoblástico no grupo TE; já no grupo controle a imunorreatividade foi fraca nas CTBs, porém positiva (p<0,001). O ER-β e PR foram imunoexpressos em ambos os grupos, os quais não mostraram diferença estatística. Também constatou-se decréscimo das CTBs no grupo TE, porém essa característica foi observada independente dos receptores estudados (p=0,013). Podemos concluir que as alterações vasculares, bem como os infiltrados inflamatórios, são sugestivas de placentite, e as alterações encontradas no epitélio são condizentes à maturidade e expulsão da placenta no momento do parto. A diferença entre ER-α e ER-β possivelmente estão relacionadas aos mecanismos fisiológicos no momento do parto, e a ausência de ER-α supostamente está associada aos receptores de PR, sugerindo uma regulação hormonal negativa.
Due to its easy management, the ovine species are na experimental model for studies in areas such as surgery, obstetrics and animal reproduction. The embryo transfer practice has be perfected saiming the selecton of genetically superior animals. The placental structures enables gas exchanges, metabolic exchanges and transporto of nutrientes, becoming responsible for the fetal development. The objective of this study was to relat the ET technique to the morphological changes, and to analyze estrogen alpha (ER-α), beta (ER-β) and progesterone (PR) receptors. 20 animals divided in two groups, 10 (ET) and 10 (control), were used 4 cotyledons from each animal were collected and the material was fixed in buffered formaline with pH 7,4 for inclusion in Paraplast® and glutaraldehyde for historesin. The samples were sectioned in 4 µm and stained with HE, which after previous analyses were forwarded to specific staning, PAS, masson’s trichrome, von kossa and Perl. For the mophometric analyses, three samples of each animal form both groups were used, they were analysed from base to the apex of the microvillous at the magnification of 400X ib 10 fields. For the receptors study, six cotyledons of six animals from both groups were used, they analyzed and counted in 10 fields per sample by three observers. In this study, arterial and venous thrombus, sclrerosis, fibrina adhesion, endotelial thickening, hyalinizing vessels and inflammatory infiltrades, were observed, such alterations were apparent in primary villous trees from ET group. In cotyledondary epithelium region, accumulation of hemosiderin, binucleate trophoblastic cells (BTC), micronucleus, cytoplasmatic and nuclear presence of calcium phosphate was observed in the epithelium and wich was also indentified in the corium region, and spilled blood was observed in the intervillous space. Those characteristics were found in both groups. However, there were statical diferences between the measurements M2 and M3 (p<0,05) in the morphometric analysis, although there was no statistical difference between the groups. For the receptor studied, only ER-α was absent from the trophoblastic epithelium in the ET group; in the control group, the immunoreactivity was weak in the BTC, although positive (p<0,001). ER-α and PR were immunoexpressed in both groups, wich did no show statistical difference. A decrease of BTC was detected in the ET group, this characteristic was observed regardless of the receptors studied (p=0,013). We can conclude that vascular alterations, as well as inflammatory infiltrades, suggest placentitis, and the alterations found in the epithelium are consistente with maturity and expulsion of the placenta at moment of the birth. The difference between ER-α and ER-β is possibly related to physiological mechanisms at the moment of parturition, and absence of ER-α is supposedly associated to PR receptors, suggesting down-regulation.
Due to its easy management, the ovine species are na experimental model for studies in areas such as surgery, obstetrics and animal reproduction. The embryo transfer practice has be perfected saiming the selecton of genetically superior animals. The placental structures enables gas exchanges, metabolic exchanges and transporto of nutrientes, becoming responsible for the fetal development. The objective of this study was to relat the ET technique to the morphological changes, and to analyze estrogen alpha (ER-α), beta (ER-β) and progesterone (PR) receptors. 20 animals divided in two groups, 10 (ET) and 10 (control), were used 4 cotyledons from each animal were collected and the material was fixed in buffered formaline with pH 7,4 for inclusion in Paraplast® and glutaraldehyde for historesin. The samples were sectioned in 4 µm and stained with HE, which after previous analyses were forwarded to specific staning, PAS, masson’s trichrome, von kossa and Perl. For the mophometric analyses, three samples of each animal form both groups were used, they were analysed from base to the apex of the microvillous at the magnification of 400X ib 10 fields. For the receptors study, six cotyledons of six animals from both groups were used, they analyzed and counted in 10 fields per sample by three observers. In this study, arterial and venous thrombus, sclrerosis, fibrina adhesion, endotelial thickening, hyalinizing vessels and inflammatory infiltrades, were observed, such alterations were apparent in primary villous trees from ET group. In cotyledondary epithelium region, accumulation of hemosiderin, binucleate trophoblastic cells (BTC), micronucleus, cytoplasmatic and nuclear presence of calcium phosphate was observed in the epithelium and wich was also indentified in the corium region, and spilled blood was observed in the intervillous space. Those characteristics were found in both groups. However, there were statical diferences between the measurements M2 and M3 (p<0,05) in the morphometric analysis, although there was no statistical difference between the groups. For the receptor studied, only ER-α was absent from the trophoblastic epithelium in the ET group; in the control group, the immunoreactivity was weak in the BTC, although positive (p<0,001). ER-α and PR were immunoexpressed in both groups, wich did no show statistical difference. A decrease of BTC was detected in the ET group, this characteristic was observed regardless of the receptors studied (p=0,013). We can conclude that vascular alterations, as well as inflammatory infiltrades, suggest placentitis, and the alterations found in the epithelium are consistente with maturity and expulsion of the placenta at moment of the birth. The difference between ER-α and ER-β is possibly related to physiological mechanisms at the moment of parturition, and absence of ER-α is supposedly associated to PR receptors, suggesting down-regulation.
Descrição
Palavras-chave
Placenta, Epitélio coriônico, Vascularização, Células binucleadas, Receptores hormonais e ovelhas, Chorionic epithelium, Vascularization, Binucleate cells, Hormonal receptors and sheep