PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Fonseca V.U., Papa P.C. & Campos D.B. 2012. [Potential involvement of adinopectin and its receptors in the modulation of steroidogenesis in corpus luteum of bitches during diestrus.] Potencial envolvimento da adiponectina e seus receptores na modulação da esteroidogênese em corpo lúteo de cadelas ao longo do diestro. Pesquisa Veterinária Brasileira 32(10):1055-1060. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva 87, SP 05508-270, Brazil. E-mail: uemura@usp.br In the estrous cycle of bitches, the luteal phase or diestrus includes a period ranging from 60 to 100 days in non-pregnant animals, characterized by elevated serum progesterone during the first 20 days post-ovulation (p.o). Adiponectin is the most abundant protein secreted by adipose tissue, but plasma concentration decreases significantly in metabolic disorders like insulin resistance and diabetes mellitus type 2, described as related changes in some bitches in diestrus. The aim of this study was to determine the expression and immunolocalization of the adiponectin system (adiponectin, and adipoR1 adipoR2) in the corpus luteum during diestrus, and correlate it to hormonal profile of 17β-estradiol and progesterone, as well as the expression of a gene target of the system, the PPAR-γ. For the study, corpora lutea were collected from 28 dogs during ovariosalpingohysterectomy on days 10, 20, 30, 40, 50, 60 and 70 post ovulation (day zero of ovulation was considered the day when the plasma progesterone concentration reached 5ng/mL). The corpora lutea were evaluated by immunohistochemistry for adiponectin, adipoR1 and adipoR2 and mRNA expression of PPAR-γ by real-time PCR. Statistical analysis of gene expression was performed with ANOVA followed by Newman-Keuls multiple comparisons. Adiponectin positive signal was stronger during the first 20 days p.o, time of the regency of progesterone; there was a gradual adiponectin and progesterone decline after this period, coinciding with the rise of 17β-estradiol, whose peak was near the 40 days p.o. The markedly adiponectin decrease occurred after 50 days p.o. The signal of adipoR1 was markedly evident at 40 days p.o and that of adipoR2 up to 50 days p.o, declining afterwards. We observed higher expression of PPAR-γ gene at 10, 30 and 70 days p.o. These results show that adiponectin and its receptors protein expression is altered during the diestrus and that these changes may be related to hormonal changes and expression of PPAR-γ, participating in the physiological mechanism of development, maintenance, activity and luteal regression in bitches.

• Corpus luteum adiponectin adipoR1 adipoR2 PPAR-γ Corpo lúteo adiponectina

Abstract in Portuguese:

RESUMO.- Fonseca V.U., Papa P.C. & Campos D.B. 2012. [Potential involvement of adinopectin and its receptors in the modulation of steroidogenesis in corpus luteum of bitches during diestrus.] Potencial envolvimento da adiponectina e seus receptores na modulação da esteroidogênese em corpo lúteo de cadelas ao longo do diestro. Pesquisa Veterinária Brasileira 32(10):1055-1060. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva 87, SP 05508-270, Brazil. E-mail: uemura@usp.br No ciclo estral de cadelas a fase luteínica, denominada diestro, compreende um período que varia de 60 a 100 dias em animais não-prenhes, caracterizado pela elevação plasmática de progesterona nos primeiros 20 dias pós ovulação (p.o). A adiponectina é a mais abundante proteína secretada pelo tecido adiposo, porém sua concentração plasmática diminui significativamente em alterações metabólicas como resistência insulínica e Diabetes mellitus tipo 2, alterações descritas como relacionadas em algumas cadelas com o período de diestro. O objetivo do estudo foi determinar a expressão e imunolocalização do sistema adiponectina (adiponectina e seus receptores, adipoR1 e adipoR2) no corpo lúteo de cadelas ao longo do diestro, correlacionando-o ao perfil hormonal de 17β-estradiol e progesterona, assim como à expressão de um dos genes alvo do sistema, o PPAR-γ. Para realização do estudo foram coletados corpos lúteos de 28 cadelas durante ovariosalpingohisterectomia de eleição nos dias 10, 20, 30, 40, 50, 60 e 70 pós ovulação (o dia zero da ovulação foi considerado aquele no qual a concentração plasmática de progesterona atingiu 5ng/mL). Os corpos lúteos foram avaliados por imunohistoquímica para adiponectina e seus receptores e a expressão do RNAm do PPAR-γ por PCR em tempo real. A análise estatística da avaliação gênica foi realizada com o teste ANOVA, seguido por comparação múltipla Newman-Keuls. O sinal da adiponectina apresentou-se mais intenso até os primeiros 20 dias p.o, momento de regência da progesterona; houve queda gradativa após este período, coincidindo com a ascensão do 17β-estradiol, cujo pico foi notado próximo do dia 40 p.o. A queda marcante da adiponectina ocorreu após 50 dias p.o. O sinal do adipoR1 mostrou-se bem evidente até os 40 dias p.o e o do adipoR2 até os 50 dias p. o, decaindo posteriormente. Foi observada maior expressão do gene PPAR-γ aos 10, 30 e 70 dias p.o. Estes resultados mostram que a expressão protéica da adiponectina e de seus receptores se altera ao longo do diestro e que estas alterações podem estar relacionados às alterações hormonais e expressão do PPAR- γ, participando do mecanismo fisiológico de desenvolvimento, manutenção, atividade e regressão luteínica em cadelas.

Abstract in English:

ABSTRACT.- Miranda-Moura M.T.M., Fonseca V.U., Silva N.B., Freitas M.L., Almeida O.B., Rocha H.A.O., Papa P.C. & Moura C.E.B. 2010. Morphological features and vascularization study of caprine cyclic corpus luteum. Pesquisa Veterinária Brasileira 30(4):351-357. Departamento de Morfologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Av. Salgado Filho s/n, Campus Lagoa Nova, Natal, RN 59072-970, Brazil. E-mail: mouraeduard@cb.ufrn.br Corpus luteum is a temporary endocrine gland that regulates either the estrous cycle and pregnancy. It presents extreme dependency on the adequate blood supply. This work aims to evaluate goat corpus luteum (CL) vascular density (VD) over the estrous cycle. For that purpose, 20 females were submitted to estrus synchronization/ovulation treatment using a medroxyprogesterone intra-vaginal sponge as well as intramuscular (IM) application of cloprostenol and equine chorionic gonadotrophine (eCG). After sponge removal, estrus was identified at about 72hs. Once treatment was over, female goats were then subdivided into 4 groups (n=5 each) and slaughtered on days 2, 12, 16 and 22 after ovulation (p.o). Ovaries were collected, withdrawn and weighted. CL and ovaries had size and area recorded. Blood samples were collected and the plasma progesterone (P4) was measured through RIA commercial kits. The VD was 24.42±6.66, 36.26±5.61, 8.59±2.2 and 3.97±1.12 vessels/mm2 for days 2, 12, 16 and 22 p.o, respectively. Progesterone plasma concentrations were 0.49±0.08, 2.63±0.66, 0.61±0.14 and 0.22±0.04ng/ml for days 2, 12, 16 e 22 p.o, respectively. Studied parameters were affected by the estrous cycle phase. Values greater than 12 p.o were observed. In the present work we observed that ovulation occurred predominantly in the right ovary (70% of the animals), which in turn presented bigger measures than the contra lateral one. There is a meaningful relationship between the weight and size of the ovary and these of CL (r=0.87, r=0.70, respectively, p<0.05). It is possible to conclude that morphology of goat’s ovaries and plasma progesterone concentration changed according to estrous cycle stages. We propose these parameters can be used as indicators of CL functional activity.

• Corpus luteum progesterone corpo luteo progesterona

Abstract in Portuguese:

RESUMO.- Miranda-Moura M.T.M., Fonseca V.U., Silva N.B., Freitas M.L., Almeida O.B., Rocha H.A.O., Papa P.C. & Moura C.E.B. 2010. Morphological features and vascularization study of caprine cyclic corpus luteum. Pesquisa Veterinária Brasileira 30(4):351-357. Departamento de Morfologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Av. Salgado Filho s/n, Campus Lagoa Nova, Natal, RN 59072-970, Brazil. E-mail: mouraeduard@cb.ufrn.br O corpo lúteo é uma glândula endócrina temporária que regula tanto o ciclo estral quanto a prenhez, apresentando extrema dependência de aporte sanguíneo adequado. Objetivaram-se avaliar mudanças morfométricas dos ovários e densidade vascular (DV) dos corpos lúteos (CL) de cabras ao longo do ciclo estral (AOLC). Vinte animais foram submetidos ao tratamento para indução/sincronização do estro, usando esponjas intravaginais com medroxiprogesterona, associadas a aplicações intramusculares de cloprostenol e gonadotrofina coriônica eqüina. Após remoção das esponjas, o estro foi identificado em aproximadamente de 72h. Concluído o tratamento, as cabras foram subdivididas em 4 grupos (n=5 cada) para abate nos dias 2, 12, 16 e 22 após ovulação (p.o.). Posteriormente, foram retirados os ovários e realizadas as mensurações de peso, tamanho e área do órgão e dos CL. Amostras de sangue foram coletadas e a progesterona sérica (P4) mensurada utilizando-se RIA convencional. A DV média dos CL AOLC foi 24,42±6,66; 36,26±5,61; 8,59±2,2 e 3,97±1,12 vasos/mm2 para os dias 2, 12, 16 e 22 p.o., respectivamente. A concentração média de P4 foi de 0,49±0,08; 2,63±0,66; 0,61±0,14 e 0,22±0,04ng/ml para os dias 2, 12, 16 e 22 p.o., respectivamente. Os parâmetros em estudo também se mostraram afetados pela fase do ciclo estral, sendo observados os maiores (p < 0,05) valores no dia 12 p.o. Neste experimento, a ovulação ocorreu predominantemente no ovário direito (70% dos animais), o qual apresentou medidas maiores que o contralateral. Observou-se ainda alta correlação significativa entre o peso do ovário e o do CL (r=0,87; p<0,05) e entre o tamanho destes órgãos (r=0,70; p<0,05). Conclui-se que, a morfologia dos ovários de cabras e a concentração sérica de progesterona variam em função da fase do ciclo estral e podem ser utilizadas como parâmetro na avaliação funcional do órgão.

Abstract in English:

ABSTRACT.- La Paz M.N., Fonseca V.U., Campos D.B., Artoni L.P., Sousa L.M.M.C. & Papa P.C. 2007. [In vitro progesterone production from bovine corpus luteum throughout gestation.] Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação. Pesquisa Veterinária Brasileira 27(9):370376. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva, 87, SP 05508-270, Brazil. E-mail: ppapa@usp.br The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20oC until processing. Progesterone was measured by RIA and protein content by Lowry’s method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern in vitro resembled what occurs in vivo.

• Corpus luteum gestation progesterone cell culture

Abstract in Portuguese:

ABSTRACT.- La Paz M.N., Fonseca V.U., Campos D.B., Artoni L.P., Sousa L.M.M.C. & Papa P.C. 2007. [In vitro progesterone production from bovine corpus luteum throughout gestation.] Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação. Pesquisa Veterinária Brasileira 27(9):370376. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva, 87, SP 05508-270, Brazil. E-mail: ppapa@usp.br The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20oC until processing. Progesterone was measured by RIA and protein content by Lowry’s method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern in vitro resembled what occurs in vivo.