Vacas leiteira de alta produção têm apresentado declínio da eficiência reprodutiva. Essa redução é devido a causas multifatoriais, entre elas a baixa concentração de estradiol (E2) no proestro e a baixa concentração de progesterona (P4) no ciclo estral subsequentente. O objetivo deste trabalho foi comparar o uso de gonadotrofina exógena na dinâmica folicular e na taxa de prenhez de vacas submetidas ao protocolo Ovsynch (Experimento 1) ou a protocolos utilizando P4 e E2 (Experimento 2). No Experimento 1, animais de primeiro serviço foram pré-sincronizados com dois protocolos (Presynch ou Double-Ovsynch). Já os animais de segundo ou mais serviços foram resincronizados com o protocolo Resynch. Os animais receberam GnRH (1º GnRH), seguido 7 dias depois pela adiministração de prostaglandina F2α (PGF2α). Nesse momento os animais foram divididos homogeneamente por paridade e número de inseminação em um de dois tratamentos: sem FSH (Ovsynch, n = 561) ou com FSH (Ovsynch + FSH, n = 571). O segundo GnRH (2º GnRH) foi administrado 56 horas após a PGF2α e a inseminação em tempo fixo foi realizada 16 horas após. Amostras de sangue foram colhidas no 1º e no 2º GnRH, na PGF2α e 6 e 13 dias após o 2º GnRH para dosagem de P4. Ainda, no 2º GnRH dosou-se também E2 No Experimento 2, os animais foram sincronizados no dia 0 com um dispositivo de P4 associado a 2 mg de benzoato de estradiol. Oito dias após o dispositivo foi removido e os animais receberam uma dose de PGF2α. Neste mesmo momento, as vacas foram divididas homogeneamente por paridade, número de serviços prévios, escore de condição corporal e presença de CL no inicio do protocolo em três tratamentos: Controle (sem tratamento adicional; n = 232); eCG (400 UI de eCG; n = 232) e FSH (20 mg de FSH; n = 230). Todos os animais receberam GnRH e foram insemados 56h após a retirada do dispositivo. Foram colhidas amostras de sangue a cada 48h do dia 11 ao dia 22. No Experimento 1, não houve efeito do FSH na concetração sérica de E2 no 2º GnRH (P = 0,88), no tamanho do maior folículo no 2 º GnRH (P = 0,63), na taxa de ovulação ao 2º GnRH (P = 0,69) ou na concentração sérica de P4 no 6º (P = 0,15) e 13º (P = 0,36) dia após o 2º GnRH. A taxa de prenhez foi semelhante (P> 0,05) entre os animais tratados com Ovsynch (36,2%) e Ovsynch + FSH (39,1%). No Experimento 2, os tratamentos não alteraram o diâmetro do folículo ovulatório (P = 0,15), o intervalo entre a remoção do dispositivo de P4 e a ovulação (P = 0,30) e a taxa de ovulação (P = 0,44). Não houve efeito de tratamento na concentração sérica de P4 (P = 0,15). A taxa de prenhez foi diferente entre os tratamentos aos 30 dias após a IATF (Controle = 28,0a vs FSH = 18,7b vs eCG = 29,7a %; P = 0,01), mas não aos 60 dias (Controle = 21,6 vs FSH = 16,1 vs eCG = 24,1%; P = 0,08) e na perda de gestacional (Controle = 18,8 vs FSH = 14,0 vs eCG = 18,4%; P = 0,39). Assim, o tratamento com FSH não estimulou o crescimento folicular final e a função luteínica de vacas leiteiras de alta produção sincronizadas com os protocolos Ovsynch e P4/E2. / Fertility in high-producing dairy cows has decreased over the years, which has been associated with reduced estradiol (E2) concentrations during proestrus and suboptimal progesterone (P4) concentrations during early stages of gestation. The objectives of the present study were to evaluate the effects of exogenous gonadotropins on follicular dynamics and risk of pregnancy per artificial insemination (P/AI) in cows subjected to the Ovsynch protocol (Experiment 1) or to a P4/E2-based timed AI protocol (experiment 2). In experiment 1, cows were enrolled in the Ovsynch protocol (GnRH, 7 d PGF2α, 56 h GnRH, 16 h IA) either after presynchronization (Presynch or Double-Ovsynch; first AI postpartum) or 32 d after previous AI. At the PGF2α injection, cows were blocked by parity and number of AI and, within each block, randomly allocated to eitherreceive 20 mg of FSH at the moment of the PGF2α(Ovsynch + FSH, n = 571) or to remain as untreated control (Ovsynch, n = 561). Blood was sampled at the 1st and 2nd GnRH and PGF2α injections, as well as on d 6 and 13 after the 2nd GnRH to access P4 and E2 (at the 2nd GnRH only) concentrations. In experiment 2, cows were received a P4 device and 2 mg of estradiol benzoate. The device was removed 8 d later concurrently with an injection of PGF2α, followed by an injection of GnRH and AI at 56 h.At the PGF2α injection, cows were blocked by parity, number of AI, body condition score, and presence of a CL at device insertion.Within each block, cows were randomly allocated to receive either 20 mg of FSH (FSH, n = 230), 400 IU of eCG (eCG, n = 232), or no additional treatment at the moment of the PGF2α (Control, n = 232). Blood was sampled at every 48 h from 1 to 12 d after AI. In experiment 1, there was no effect of FSH on serum E2 at the 2nd GnRH (P = 0.88), follicle diameter at the 2nd GnRH (P = 0.63), ovulatory response to the 2nd GnRH (P = 0.69), or serum P4 on d 6 (P = 0.15) and 13 (P = 0.36) after the 2nd GnRH. Also, P/AI was similar (P > 0.05) between Ovsynch (36.2%) and Ovsynch + FSH (39.1%). In experiment 2, treatment did not affect ovulatory diameter (P = 0.15), interval from P4 device removal and ovulation (P = 0.30), ovulatory response to the 2nd GnRH (P = 0.44), and serum P4 (P = 0.15). Interestingly, treatment with FSH reduced (P = 0.01) P/AI on d 30 (Control = 28.0a vs. FSH = 18.7b vs. eCG = 29.7a %), but not on d 60after timed AI (Control = 21.6 vs. FSH = 16.1 vs. eCG = 24.1%; P = 0.08).The risk of pregnancy loss was not affected by treatment (Control = 18.8 vs. FSH = 14.0 vs. eCG = 18.4%; P = 0.39). In conclusion, treatment with FSH failed to enhance the final growth of the ovulatory follicle and did not improve luteal function after AI in high-producing dairy cows synchronized with either the Ovsynch or P4/E2-based timed AI protocols.

O corpo lúteo (CL) canino apresenta períodos regulares de formação, atividade e regressão, marcados por intensa remodelação tecidual, o que depende diretamente de aporte energético, em alguns casos mediado pela insulina. Além de seu papel metabólico, a insulina, através de diferentes genes, pode desempenhar um papel fundamental na regulação da esteroidogênese, e consequentemente nas funções do CL de cadelas cíclicas. Nosso objetivo na primeira parte experimental foi mapear os genes diferencialmente expressos no CL, em diferentes estágios do diestro, diretamente relacionados à sinalização insulínica e a esteroidogênese no CL canino, caracterizando sua expressão gênica e proteica. A via secundária de captação de glicose também decorrente da sinalização insulínica foi abordada. Cadelas não gestantes foram submetidas à ovariosalpingohisterectomia a cada 10 dias entre os dias 10 e 60 (n=5/grupo) após a ovulação. Os CL coletados foram utilizados para sequenciamento de RNA (RNA-Seq) e validação por PCR em tempo real, e proteica por Western blotting e imunofluorescência. Na segunda parte experimental, através de cultivo celular, quantificamos a expressão dos genes relacionados à esteroidogênese após estímulo insulínico, e realizamos o bloqueio das vias phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase 14 (MAPK14) e mitogen-activated protein kinase 1(MAP2K1) para mensuração da produção de esteroides (n=4/grupo). Foram identificados sete genes diferencialmente expressos relacionados à sinalização insulínica: insulin receptor substrate (IRS1), phosphoinositide-3-kinase regulatory subunit 3 (PI3KR3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PI3KCG), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase 13 (MAPK13), mitogen-activated protein kinase 14 (MAPK14) e suppressor of cytokine signaling 1 (SOCS1), e dois genes, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) e hydroxy-delta-5-steroid dehydrogenase, 3 beta (HSD3B), identificados como envolvidos com a esteroidogênese. A via secundária de captação de glicose mostrou que adenylyl cyclase-associated protein (CAP1), CRK proto-oncogene, adaptor protein (CRKII) apresentaram aumento de sua expressão no período em que ocorre o aumento da produção de progesterona (P4), diferente de member of RAS oncogene family (RAP) e ras homolog family member Q (RHOQ) que apresentaram menor expressão gênica nos dias 40, coincidindo com o aumento de estradiol (E2) no diestro. Nos experimentos em cultivo celular, sob estímulo insulínico, a expressão de CYP19A1 não apresentou diferença quando as células eram provenientes do dia 20, diferentemente do dia 40, no qual houve aumento de expressão no grupo tratado com insulina. Em relação à expressão de HSD3B, houve aumento de expressão no dia 20 e 40. A produção de P4 apresentou diminuição com o bloqueio de PI3K, MAPK14 e MAP2K1, enquanto que a produção do E2 apresentou diminuição nos bloqueios com PI3K e MAPK14, e não houve diferença de produção com o bloqueio de MAP2K1. Em conjunto, estes dados sugerem que MAPK e PI3K podem modular a esteroidogênese no CL canino, provavelmente via expressão de HSD3B e CYP19A1. A ativação da via CAP-CrKII-RHOQ-RAP, não esta envolvida neste processo. Concluímos que a insulina é capaz de modular a esteroidogênese no CL canino e que a resposta hormonal ao estímulo insulínico depende do dia do diestro. / The canine corpus luteum (CL) has regular periods of formation, activity and regression marked by intense tissue remodeling, which depends directly on energy supply in some cases mediated by insulin. In addition to its metabolic role, insulin, through different genes, may play a key role in the regulation of steroidogenesis, and consequently in the CL functions of cyclic bitches. Our objective in the first experimental part was to map the differentially expressed genes in CL, at different stages of the diestrus, directly related to insulin signaling and steroidogenesis in canine CL, characterizing their gene and protein expression. The secondary pathway of glucose uptake also resulting from insulin signaling was addressed. Non-pregnant dogs were submitted to ovariosalpingohisterectomy every 10 days between days 10 and 60 (n=5/group) post- ovulation. The collected CL was used for RNA sequencing (RNA-Seq), validation by real-time PCR and protein for Western blotting and immunofluorescence. In the second experimental part, through cell culture we identified different responses of luteal cells after insulin stimulation under the expression of differentially expressed steroidogenesis genes. We also performed phosphoinositide 3-kinase (PI3K), Mitogen-activated protein kinase 14 (MAPK14) and mitogen-activated protein kina 1 (MAP2K1) pathway blockade to measure steroids production (n = 4/group). Seven differentially expressed genes related to insulin signaling were identified: insulin receptor substrate IRS1), phosphoinositide-3-kinase regulatory subunit 3 (PI3KR3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PI3KCG), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase 13 (MAPK13), mitogen-activated protein kinase 14 (MAPK14) and suppressor of cytokine signaling 1 (SOCS1), in addition to two genes, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta (HSD3B), identified as being involved with steroidogenesis. The secondary glucose uptake pathway showed that adenylyl cyclase-associated protein 1 (CAP1) and CRK Proto-Oncogene, Adaptor Protein (CRKII) increased expression in the period of increased production of progesterone (P4). different than member Of RAS Oncogene Family (RAP) and ras homolog family member Q (RHOQ) showed less gene expression on days 40 p.o., coinciding with the increase of estradiol (E2). In the cell culture experiments under insulin stimulation, the expression of CYP19A1 did not present difference when the cells were coming from day 20, unlike day 40, in which there was increased expression in the group treated with insulin.. HSD3B expression increased on days 20 and 40. The production of P4 presented a decrease with PI3K, MAPK14 and MAP2K1, while that production of E2 decreased with PI3K and MAPK14 blockade, and did not alter with MAP2K1 blockade. Together, these data suggest that MAPK and PI3K can modulate steroidogenesis in the canine CL, probably via HSD3B and CYP19A1 expression. The activation of the CAP-CrKII-RHOQ-RAP pathway is not involved in this process. We conclude that insulin is able modulate steroidogenesis in canine CL and that hormonal response to insulin stimulus depends on the day of the diestrus.

O objetivo deste estudo foi determinar os efeitos da aplicação de hCG no quarto dia após a inseminação a tempo fixo (IATF) sobre o tamanho do corpo lúteo (CL) existente, a indução de CL acessórios, a concentração de progesterona (P4) sérica e taxa de prenhez de vacas de corte em lactação. Nos três experimentos, vacas de corte multíparas (n=569), cruza Bos taurus, com período pós-parto entre 45 e 70 dias, foram sincronizadas com a administração de 2mg de benzoato de estradiol i.m. e a inserção de um dispositivo intravaginal contendo 0,750g de P4 (Dia -9). Sete dias após, administraram-se 150 μg de D-cloprostenol e 0,5 mg de cipionato de estradiol, i.m., no momento da retirada do dispositivo (Dia -2). Sessenta vacas de corte em lactação foram divididas aleatoriamente em dois grupos: hCG (n=30) tratadas com 1500UI i.m. de hCG e Controle (n=30) injetadas com 1,5 mL de solução salina i.m. no dia 4 depois do estro (Dia 0). Realizaram-se coletas sanguíneas dos animais para mensuração da concentração sérica de P4 nos Dias 4, 7, 10 e 14 do ciclo estral. Para acompanhamento da dinâmica ovariana, sessenta animais foram divididos aleatoriamente nos grupos hCG (n=30) ou Controle (n=30) e receberam o mesmo protocolo hormonal como citado anteriormente. No Dia 0, os ovários foram examinados por ultrassonografia transretal para determinar o diâmetro do folículo ovulatório; no Dia 4, o diâmetro do FD e presença do CL e no Dia 7 para mensurar a área luteal do CL e a presença de CL acessório. Quinhentas e sessenta e nove vacas (hCG, n= 269 e Controle, n= 300) foram inseminadas a tempo fixo 52-56 horas depois da retirada dos implantes de P4 e tratadas no Dia 4, conforme o protocolo descrito. O diagnóstico de prenhez foi realizado por ultrassonografia 30 dias após a IATF. Os resultados observados, mostraram que vacas tratadas com hCG no Dia 4 apresentaram maiores concentrações sérica de P4 no Dia 7, comparadas com as do grupo Controle (4,45 vs. 3,37ng/mL, respectivamente; p<0,05), mas níveis semelhantes de P4 nos Dias 10 e 14. Os animais do grupo hCG apresentaram CL com maior (p<0,01) área luteal no Dia 7, em relação ao grupo Controle (3,52 cm² vs. 2,66 cm², respectivamente) e uma incidência de 29,6 % de indução da ovulação do FD presente no Dia 4. Observou-se uma taxa de prenhez maior (p=0,071) no grupo tratado com hCG (53,9%) comparando-se com as vacas não tratadas (46,3%). Conclui-se que a administração de hCG no quarto dia do ciclo estral promove o aumento da área do CL, melhora função luteal, pode induzir a formação de CL acessório e tende a aumentar a taxa de prenhez de vacas de corte em lactação. / The aim of this study was to determine the effects of injection of hCG on the fourth day after the TAI on the size of the existing corpus luteum (CL), the induction of CL accessories, the concentration of progesterone (P4) levels and the possibility of increasing the rate of pregnancy of lactating beef cows subjected to synchronization of estrus and ovulation. Multiparous beef cows (n= 569), Bos taurus crossbreed with postpartum between 45 and 70 days were synchronized by administration of 2mg of estradiol benzoate im and the insertion of an intravaginal device containing 0,750g of P4 (Day -9). Seven days later, were administered 150mg of D- cloprostenol 0.5 mg of estradiol cypionate, im, at the time of device removal (Day -2). Sixty lactating beef cows were randomly divided into two groups: hCG (n= 30) treated with im 1500UI hCG and Control (n= 30) injected with 1.5 ml of saline im on day 4 after estrus (Day 0). There were blood collections of animals for measurement by radioimmunoassay of serum P4 on Days 4, 7, 10 and 14 of the estrous cycle. To evaluate the ovarian dinamics, sixty animals (hCG, n= 30 and Control, n = 30) received the same hormonal protocol and were uniformly distributed into groups according to the diameter of the ovulatory follicles on day 0 (estrus). On Day 0, the ovaries were examined by transrectal ultrasonography to determine the diameter of the ovulatory follicle, on Day 4, the diameter of the dominant follicle (DF) and the presence of CL on Day 7 to measure the area of the luteal CL and the presence of accessory CL. Five hundred and sixty-nine cows (hCG, n= 269 and Control, n= 300) were inseminated at a fixed time 52 to 56 hours after removal of the P4 devices treated on Day 4, according to the protocol described previously. The diagnosis of pregnancy were performed by ultrasonography 30 days after TAI. The results demonstrate that cows treated with hCG on Day 4 showed higher serum concentrations of P4 on Day 7, compared with the control group (4.45 vs. 3.37ng/mL, respectively, (p<0.05), but similar levels of P4 on Days 10 and 14. The animals of hCG group had greater (p<0,01) luteal area on Day 7, compared to the Control group (3.52cm² vs. 2.66cm², respectively) and a 29.6% incidence of induction DF ovulation present on Day 4. We observed a higher rate of pregnancy (p= 0.071) in the group treated with hCG (53.9%) comparing with untreated cows (46.3%). We conclude that administration of hCG on the fourth day of the estrous cycle increase the CL area, improves luteal function, can induce the formation of accessory CL and tends to increase the pregnancy rate of lactating beef cows.

Orientador: Maria Denise Lopes / Resumo: O objetivo desse estudo é obter conhecimentos mais profundos sobre os mecanismos regulatórios da manutenção e principalmente do término da função lútea em cadelas gestantes e não gestantes. O perfil de P4 é muito semelhante em cadelas gestantes e não gestantes, porém começa a divergir aproximadamente no dia 60 da fase lútea. Nesse momento, evidencia-se um declínio abrupto de P4 nas cadelas gestantes, sinalizando a luteólise pré-parto,com aumento da PGF2α e grande atividade apoptótica no interior do corpo lúteo (CL). Diferente do mecanismo de cadelas não gestantes, em que a regressão lútea é passiva e pré-programada. No presente trabalho foi validada a expressão dos seguintes fatores angiogênicos - VEGFA, IGFBP5, Endotelina, THBS2 e TGFB1 em CLs de cadelas cíclicas e gestantes. Para tal, foi realizado o acompanhamento do ciclo estral de fêmeas em diestro cíclico (n=20) e as fêmeas do grupo gestante foram inseminadas artificialmente (n=20). Todas as fêmeas foram submetidas a ovariohisterectomia (OSH) nos dias 10, 20, 40 e 60 após a onda pré-ovulatória de LH. Os CLs coletados foram submetidos às técnicas de qRT-PCR e imunofluorescência. Para análise estatística, aplicou-se o teste t de student, sendo os dados analisados pelo programa GraphPadPrism 6 e considerado significativo quando p<0,05. Os resultados mostraram que os fatores angiogênicos selecionados foram diferencialmente expressos nos dias 40 e 60, especialmente no grupo das cadelas gestantes. Nos outros momentos avaliado... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study is to obtain a deeper understanding of regulatory mechanisms of maintenance and especially of the end of the luteal function in pregnant and non-pregnant bitches. The profile of P4 is very similar in pregnant and non-pregnant bitches, but it begins to diverge approximately on day 60 of the luteal phase. At this moment, an abrupt decline of P4 is evidenced in the pregnant bitches, signaling the prepartum luteolysis: associated to an increase of the PGF2α and great apoptotic activity inside the corpus luteum (CL). Differently from non-pregnant bitches, where luteal regression is passive and preprogrammed. In the present work, it was validated the expression of the main angiogenic factors - VEGFA, IGFBP5, Endothelin, THBS2 and TGFB1 in CLs of cyclic and pregnant bitches. For this, the estral cycle of females in cyclic diestrus (n = 20) was monitored and the females of the pregnant group were artificially inseminated (n = 20). All females were submitted to ovariohysterectomy (OSH) on days 10, 20, 40 and 60 after the preovulatory LH surge. The collected CLs were submitted to qRT-PCR and immunofluorescence techniques. For statistical analysis, the student's t-test was applied, the data analyzed by the program GraphPadPrism 6 and it was considered significant when p <0.05. The results showed that the selected angiogenic factors were differentially expressed on days 40 and 60, especially in the group of pregnant bitches. No differences were observed in the... (Complete abstract click electronic access below) / Mestre

O uso de biotecnologias pode ser um método eficaz para melhorar a eficiência da reprodução e aumentar a produção de animais geneticamente superiores. O tratamento superovulatório é uma técnica comum utilizada com o objetivo de difundir o material genético desejado, embora seu uso em búfalos ainda apresente limitações, principalmente relacionada à baixa taxa de recuperação de embriões. O corpo lúteo (CL) é uma glândula reprodutiva transitória que produz progesterona, requerida para o estabelecimento e manutenção da prenhez e regulação do ciclo reprodutivo. O desenvolvimento e as funções do corpo lúteo são afetados pela hiperestimulação ovariana. As células luteínicas de animais superovulados apresentam características compatíveis com alta síntese de proteína. O fator de crescimento vascular endotelial (VEGF) e o fator de crescimento fibroblástico básico (bFGF) são reguladores importantes do desenvolvimento e função do CL, e também são afetados pelo tratamento superovulatorio. Vários estudos sugerem que o LH (hormônio luteinizante) e hCG (gonadotrofina coriônica humana) modulam a expressão dos fatores de crescimento. Este trabalho teve como objetivo avaliar a expressão gênica e protéica dos sistemas VEGF e bFGF em corpo lúteo cíclico de búfalas não tratadas e superovuladas. Foram utilizados vinte corporea lutea (CLL) divididos em 5 grupos de acordo com o estágio do ciclo estral (2, 6, 12, 17 e 26 dias após ovulação p.o.) e o grupo 6 era composto de CLL de animais superovulados no dia 6 p.o. Os CLL foram coletados em abatedouro, dissecados e congelados imediatamente em nitrogênio líquido para posterior extração de proteína e de mRNA. A análise protéica do VEGF, KDR, bFGF, FGFR-2 e FGFR-3 foi determinada por western blotting, enquanto a análise da expressão gênica de VEGF, KDR, Flt-1, bFGF, FGFR-1 a 4, por RT-PCR em tempo real. No CL de búfalas superovuladas observou-se maior expressão protéica dos sistemas VEGF e bFGF, em relação aos animais não tratados (p<0,05). Por outro lado, a expressão do mRNA de todos os genes estudados decresceu (sistema VEGF-A p<0.001, bFGF, FGFR-1 e FGFR-3, p<0.05) ou apresentou tendência a decrescer (FGFR-2 e FGFR-4, p<0.1) nos animais superovulados. Durante o ciclo estral, a expressão protéica do VEGF não variou, apesar da expressão de todas as outras proteínas e mRNA estudados apresetarem variações de acordo com a fase do ciclo estral. Os resultados obtidos sugerem que o tratamento superovulatório aumenta a taxa de tradução dos fatores angiogênicos no CL de búfalas, e que a expressão dos sistemas VEGF e bFGF ao longo do ciclo estral é tempo-dependente, indicando um papel importante destes fatores na regulação das funções do CL. / Biotechniques can be an effective way of improving reproduction efficiency and enhancing the production of genetically superior animals. The superovulatory treatment is a common technique aiming to spread desired genetical material, although its use in buffalos still presents limitations, mainly the low embryo recovery rate. The corpus luteum (CL) is a temporary endocrine gland that produces progesterone (P), which is required to the establishment and maintenance of pregnancy and regulation of reproductive cycle. CL development and function are affected by ovarian hyperestimulation. The luteal cells of superovulated animals are described to show characteristics compatible with higher protein synthesis. The vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are important regulators of CL and are also affected by superovulatory treatment. Several studies suggest that LH (luteinizing hormone) and hCG (human chorionic gonadotropin) modulate expression of growth factors. The aim of this work was to access gene and protein expression of VEGF and bFGF systems in cyclic CL of nontreated and superovulated water buffalos. Twenty water buffaloes corpora lutea (CLL) were divided in five groups according to estrous cycle stage (days 2, 6, 12, 17 and 26 after ovulation - p.o.) and the sixth group was composed by superovulated CLL from day 6 p.o. The CLL were collected at the slaughterhouse, dissected and frozen immediately in liquid nitrogen for posterior protein and mRNA extraction. Protein expression of VEGF and its receptors KDR and Flt-1 as well as bFGF and its receptors FGFR-2 and FGFR-3 was measured by western blotting (WB). For the relative gene expression of VEGF, KDR, Flt-1, bFGF, FGFR-1 to 4, we used real time RT-PCR. VEGF and bFGF systems protein expression showed an increase (p <0.05) in superovulated CLL compared to non-treated CLL on day 6 after p.o. On the other hand, mRNA expression from all studied genes was decreased (VEGF-A system p<0.001, bFGF, FGFR-1 and FGFR-3, p<0.05) or tended to decrease (FGFR-2 and FGFR-4, p<0.1) in superovulated CLL. In estrous cycle CLL VEGF protein did not show a time dependent expression although all other proteins and mRNAs expression were dependent on estrous cycle stage. These results indicate that the superovulatory treatment increased transduction rate of angiogenic factors in CL and that VEGF and bFGF systems are expressed in a time-dependent manner during the estrous cycle, indicating that these growth factors are important regulators of CL function.

O corpo lúteo (CL) canino apresenta períodos regulares de formação, atividade e regressão, marcados por intensa remodelação tecidual, o que depende diretamente de aporte energético, em alguns casos mediado pela insulina. Além de seu papel metabólico, a insulina, através de diferentes genes, pode desempenhar um papel fundamental na regulação da esteroidogênese, e consequentemente nas funções do CL de cadelas cíclicas. Nosso objetivo na primeira parte experimental foi mapear os genes diferencialmente expressos no CL, em diferentes estágios do diestro, diretamente relacionados à sinalização insulínica e a esteroidogênese no CL canino, caracterizando sua expressão gênica e proteica. A via secundária de captação de glicose também decorrente da sinalização insulínica foi abordada. Cadelas não gestantes foram submetidas à ovariosalpingohisterectomia a cada 10 dias entre os dias 10 e 60 (n=5/grupo) após a ovulação. Os CL coletados foram utilizados para sequenciamento de RNA (RNA-Seq) e validação por PCR em tempo real, e proteica por Western blotting e imunofluorescência. Na segunda parte experimental, através de cultivo celular, quantificamos a expressão dos genes relacionados à esteroidogênese após estímulo insulínico, e realizamos o bloqueio das vias phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase 14 (MAPK14) e mitogen-activated protein kinase 1(MAP2K1) para mensuração da produção de esteroides (n=4/grupo). Foram identificados sete genes diferencialmente expressos relacionados à sinalização insulínica: insulin receptor substrate (IRS1), phosphoinositide-3-kinase regulatory subunit 3 (PI3KR3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PI3KCG), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase 13 (MAPK13), mitogen-activated protein kinase 14 (MAPK14) e suppressor of cytokine signaling 1 (SOCS1), e dois genes, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) e hydroxy-delta-5-steroid dehydrogenase, 3 beta (HSD3B), identificados como envolvidos com a esteroidogênese. A via secundária de captação de glicose mostrou que adenylyl cyclase-associated protein (CAP1), CRK proto-oncogene, adaptor protein (CRKII) apresentaram aumento de sua expressão no período em que ocorre o aumento da produção de progesterona (P4), diferente de member of RAS oncogene family (RAP) e ras homolog family member Q (RHOQ) que apresentaram menor expressão gênica nos dias 40, coincidindo com o aumento de estradiol (E2) no diestro. Nos experimentos em cultivo celular, sob estímulo insulínico, a expressão de CYP19A1 não apresentou diferença quando as células eram provenientes do dia 20, diferentemente do dia 40, no qual houve aumento de expressão no grupo tratado com insulina. Em relação à expressão de HSD3B, houve aumento de expressão no dia 20 e 40. A produção de P4 apresentou diminuição com o bloqueio de PI3K, MAPK14 e MAP2K1, enquanto que a produção do E2 apresentou diminuição nos bloqueios com PI3K e MAPK14, e não houve diferença de produção com o bloqueio de MAP2K1. Em conjunto, estes dados sugerem que MAPK e PI3K podem modular a esteroidogênese no CL canino, provavelmente via expressão de HSD3B e CYP19A1. A ativação da via CAP-CrKII-RHOQ-RAP, não esta envolvida neste processo. Concluímos que a insulina é capaz de modular a esteroidogênese no CL canino e que a resposta hormonal ao estímulo insulínico depende do dia do diestro. / The canine corpus luteum (CL) has regular periods of formation, activity and regression marked by intense tissue remodeling, which depends directly on energy supply in some cases mediated by insulin. In addition to its metabolic role, insulin, through different genes, may play a key role in the regulation of steroidogenesis, and consequently in the CL functions of cyclic bitches. Our objective in the first experimental part was to map the differentially expressed genes in CL, at different stages of the diestrus, directly related to insulin signaling and steroidogenesis in canine CL, characterizing their gene and protein expression. The secondary pathway of glucose uptake also resulting from insulin signaling was addressed. Non-pregnant dogs were submitted to ovariosalpingohisterectomy every 10 days between days 10 and 60 (n=5/group) post- ovulation. The collected CL was used for RNA sequencing (RNA-Seq), validation by real-time PCR and protein for Western blotting and immunofluorescence. In the second experimental part, through cell culture we identified different responses of luteal cells after insulin stimulation under the expression of differentially expressed steroidogenesis genes. We also performed phosphoinositide 3-kinase (PI3K), Mitogen-activated protein kinase 14 (MAPK14) and mitogen-activated protein kina 1 (MAP2K1) pathway blockade to measure steroids production (n = 4/group). Seven differentially expressed genes related to insulin signaling were identified: insulin receptor substrate IRS1), phosphoinositide-3-kinase regulatory subunit 3 (PI3KR3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PI3KCG), mitogen-activated protein kinase 9 (MAPK9), mitogen-activated protein kinase 13 (MAPK13), mitogen-activated protein kinase 14 (MAPK14) and suppressor of cytokine signaling 1 (SOCS1), in addition to two genes, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta (HSD3B), identified as being involved with steroidogenesis. The secondary glucose uptake pathway showed that adenylyl cyclase-associated protein 1 (CAP1) and CRK Proto-Oncogene, Adaptor Protein (CRKII) increased expression in the period of increased production of progesterone (P4). different than member Of RAS Oncogene Family (RAP) and ras homolog family member Q (RHOQ) showed less gene expression on days 40 p.o., coinciding with the increase of estradiol (E2). In the cell culture experiments under insulin stimulation, the expression of CYP19A1 did not present difference when the cells were coming from day 20, unlike day 40, in which there was increased expression in the group treated with insulin.. HSD3B expression increased on days 20 and 40. The production of P4 presented a decrease with PI3K, MAPK14 and MAP2K1, while that production of E2 decreased with PI3K and MAPK14 blockade, and did not alter with MAP2K1 blockade. Together, these data suggest that MAPK and PI3K can modulate steroidogenesis in the canine CL, probably via HSD3B and CYP19A1 expression. The activation of the CAP-CrKII-RHOQ-RAP pathway is not involved in this process. We conclude that insulin is able modulate steroidogenesis in canine CL and that hormonal response to insulin stimulus depends on the day of the diestrus.

Orientador: José Antonio Dell´Aqua Junior / Resumo: O objetivo do presente estudo foi avaliar o fluxo sanguíneo luteal e os efeitos colaterais em jumentas após administração de dois agentes luteolíticos. Cinco dias após a ovulação, oito fêmeas asininas foram randomizadas em crossover design, em dois grupos experimentais. No grupo 1 (GI) foi utilizado Dinoprost Trometamina, e no grupo 2 (G2) Cloprostenol sódico. Foram realizados exames ultrassonográficos modo B e modo Doppler 15 minutos antes (-15) da administração dos análogos da PGF2α, e nos tempos 0, 15, 30, 45 e 60 minutos e 2, 3, 4, 5, 6, 7, 8, 12 e 24 horas pós-aplicação. Já os efeitos colaterais foram observados nos tempos 0, 15, 30, 45 e 60 minutos pós-administração dos agentes luteolíticos. O fluxo sanguíneo e a área do corpo lúteo reduziram gradativamente durante as primeiras 24 horas em ambos os grupos. Em relação a observação dos efeitos colaterai, a aplicação de Dinoprost Trometamina provocou um maior grau de sudorese, enquanto que, após aplicação de Cloprostenol Sódico, maior desconforto abdominal e diarreia foram observados como efeitos colaterais. Conclui-se que ambos os tratamentos (dinoprost e cloprostenol) são eficientes na promoção da luteólise, entretanto os efeitos colaterais diferem em resposta à administração de cada ativo. Novos estudos se fazem necessários, com redução das doses luteolíticas efetivas visando minimizar os efeitos colaterais observados. / Abstract: The aim of the study was to evaluate the blood flow and side effect in jennies after treatment with two luteolytic drugs. Five days after ovulation, eight jennies included were randomized in crossover design into two experimental groups. In group 1 (GI) Dinoprost Tromethamine was used, and in group 2 (G2) Cloprostenol sodium. Mode B and Doppler ultrasound examinations were performed 15 minutes before (-15) administration of PGF2α, and at times 0, 15, 30, 45 and 60 minutes and 2, 3, 4, 5, 6, 7, 8, 12 and 24 hours after application. Side effects were observed at 0, 15, 30, 45 and 60 minutes after luteolytic agents administration. Blood flow and corpus luteum area decreased gradually during the first 24 hours in both groups. Regarding the observation of side effects, G1 presented major score of sweating, while in G2, greater abdominal discomfort and diarrhea was evidenced as a major side effect. It is concluded that both treatments (dinoprost and cloprostenol) are efficient in promoting luteolysis, however side effects differ between each active. Further studies are needed to determine whether low doses may be effective in inducing luteolysis to minimize side effects. Keywords: jennies, corpus luteum, Doppler ultrasonography, prostaglandins. / Mestre

<p>Extracellular matrix degrading proteases from the plasminogen (plg) activator (PA) and the matrix metalloproteinase (MMP) systems have been implicated as important mediators of ovulation and corpus luteum (CL) formation and regression. The aim of this thesis was to investigate the expression and regulation of PAs and MMPs in the ovary and to examine their functional roles for CL formation and function. </p><p> The expression of membrane-type MMP-1 (MT1-MMP) and its substrate gelatinase A (MMP-2) mRNAs was studied during pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-induced ovulation in immature rats. These proteases were coordinately regulated so that both were highly expressed in the theca cells of large preovulatory follicles. This suggests that MT1-MMP activates gelatinase A in preovulatory follicles to degrade the follicular wall during ovulation. </p><p> In pseudopregnant (psp) rats, MT1-MMP mRNA was expressed in the CL throughout the luteal phase. Tissue inhibitor of metalloproteases type-1 (TIMP-1) mRNA was expressed during CL formation and regression. MMP-2 and collagenase-3 mRNAs were expressed during CL formation and regression, respectively. When the luteal phase was artificially prolonged or shortened, TIMP-1 and collagenase-3 mRNAs were induced only after the serum progesterone levels had decreased, indicating a close association with luteolysis in the rat. </p><p> In psp mice, the expression of mRNAs coding for both PAs, seven MMPs, and five protease inhibitors was studied. Most of the studied molecules were coordinately expressed during formation or regression of the CL. However, uPA, MT1-MMP, and TIMP-3 mRNAs were expressed throughout the luteal phase. The role of uPA was examined in psp uPA deficient mice. These mice displayed no abnormalities in luteal function or vascularity. The role of uPA is thus either not essential or can be compensated by other proteases in the absence of uPA. </p><p> In order to control the timing of the CL formation, a mouse model for PMSG/hCG-induced CL formation was developed. Five different protocols were evaluated. One of them provided CL that were stable for six days. In that protocol the mice were treated with prolactin (PRL), twice daily from day 2 of CL life onward. The expression of the steroid acute regulatory protein (StAR) mRNA in the psp CL was also characterized to assess its use as a molecular marker for CL development and regression. It was highly expressed in the forming and functional CL and downregulated at a late stage of CL regression.</p><p> The functional role of plg and MMPs for CL formation and function was investigated in plg deficient mice treated with the MMP inhibitor galardin (GM6001). Both psp mice and PMSG/hCG +PRL-induced CL formation were used. Several molecular markers for CL development and regression were used to evaluate the health status of the CL. Our data showed that healthy and vascularized CL formed even in plg deficient mice treated with the inhibitor. However, serum progesterone levels were significantly reduced in these mice, an effect that was mainly attributable to the plg deficiency. In conclusion, neither plg nor MMPs, alone or in combination, seem to be essential for the development of a functional CL.</p>

Extracellular matrix degrading proteases from the plasminogen (plg) activator (PA) and the matrix metalloproteinase (MMP) systems have been implicated as important mediators of ovulation and corpus luteum (CL) formation and regression. The aim of this thesis was to investigate the expression and regulation of PAs and MMPs in the ovary and to examine their functional roles for CL formation and function. The expression of membrane-type MMP-1 (MT1-MMP) and its substrate gelatinase A (MMP-2) mRNAs was studied during pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-induced ovulation in immature rats. These proteases were coordinately regulated so that both were highly expressed in the theca cells of large preovulatory follicles. This suggests that MT1-MMP activates gelatinase A in preovulatory follicles to degrade the follicular wall during ovulation. In pseudopregnant (psp) rats, MT1-MMP mRNA was expressed in the CL throughout the luteal phase. Tissue inhibitor of metalloproteases type-1 (TIMP-1) mRNA was expressed during CL formation and regression. MMP-2 and collagenase-3 mRNAs were expressed during CL formation and regression, respectively. When the luteal phase was artificially prolonged or shortened, TIMP-1 and collagenase-3 mRNAs were induced only after the serum progesterone levels had decreased, indicating a close association with luteolysis in the rat. In psp mice, the expression of mRNAs coding for both PAs, seven MMPs, and five protease inhibitors was studied. Most of the studied molecules were coordinately expressed during formation or regression of the CL. However, uPA, MT1-MMP, and TIMP-3 mRNAs were expressed throughout the luteal phase. The role of uPA was examined in psp uPA deficient mice. These mice displayed no abnormalities in luteal function or vascularity. The role of uPA is thus either not essential or can be compensated by other proteases in the absence of uPA. In order to control the timing of the CL formation, a mouse model for PMSG/hCG-induced CL formation was developed. Five different protocols were evaluated. One of them provided CL that were stable for six days. In that protocol the mice were treated with prolactin (PRL), twice daily from day 2 of CL life onward. The expression of the steroid acute regulatory protein (StAR) mRNA in the psp CL was also characterized to assess its use as a molecular marker for CL development and regression. It was highly expressed in the forming and functional CL and downregulated at a late stage of CL regression. The functional role of plg and MMPs for CL formation and function was investigated in plg deficient mice treated with the MMP inhibitor galardin (GM6001). Both psp mice and PMSG/hCG +PRL-induced CL formation were used. Several molecular markers for CL development and regression were used to evaluate the health status of the CL. Our data showed that healthy and vascularized CL formed even in plg deficient mice treated with the inhibitor. However, serum progesterone levels were significantly reduced in these mice, an effect that was mainly attributable to the plg deficiency. In conclusion, neither plg nor MMPs, alone or in combination, seem to be essential for the development of a functional CL.

Measurements of ovarian, corpus luteum, follicles of Lithuanian Black and White and German Black and White breed cows made by ultrasound scanner were revised. Effect of different factors, such as age, breed and season determining persistence of corpus luteum was statistically evaluated. Validation of treatment efficacy of persistent corpus luteum was carried out based on testing of progesterone concentration in peripheral blood plasma.