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Master's Dissertation
DOI
10.11606/D.10.2014.tde-08012015-133713
Document
Author
Full name
Antenor Pereira Bonfim Neto
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Papa, Paula de Carvalho (President)
Binelli, Mario
Lopes, Maria Denise
Title in Portuguese
O papel do 17β-estradiol no processo luteolítico de cadelas não prenhes
Keywords in Portuguese
Cadela
Corpo lúteo
Diestro
Estradiol
Luteólise
Abstract in Portuguese
O 17β-estradiol (E2) desempenha um papel importante na função reprodutora e na fertilidade feminina, porém, sua ação é estendida para a maioria dos tecidos. Sabendo que o E2 tem funções pleiotrópicas em diferentes tecidos e órgãos, e que pode estar envolvido tanto na proliferação como na morte celular, nossa hipótese é que o E2 seja um dos iniciadores de regressão luteínica em cadelas não prenhes. Para testar nossa hipótese, foram usados corpos lúteos (CL) provenientes de 28 cadelas nos dias 10, 20, 30, 40, 50, 60, 70 e >70 após a ovulação (n = 4/grupo) para os experimentos ex vivo. Nesta etapa foram analisadas as proteínas pró-apoptóticas (CASPASES, 3, 8, 9 e BAX) por imuno-histoquímica e western blotting, além da expressão dos genes CASP3, 8, 9, BAX, FAS, MKI67, ESR1, ESR2m por PCR em tempo real. Na segunda etapa deste trabalho, utilizamos CL de 12 cadelas nos dias 20, 40 e 60 após a ovulação (n=4 por grupo), cujas células foram cultivadas e divididas em seis tratamentos: Controle, E2 (tratado com E2), bloqueador de ERα (tratado com MPP), bloqueador de ERβ (tratado com PHTPP), E2 + bloqueador de ERα (tratado com E2 + MPP) e E2 + bloqueador de ERβ (tratado com E2 + PHTPP). Foram avaliados os mesmos genes do experimento ex vivo, bem como os genes CYP19A1, CYP11A1, HSD3B1 e SLC2A4. De modo geral a expressão dos fatores pró-apoptóticos foi mais alta a partir do dia 40 e atingiu valores máximos nos dias 60 e 70 após a ovulação, assim como a expressão de ERS2. Essa correlação foi observada também nas células do grupo E2 + bloqueador de ERα, que também apresentaram regulação negativa de HSD3B1. Quando do bloqueio do ERβ, as células luteínicas responderam com aumento dos genes relacionados à esteroidogênese e à proliferação celular, principalmente quando oriundas dos dias 20 e 40 p.o. Dessa forma, conclui-se que o bloqueio do ERα levou ao aumento dos genes pró-apoptóticos, e o bloqueio do ERβ possibilitou aumento dos genes luteotróficos. Estes achados confirmam o papel pleiotrópico do estradiol no CL canino e incluem este hormônio, assim como o balanço entre seus receptores, dentre os atores principais da regulação da meia vida do CL canino.
Title in English
The role of 17b-estradiol in the luteolytic process of non-pregnant bitches
Keywords in English
Bitch
Corpus luteum
Diestrus
Estradiol
Luteolysis
Abstract in English
The 17β-estradiol (E2) plays an important role in the reproductive function and female fertility, however, its action is extended to most tissues. Knowing that E2 has pleiotropic roles in different organs and tissues, and that might be involved in both cell death and proliferation, our hypothesis is that E2 is one of the triggers of luteal regression in non-pregnant bitches. To test our hypothesis, corpora lutea (CL) from 28 dogs on days 10, 20, 30, 40, 50, 60, 70 and >70 after ovulation (n = 4/group) were used for ex vivo experiments. Pro-apoptotic proteins expression (CASPASES, 3, 8, 9 e BAX) were analyzed by immunohistochemistry and western blotting, and the gene expression of CASP3, 8, 9, BAX, FAS, MKI67, ESR1 and ESR2 were analyzed by real-time PCR. In the second step of this study, CL from 12 bitches on days 20, 40 and 60 (n = 4 per group) were used. Luteal cells were isolated and divided into six treatments: Control, E2 (treated with E2), ERα Blocker (treated with MPP), ERβ Blocker (treated with PHTPP), ERα + E2 Blocker (treated with E2 + MPP) and E2 + ERβ Blocker (treated with E2 + PHTPP). The expression of the same genes from the in vivo experiment was evaluated, as well as that of CYP19A1, CYP11A1, HSD3B1 and SLC2A4. In general, the expression of pro-apoptotic factors was higher from day 40 and reached highest expression on days 60 and 70 after ovulation, coinciding with increasing in expression of ERS2. This correlation was also observed in the cells from the group E2 + ERα blockade, which also showed HSD3B1down-regulation. When ERβ was blocked, cells of days 20 and 40 post ovulation responded increasing the expression of steroidogenesis and proliferation related genes. Thus, we deduce that the ERα blockade promotes the increase of pro-apoptotic and that o ERβ of luteotrophic genes expression. Our findings confirm the pleiotropic role of estradiol in canine CL and include this hormone, as well as the balance between its two receptors, among the factors controlling canine CL lifespan.
 
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Publishing Date
2015-03-26
 
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