Objetivo: Avaliar marcadores de reserva ovariana e a presença de anticorpo anti-corpo lúteo (anti-CoL) em pacientes com arterite de Takayasu (AT) e possível associação com parâmetros clínicos, laboratoriais e uso de imunossupressores. Métodos: 20 pacientes com AT e 24 controles saudáveis foram avaliados para anti-CoL (immunoblot). A reserva ovariana foi avaliada por: hormônio folículo estimulante (FSH), hormônio luteinizante (LH), estradiol, hormônio anti-Mülleriano (HAM) e contagem de folículos antrais (CFA). HAM foi dosado por ELISA utilizando dois diferentes testes. Dados demográficos, obstétricos, alterações menstruais, aspectos clínicos, imagens vasculares e tratamento foram também analisados. Resultados: A média da idade atual foi similar em pacientes e controles (31,2 ± 6,1 vs. 30,4 ± 6,9 anos, p = 0,69). As frequências de HAM baixo foram idênticas em pacientes com AT com ambos os testes de ELISA e maiores quando comparadas ao grupo controle (50% vs.17%, p=0,02, 50% vs. 19%, p=0,048). Observou-se uma correlação positiva entre os dois testes de ELISA em pacientes (r=0,93, p < 0,0001) e em controles saudáveis (r=0,93, p < 0,0001). Pacientes com AT apresentaram menor CFA (11 vs. 16, p=0,13) e maior frequência de CFA reduzida (41% vs. 22%, p=0,29), contudo sem significância estatística. Não foram encontradas diferenças entre os dois grupos em relação às outras características demográficas e clínicas, dados obstétricos e demais parâmetros da reserva ovariana (p > 0,05). Anti-CoL foi observado apenas em uma paciente com AT (5% vs. 0%, p = 0,45). Avaliação adicional das mulheres com AT comparando as com baixos níveis de HAM ( < 1,0 ng/mL) versus aquelas com níveis de HAM QRUPD ng/mL) não mostrou diferença entre os dois grupos em relação a duração da doença, atividade de doença, provas de fase aguda, exames de imagem vascular e tratamento (p > 0,05). Conclusão: O presente estudo foi o primeiro a sugerir que as pacientes com AT podem apresentar reserva ovariana diminuída / Objective: To assess ovarian reserve markers and anti-corpus luteum antibodies (anti-CoL) in Takayasu arteritis (TA) patients and a possible association with clinical and laboratory parameters and the use of immunosuppressive drugs. Methods: 20 TA and 24 healthy controls were evaluated for anti-CoL (immunoblot). Ovarian reserve was assessed by: follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, antiMüllerian hormone (AMH) and antral follicle count (AFC). AMH was measured by ELISA using two different kits. Demographical data, menstrual abnormalities, obstetric data, clinical features, vascular imaging and treatment were also analyzed. Results: The mean current age was similar in TA patients and controls (31.2 6.1 vs. 30.4 6.9 years, p=0.69). The frequencies of decreased levels of AMH in TA patients were identical using both kits and higher when compared to controls (50% vs. 17%, p=0.02; 50% vs. 19%, p=0.048). A positive correlation was observed between the two kits in TA patients (r=+0.93; p < 0.0001) and in healthy controls (r=+0.93; p < 0.0001). The apparent lower AFC (11 vs. 16, p=0.13) and the higher frequency of low AFC (41% vs. 22%, p=0.29) in TA compared to controls did not reach statistical significance. No differences between the two groups were found concerning other demographic and clinical characteristics, obstetric data and other parameters of ovarian reserve (p > 0.05). Anti-CoL was solely observed in TA patients (5% vs. 0%, p=0.45). Further evaluation of TA patients comparing patients with low AMH levels ( < 1.0ng/mL) versus normal AMH levels (.- 1.0ng/mL) revelead no differences regarding disease duration, disease activity, acute phase reactants, vascular imaging and treatment between these two groups (p > 0.05). Conclusions: The present study was the first to suggest that TA patients may have diminished ovarian reserve

Objetivo: Avaliar marcadores de reserva ovariana e a presença de anticorpo anti-corpo lúteo (anti-CoL) em pacientes com arterite de Takayasu (AT) e possível associação com parâmetros clínicos, laboratoriais e uso de imunossupressores. Métodos: 20 pacientes com AT e 24 controles saudáveis foram avaliados para anti-CoL (immunoblot). A reserva ovariana foi avaliada por: hormônio folículo estimulante (FSH), hormônio luteinizante (LH), estradiol, hormônio anti-Mülleriano (HAM) e contagem de folículos antrais (CFA). HAM foi dosado por ELISA utilizando dois diferentes testes. Dados demográficos, obstétricos, alterações menstruais, aspectos clínicos, imagens vasculares e tratamento foram também analisados. Resultados: A média da idade atual foi similar em pacientes e controles (31,2 ± 6,1 vs. 30,4 ± 6,9 anos, p = 0,69). As frequências de HAM baixo foram idênticas em pacientes com AT com ambos os testes de ELISA e maiores quando comparadas ao grupo controle (50% vs.17%, p=0,02, 50% vs. 19%, p=0,048). Observou-se uma correlação positiva entre os dois testes de ELISA em pacientes (r=0,93, p < 0,0001) e em controles saudáveis (r=0,93, p < 0,0001). Pacientes com AT apresentaram menor CFA (11 vs. 16, p=0,13) e maior frequência de CFA reduzida (41% vs. 22%, p=0,29), contudo sem significância estatística. Não foram encontradas diferenças entre os dois grupos em relação às outras características demográficas e clínicas, dados obstétricos e demais parâmetros da reserva ovariana (p > 0,05). Anti-CoL foi observado apenas em uma paciente com AT (5% vs. 0%, p = 0,45). Avaliação adicional das mulheres com AT comparando as com baixos níveis de HAM ( < 1,0 ng/mL) versus aquelas com níveis de HAM QRUPD ng/mL) não mostrou diferença entre os dois grupos em relação a duração da doença, atividade de doença, provas de fase aguda, exames de imagem vascular e tratamento (p > 0,05). Conclusão: O presente estudo foi o primeiro a sugerir que as pacientes com AT podem apresentar reserva ovariana diminuída / Objective: To assess ovarian reserve markers and anti-corpus luteum antibodies (anti-CoL) in Takayasu arteritis (TA) patients and a possible association with clinical and laboratory parameters and the use of immunosuppressive drugs. Methods: 20 TA and 24 healthy controls were evaluated for anti-CoL (immunoblot). Ovarian reserve was assessed by: follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, antiMüllerian hormone (AMH) and antral follicle count (AFC). AMH was measured by ELISA using two different kits. Demographical data, menstrual abnormalities, obstetric data, clinical features, vascular imaging and treatment were also analyzed. Results: The mean current age was similar in TA patients and controls (31.2 6.1 vs. 30.4 6.9 years, p=0.69). The frequencies of decreased levels of AMH in TA patients were identical using both kits and higher when compared to controls (50% vs. 17%, p=0.02; 50% vs. 19%, p=0.048). A positive correlation was observed between the two kits in TA patients (r=+0.93; p < 0.0001) and in healthy controls (r=+0.93; p < 0.0001). The apparent lower AFC (11 vs. 16, p=0.13) and the higher frequency of low AFC (41% vs. 22%, p=0.29) in TA compared to controls did not reach statistical significance. No differences between the two groups were found concerning other demographic and clinical characteristics, obstetric data and other parameters of ovarian reserve (p > 0.05). Anti-CoL was solely observed in TA patients (5% vs. 0%, p=0.45). Further evaluation of TA patients comparing patients with low AMH levels ( < 1.0ng/mL) versus normal AMH levels (.- 1.0ng/mL) revelead no differences regarding disease duration, disease activity, acute phase reactants, vascular imaging and treatment between these two groups (p > 0.05). Conclusions: The present study was the first to suggest that TA patients may have diminished ovarian reserve

Résumé L’approvisionnement en cholestérol est un facteur limitant la stéroïdogenèse ovarienne. Pour cette raison, la majorité du cholestérol requis pour la synthèse des stéroïdes est importé de la circulation via les récepteurs des lipoprotéines de haute (HDL) et de basse densité (LDL) nommés scavenger receptor (SR-BI) et low-density lipoprotein receptor (LDLr). L’ARN messager de SR-BI est exprimé dans les ovaires de porcs durant toutes les étapes de la folliculogenèse ainsi que dans le corps jaune (CL). L’expression de la protéine SR-BI a également été détectée dans les follicules de souris lors du cycle œstral. Chez les deux espèces, l’expression est concentrée dans le cytoplasme et en périphérie des cellules du follicule. Les gonadotrophines induisent l'expression de SR-BI dans les cellules de la granulosa porcines, avec une expression cytoplasmique qui augmente durant la période périovulatoire, et avec une migration aux périphéries cellulaires durant la maturation du CL. Une conformation de 82 kDa de SR-BI est fortement exprimée dans le CL porcin, avec une conformation moins abondante de 57 kDa. Les différences entre les conformations sont attribuables à la glycosylation. La culture in vitro de follicules porcins avec des gonatrophines chorioniques humaines (hCG) a induit une hausse de régulation dépendante du temps du SR-BI de 82 kDa dans les cellules du granulosa. SR-BI et LDLr ont été exprimés réciproquement, avec LDLr étant le plus élévé dans les cellules folliculaires du granulosa et diminuant précipitamment avec la formation du CL. Pour explorer plus en détail les mécanismes d’approvisionnement en cholestérol de la stéroïdogenèse ovarienne, nous avons examiné des souris soumis à un traitement de désaccouplement de l'ovulation, et des souris portant la mutation nulle du gène Scarb1 (SR-BI-/-). Les résultats ont démontré que des ovocytes enfermés dans des structures lutéinisées expriment SR-BI. Les souris SR-BI-/ - présentaient de petits CLs, et de large follicules avec des cellules de thèque hypertrophiées et des kystes folliculaires avec des cavités remplies de sang et une diminution de 50% du niveau de progestérone dans le sérum. Les souris SR-BI-/ - traitées avec une combinaison de 20 g / g de mevinoline et 100  g / g de chloroquine ont démontré une diminution de 43% du niveau de progestérone sérique chez le type sauvage et de 30% chez les souris SR-BI-/ -. L’expression protéique de l’enzyme limitant pour la synthèse du cholestérol, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a augmenté chez les souris SR-BI-/-. Nous avons présenté des preuves démontrant que les cellules des follicules expriment le SR-BI durant la stéroïdogenèse et que la lutéinisation augmente l’expression de SR-BI. La maturation post-transcriptionelle est caractérisée par la glycosylation. Sous des conditions normales, l’expression de LDLr est arrêtée durant la lutéinisation. Ainsi SR-BI devient le facteur principal pour l’importation du cholestérol extracellulaire. En plus, la perturbation extracellulaire du cholestérol synthétisé de novo et l’absorption par les LDLr chez les souris SR-BI-/- diminuent la fonction lutéal. L’homéostasie du cholestérol ovarien est très importante pour une lutéinisation adéquate et sa perturbation mène à une réduction, mais non à un blocage complet, de la fonction lutéal. En conclusion, l’expression de SR-BI est un facteur important, mais non essentiel, pour maintenir l’homéostasie du cholestérol ovarien et la synthèse des stéroïdes, et la lutéinisation. Un réseau de mécanismes complémentaires et compensatoires d’approvisionnement en cholestérol agit en concert pour assurer la synthèse des stéroïdes ovariens. / Abstract Ovarian cholesterol supply is rate limiting to ovarian steroidogenesis. For this reason, the majority of cholesterol required for steroid synthesis is imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating HDL and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). SR-BI protein expression in mouse ovary during estrous cycle was also detected. In both species, expression is concentrated in cytoplasm and periphery of follicular cells. Gonadotropins induce SR-BI expression in pig granulosa cells, with cytoplasmic expression increasing through the periovulatory period, with migration to the cell periphery as the CL matured. An 82-kDa form of SR-BI is strongly expressed in the pig CL, with the less abundant 57-kDa form, differences between forms are attributable to glycosylation. In vitro culture of pig follicles with human chorionic gonadotropin (hCG) induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. To further explore mechanisms of cholesterol supply to ovarian steroidogenesis, we examined mice treated to uncouple ovulation and mice bearing null mutation of the Scarb1 gene (SR-BI-/-). Results show entrapped oocytes in luteinized structures expressed SR-BI. SR-BI-/- mice displayed small corpora lutea, large follicles with theca cells hypertrophied, follicular cysts with blood filled cavities and 50% decreased in plasma progesterone. In SR-BI-/- mice, treatment with a combination of 20 g/g of mevinolin and 100 g/g of chloroquine (CHLORO) was employed to disturbed cholesterol sources. Serum progesterone was reduced by 43% in wild type and 30% in SR-BI-/- mice. The protein expression of the rate-limiting enzyme for cholesterol synthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) increased in SR-BI-/- mice. It was concluded that follicular cells express SR-BI during follicle development and luteinization causes upregulation of SR-BI expression. Posttranslational maturation is characterized by glycosylation. Under normal conditions expression of the LDLr (low density lipoprotein recepors) is extinguished during luteinization such that SR-BI becomes the principal means of importation of extracellular cholesterol. Further, perturbation of cholesterol de novo synthesis and uptake from LDLr in SR-BI-/- mice leads to a reduction of luteal function. Ovarian cholesterol homeostasis is central to adequate luteinization, and its perturbation leads to reduction, but not to complete impairment, of luteal function. We conclude that SR-BI expression is an important but not essential factor in maintaining ovarian cholesterol homeostasis, steroid synthesis and luteinization. A network of complementary and compensatory cholesterol supply mechanisms act in concert to assure ovarian steroid synthesis. / Studies were funded by Colegio de Postgraduados, México. CONACyT, México. SRE, México. Ministère de l’Éducation du Québec, University of Montreal and an Operating Grant to B.D. Murphy from the Canadian Institutes of Health Research.

In ruminants, prostaglandin F2 alpha (PGF2α) is released from the endometrium in a pulsatile pattern at the time of luteolysis. The luteolytic PGF2α pulses are transported from the uterus to the corpus luteum (CL) through the utero-ovarian plexus (UOP) to cause luteolysis. At the time of establishment of pregnancy, interferon tau (IFNT) secreted by the conceptus suppresses the pulsatile release of PGF2α and thereby rescues the CL and maintains its secretion of progesterone. However, basal concentrations of PGF2α are higher in pregnant ewes than in cyclic ewes. The pulsatile release of PGF2α likely requires selective carrier-mediated transport and cannot be supported by a simple diffusion mechanism. The molecular and functional aspects of carrier mediated transport of PGF2α from the uterus to the ovary through the utero- ovarian plexus (UOP) at the time of luteolysis and recognition/establishment of pregnancy are largely unknown ruminants. Results indicate that intrauterine inhibition of (PGT) prevents the pulsatile release of PGF2α independently of spatial expressions of estrogen receptor (ESR-1) and oxytocin receptor (OXTR) proteins by the endometrium at the time of luteolysis in sheep. PGT protein is expressed in the UOP during the estrous cycle and pharmacological inhibition of PGT prevents transport of luteolytic PGF2α pulse through the UOP in sheep. IFNT activates novel JAK-SRC-EGFR-RAS-RAF-ERK1/2-EGR-1 signaling modules in endometrial luminal epithelial (LE) cells and regulates PGT- mediated release of PGF2α through these novel cell-signaling pathways. IFNT stimulates ERK1/2 pathways in endometrial LE cells and inhibition of ERK1/2 inhibits IFNT action and restores spatial expression of OXTR and ESR-1 proteins in endometrial LE cells and restores endometrial luteolytic pulses of PGF2α in sheep. Collectively, the results of the present study provide the first evidence to indicate that transport of endometrial luteolytic PGF2α pulses from the uterus to the ovary through the UOP is controlled by a PGT-mediated mechanism in sheep, new mechanistic insight into molecular mechanisms regulating cellular and compartmental transport of PGF2α at the time of luteolysis, and new mechanistic understanding of IFNT action and release of PGF2α from the endometrial LE cells and thus opens a new arena of research in IFNT signaling and PGT function.

O objetivo deste estudo foi avaliar o efeito da ressincronização da ovulação, iniciada 24 dias após a primeira IATF, sobre a área do corpo lúteo (CL), a concentração plasmática de progesterona (P4) e a taxa de prenhez. Exp.1 526 novilhas Brangus com idades entre 24 e 26 meses, foram submetidas a um programa de IATF no início da estação de acasalamento. O protocolo de sincronização para a primeira IATF começou com a inserção de um implante intra-vaginal contendo 750 mg de P4 e a administração de 2 mg de benzoato de estradiol (BE) intramuscular (i.m.) no dia -9 (D-9). Depois de sete dias (D-2), os implantes de P4 foram removidos, e 150 μg de D-cloprostenol (PGF), i.m., e 1 mg de cipionato de estradiol (CE), i.m., foram administrados. A IATF foi realizada entre 48 e 54 horas após a remoção do implante de P4 (D0). Vinte e quatro dias após a primeira IATF (D24), as novilhas foram divididas aleatoriamente nos seguintes grupos experimentais: controle (n = 167, sem tratamento), BE (n = 208, 1 mg de BE, i.m.) e hCG (n = 151, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo implante intra-vaginal contendo 750 mg de P4 na D24. No dia 31 (D31), os implantes de P4 foram removidos e o diagnóstico de prenhez foi realizado por ultrassonografia. As taxas de prenhez da primeira IATF no D31 foram 58,7% (98/167), 53,4% (111/208) e 52,9% (80/151), respectivamente, para os grupos controle, BE e hCG. Novilhas diagnosticadas como não gestantes receberam 150 μg de PGF, i.m., e 1 mg de CE, i.m., sendo a segunda IATF realizada 48 a 54 horas após a remoção do implante (D33). No D31, os subgrupos de novilhas prenhes de cada grupo experimental foram aleatoriamente divididos, sendo realizado exame por ultrassonografia para determinar a área do CL e coleta de uma amostra de sangue para determinar a concentração sérica de P4: Controle (n = 13), BE (n = 26), e hCG (n = 24). A área de CL foi significativamente maior (P<0,05) no grupo hCG (3,42±0,76 cm2), em comparação aos grupos de BE (2,44±0,57 cm2) e controle (2,61±0,61 cm2). Da mesma forma, a concentração sérica de P4 foi significativamente maior (P<0,05) no grupo hCG (12,43±3,48 ng/ml) em comparação aos grupos BE (6,92±3,04 ng/ml) e controle (7,29±2,45 ng/ml). O uso do BE e do hCG em programas de ressincronização da ovulação 24 dias após a IATF não interferiu na taxa de prenhez da primeira IATF. É provável que o mecanismo de ação do BE não afete a atividade do CL, a produção de P4, e consequentemente, não tenha efeito negativo na manutenção da prenhez em protocolos de ressincronização da ovulação. O tratamento com hCG resultou no aumento da área de CL e da produção de P4, porém, este efeito não favoreceu a taxa de prenhez da primeira IATF. Exp.2 184 novilhas Brangus com idade entre 24 a 26 meses e peso corporal médio de 361±29,2 kg foram submetidas a dois programas de IATF. O protocolo de sincronização para a primeira IATF foi o mesmo utilizado no Exp.1. Vinte e quatro dias após a primeira IATF (D24), as novilhas foram aleatoriamente divididas conforme os hormônios utilizados para ressincronização, formando os seguintes grupos experimentais: BE (n = 83, 1 mg de BE, i.m.) e hCG (n = 101, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo dispositivo intravaginal contendo 750mg de progesterona no D24. No D31, os implantes foram removidos e o diagnóstico de gestação por ultrassonografia foi realizado. As taxas de prenhez da primeira IATF no D31 foram de 63,9% (53/83) e 64,9% (65/101), respectivamente, para os grupos BE e hCG. Novilhas diagnosticadas como não gestantes (n=66) receberam 150 μg de PGF, i.m., e 1 mg de CE, im; a segunda IATF foi realizada no D33. Trinta dias após a segunda IATF (D63), foi realizado o segundo diagnóstico de gestação. As perdas gestacionais entre o D31 e D63, das novilhas prenhes da primeira IATF foram 9,4% (5/53) e 6,2% (4/65) respectivamente para os grupos BE e hCG. As taxas de prenhez da segunda IATF foram 40,0% (12/30) e 22,2% (8/36), respectivamente, para os grupos BE e hCG. As taxas de prenhez acumulada para os grupos BE e hCG foram, respectivamente, 72,3% (60/83) e 68,3% (69/101). O uso do BE e hCG para ressincronização da ovulação 24 dias após a primeira inseminação não afetou a taxa de prenhez da primeira IATF. As taxas de prenhez obtidas na segunda IATF foram inferiores às expectativas, considerando a resposta da primeira IATF. Entretanto, as taxas de prenhez acumulada foram similares e satisfatórias para os primeiros 33 dias da estação de acasalamento. / The aim of this study was to evaluate the effect of resynchronization of ovulation, which began 24 days after the first TAI, on the corpus luteum area (CL), plasma progesterone production (P4) and pregnancy rates. Exp.1 526 Brangus heifers between 24 and 26 months of age were submitted to a TAI program at the beginning of the breeding season. The protocol synchronization for the first TAI started with the insertion of an intravaginal implant containing 750 mg progesterone (P4) and the administration of 2 mg of estradiol benzoate (EB) intramuscular (i.m.) on day -9 (D-9). After seven days (D-2), P4 implants were removed, and 150 μg D-cloprostenol (PGF), and 1 mg estradiol cypionate (EC), were administered, i.m. The TAI was carried out between 48 and 54 hours after removal of the P4 implant (D0). Twenty-four days after the first TAI (D24), heifers were divided randomly into the following groups: control (n = 167, untreated), EB (n = 208, 1 mg EB, i.m.) and hCG (n = 151, 1000 IU hCG, i.m.). Heifers of the EB and hCG groups received a new intravaginal implant containing 750 mg of P4 on D24. On day 31 (D31), P4 implants were removed and the pregnancy diagnosis was performed by ultrasonography. Pregnancy rates for the first TAI, on D31, were 58.7% (98/167), 53.4% (111/208) and 52.9% (80/151), respectively, for the control, EB and hCG groups. Non-pregnant heifers received 150 μg PGF, i.m., and 1 mg EC, i.m., and the second TAI was performed 48 to 54 hours after removal of the P4 implant (D33). On D31, subgroups of pregnant cows from each experimental groups were randomly divided to determine the surface area of the CL by ultrasound and blood samples were collected to determine P4 concentrations: control (n = 13), BE (n = 26), and hCG (n = 24). The surface area of the CL was significantly higher (P<0.05) in the hCG group (3.42±0.76 cm2) compared to the EB (2.44±0.57 cm2) and control (2.61±0.61 cm2) groups. Also, P4 concentrations were significantly higher (P<0.05) in the hCG group (12.43±3.48 ng/mL) compared to the EB groups (6.92±3.04 ng/mL) and control (7.29±2.45 ng/mL). The use of EB and hCG in ovulation resynchronization programs 24 days after TAI did not affect the pregnancy rates of the first TAI. It is likely that EB mechanism of action does not affect the activity of the CL and P4 production, consequently having no negative effect on the maintenance of pregnancy. Nevertheless, the hCG treatment on D24 increased the area of CL and P4 plasma levels, but this effect neither improves nor compromised pregnancy rate of the first TAI. Exp.2 184 aged 24-26 months Brangus heifers old with mean body weight of 361±29.2 kg were submitted to two consecutive TAI programs. The synchronization protocol to the first TAI was the same as in Exp.1. Twenty-four days after the first TAI (D24), heifers were randomly divided according to the hormones used for resynchronization, according to the following groups: BE (n = 83, 1 mg EB, i.m.) and hCG (n = 101, hCG 1000 IU, i.m.). Heifers of the EB and hCG groups received a new intravaginal device containing 750 mg of progesterone on D24. On D31, P4 implants were removed and pregnancy diagnosis was performed by ultrasonography. The first TAI pregnancy rates on D31 were 63.9% (53/83) and 64.9% (65/101), respectively, for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Pregnancy rates for the second TAI were 40.0% (12/30) and 22.2% (8/36), for the EB and hCG groups respectively. The cumulative pregnancy rates for EB and hCG groups were, respectively, 72.3% (60/83) and 68.3% (69/101). The use of hCG and EB for resynchronization of ovulation 24 days after the first insemination did not affect pregnancy rates of the first TAI. Pregnancy rates obtained in the second TAI were below expected values, considering the first TAI response. However, cumulative pregnancy rates were similar and satisfactory for the first 33 days of the breeding season.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / It is well established that locally produced factors exert pivotal roles during dominant follicle selection, oocyte maturation, ovulation and luteolysis. However, the identification of these factors and pathways involved in these processes are not yet established. In the present study, we focused on the in vivo bovine models to study reproductive physiology, which were used to identify receptors and intracellular signaling pathways involved in follicle selection and luteolysis. In the first study, it was reviewed the in vivo models used in our lab, describing and discussing the different bovine models and techniques currently used to study ovarian physiology in this mono-ovulatory specie. In a second study, it was evaluated the expression of estrogen receptors (ESRs) before (day 2 of follicular wave), during (day 3) and after (day 4) follicular deviation in cattle. ESR1 and ESR2 transcripts levels were higher in dominant (F1) than subordinate (F2) follicle after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant (an antagonist of ESRs) inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels of both ESR1 and ESR2 were not affected by fulvestrant injection. In the third study, our objective was to demonstrate the role of the transcription factor signal transducer and activator of transcription 3 (STAT3) and the nuclear receptor 5A2 (NR5A2) in luteolysis. Luteal and blood samples were collected from separate groups of cows on Day 10 of the estrous cycle 0, 2, 12, 24, and 48 hours after prostaglandin F2 alpha (PGF) treatment. Serum progesterone concentrations decreased (P < 0.05) within 2h and the histological examination of the corpus luteum at 24 and 48h after PGF treatment confirmed functional and morphological luteolysis, respectively. The abundance of STAR mRNA and protein decreased at 12h after PGF treatment. The abundance of NR5A2 mRNA and protein decreased (P < 0.05) at 12 and 24h post-PGF, respectively. Levels of STAT3 mRNA remained constant (P > 0.05) throughout the time-points evaluated. However, the abundance of phosphorylated isoform of STAT3, normalized to total STAT3, increased reaching a peak at 12h and remaining high until 48h after PGF treatment. In conclusion, bovine in vivo models provide a valuable system to study reproductive events under physiological endocrine environment while keeping intact the communication between follicular cells through autocrine and paracrine signaling, without the need to perform ovariectomy or euthanaze the animals. Our results suggest that both ESR1 and ESR2 are regulated during follicular deviation and dominance and in response to FSH treatment in cattle, ESRs are required for normal gene expression and development of the dominant follicle. PGF treatment results in decreased expression of the nuclear receptor NR5A2 and activation of STAT3 by phosphorylation in bovine luteal cells. / É bem estabelecido que fatores produzidos localmente exercem papel essencial durante a seleção do folículo dominante, maturação oocitária, ovulação e luteólise. No entanto, os fatores e vias envolvidas nestes processos não estão totalmente estabelecidos. No presente estudo, enfatizou-se o uso de modelos bovinos in vivo para o estudo da fisiologia reprodutiva, sendo aqui utilizados para identificar receptores e vias de sinalização intracelular envolvidas na seleção do folículo e luteólise. No primeiro estudo, revisaram-se os modelos in vivo utilizados em nosso laboratório, descreveram-se e discutiram-se os diferentes modelos em bovinos e técnicas atualmente utilizadas para estudar fisiologia ovariana nesta espécie monovulatória. Em um segundo estudo, avaliou-se a expressão de receptores de estradiol (ESRS) antes (dia 2 da onda folicular), durante (dia 3) e após (dia 4) a divergência folicular em bovinos. Os níveis dos transcritos ESR1 e ESR2 foram maiores no folículo dominante (F1) que no subordinado (F2) após a divergência folicular. O tratamento com FSH manteve os níveis de RNAm de ambos ESR1 e ESR2 nos folículos F2 em níveis semelhantes aos observados em folículos F1. A injeção intrafolicular de 100 uM de fulvestrant (um antagonista de ESRs) inibiu o crescimento folicular e causou uma diminuição dos níveis de RNAm de CYP19A1. Os níveis de transcritos, tanto para ESR1 e ESR2, não foram afetados pela injeção de fulvestrant. Num terceiro estudo, o nosso objetivo foi demonstrar o papel do Transdutor de sinais e ativador de transcrição 3 (STAT3) e do receptor nuclear 5A2 (NR5A2) na luteólise. Amostras de corpo lúteo (CL) e sangue foram coletadas dos grupos de vacas 0, 2, 12, 24 e 48 horas após o tratamento com prostaglandina F2 alpha (PGF) no dia 10 do ciclo estral. A concentração de progesterona sérica diminuiu (P < 0.05) em 2 horas e o exame histológico do CL às 24h e 48h após o tratamento com PGF confirmou a ocorrência de luteólise funcional e morfológica, respectivamente. A abundância de RNAm e proteína de STAR diminuiu às 12h após o tratamento com PGF. A abundância de RNAm e proteína de NR5A2 diminuiu (P < 0.05) às 12 e 24 horas pós-PGF, respectivamente. Os níveis de RNAm de STAT3 permaneceram constantes (P> 0.05) ao longo do tempo avaliado. No entanto, a abundância da isoforma fosforilada de STAT3, normalizados para STAT3 total, aumentou, atingindo um pico às 12h e permaneceu elevada até 48h após o tratamento com PGF. Em conclusão, os modelos bovinos in vivo fornecem um sistema valioso para estudar os eventos reprodutivos sob ambiente fisiológico, mantendo intacta a comunicação entre as células foliculares através de sinalização autócrina e parácrina, reduzindo a necessidade de realizar ovariectomia ou realizar a eutanásia dos animais. Nossos resultados sugerem que tanto ESR1 como ESR2 são regulados durante a divergência e dominância folicular em bovinos e em resposta ao tratamento com FSH, e ESRs são necessários para a expressão gênica e para o desenvolvimento do folículo dominante. O tratamento com PGF resulta em diminuição da expressão do receptor nuclear NR5A2 e ativação de STAT3 por fosforilação em células luteais bovinas.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to verify if prostaglandin F2α (PGF2α) administered by intravulvosubmucous (IVSM) injection induces luteolysis by reaching the corpus luteum (CL) directly by a local absorption rote, preventing its metabolism in the lungs, or after absorption and distribution via the systemic circulation. In a first trial, the estrus rate of 1,937 beef cows was monitored for 5 days (25.6% of estrus). At day 5, the cows that did not show estrus received 1/5 of the standard dose of dinoprost IVSM (5 mg; n = 1440) and resulted in 68.2% of estrus in the next 5 days. However, in a second trial, the number of heifers detected in estrus after dinoprost injected via the IVSM (47.4%; n = 97) or intramuscular route (IM; 54.7%; n = 95) at day 5 was not different (P > 0.05). Based on serum progesterone concentrations, animals treated with 5 mg dinoprost at day 5 of the estrous cycle do not present functional luteolysis regardless of the administration route. At day 10 of the estrous cycle, luteolysis was variable in cows treated with 5 mg of dinoprost. Nevertheless, luteolysis occurred in all animals treated with 25 mg dinoprost independent of the estrous cycle day. After treatment, the PGF2α concentration did not differ in serum from uterine and jugular veins. This was further confirmed by measuring the concentration of 13,14-dihydro-15-keto- PGF (PGFM) after 5 mg dinoprost injection via the IM or IVSM route. Dinoprost IM- and IVSM-administered resulted in a similar PGFM serum pattern over time, suggesting the same absorption rate for both routes. Although anatomical evidences suggest that PGF2α injected IVSM could be taken direct to the ovaries, avoiding the systemic circulation, the results do not support this hypothesis. In summary, the route of PGF2α administration (IVSM or IM) resulted in similar serum concentrations of PGF2α, PGFM, and luteolysis. Taking all the results together, the PGF2α injection via IVSM reached the systemic circulation before reaching the ovary, and the effectiveness of low doses of PGF2α was dependent on luteal phase and not on the route of administration. / O presente trabalho teve por objetivo verificar se a prostaglandina F2α (PGF2α) administrada na submucosa vulvar (IVSM) induz a luteólise ao alcançar o corpo lúteo (CL) diretamente por uma via local, evitando sua metabolização nos pulmões, ou após a absorção e distribuição através da circulação sistêmica. Em um primeiro estudo, o estro de 1937 vacas de corte foi monitorado durante 5 dias (25,6% de estro). No dia 5, as vacas que não apresentaram estro receberam 5mg de dinoprost via IVSM (1/5 da dose padrão; n=1440), resultando em 68,2% de estro nos 5 dias seguintes. Todavia, em outro experimento utilizando a mesma dose, o número de novilhas detectadas em estro após o tratamento IVSM (47,4%, n=97) ou intramuscular (IM; 54,7%, n=95) no dia 5 não diferiu entre os grupos (P>0,05). Com base na concentração sérica de progesterona, os animais tratados com 5mg de dinoprost no dia 5 do ciclo estral não apresentaram luteólise funcional, independentemente da via de administração. Após o tratamento com 5mg dinoprost IM ou IVSM no dia 10 do ciclo, 3/5 e 2/5 animais apresentaram luteólise, respectivamente. Entretanto, a luteólise ocorreu em todos os animais tratados com 25mg de dinoprost, independente do dia do ciclo estral (dia 5 ou 10). A concentração de PGF2α não diferiu no soro das veias uterina e jugular. O mesmo foi observado quanto ao padrão de 13,14-dihidro-15-ceto prostaglandina F2α (metabólito de PGF2α; PGFM) sérico ao longo do tempo após a aplicação de 5 mg de dinoprost via IM ou IVSM. Em resumo, a via de administração de PGF2α (IVSM ou IM) resultou em concentrações séricas semelhante de PGF2α, PGFM e luteólise. Embora evidências anatômicas permitam sugerir que a PGF2α injetada via IVSM possa ser transportada diretamente aos ovários, a injeção de PGF2α via IVSM atinge a circulação sistêmica antes de chegar ao ovário, e a eficácia de baixas doses de PGF2α é dependente da fase luteal e não da via de administração.

O objetivo deste estudo foi avaliar o efeito da ressincronização da ovulação, iniciada 24 dias após a primeira IATF, sobre a área do corpo lúteo (CL), a concentração plasmática de progesterona (P4) e a taxa de prenhez. Exp.1 526 novilhas Brangus com idades entre 24 e 26 meses, foram submetidas a um programa de IATF no início da estação de acasalamento. O protocolo de sincronização para a primeira IATF começou com a inserção de um implante intra-vaginal contendo 750 mg de P4 e a administração de 2 mg de benzoato de estradiol (BE) intramuscular (i.m.) no dia -9 (D-9). Depois de sete dias (D-2), os implantes de P4 foram removidos, e 150 μg de D-cloprostenol (PGF), i.m., e 1 mg de cipionato de estradiol (CE), i.m., foram administrados. A IATF foi realizada entre 48 e 54 horas após a remoção do implante de P4 (D0). Vinte e quatro dias após a primeira IATF (D24), as novilhas foram divididas aleatoriamente nos seguintes grupos experimentais: controle (n = 167, sem tratamento), BE (n = 208, 1 mg de BE, i.m.) e hCG (n = 151, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo implante intra-vaginal contendo 750 mg de P4 na D24. No dia 31 (D31), os implantes de P4 foram removidos e o diagnóstico de prenhez foi realizado por ultrassonografia. As taxas de prenhez da primeira IATF no D31 foram 58,7% (98/167), 53,4% (111/208) e 52,9% (80/151), respectivamente, para os grupos controle, BE e hCG. Novilhas diagnosticadas como não gestantes receberam 150 μg de PGF, i.m., e 1 mg de CE, i.m., sendo a segunda IATF realizada 48 a 54 horas após a remoção do implante (D33). No D31, os subgrupos de novilhas prenhes de cada grupo experimental foram aleatoriamente divididos, sendo realizado exame por ultrassonografia para determinar a área do CL e coleta de uma amostra de sangue para determinar a concentração sérica de P4: Controle (n = 13), BE (n = 26), e hCG (n = 24). A área de CL foi significativamente maior (P<0,05) no grupo hCG (3,42±0,76 cm2), em comparação aos grupos de BE (2,44±0,57 cm2) e controle (2,61±0,61 cm2). Da mesma forma, a concentração sérica de P4 foi significativamente maior (P<0,05) no grupo hCG (12,43±3,48 ng/ml) em comparação aos grupos BE (6,92±3,04 ng/ml) e controle (7,29±2,45 ng/ml). O uso do BE e do hCG em programas de ressincronização da ovulação 24 dias após a IATF não interferiu na taxa de prenhez da primeira IATF. É provável que o mecanismo de ação do BE não afete a atividade do CL, a produção de P4, e consequentemente, não tenha efeito negativo na manutenção da prenhez em protocolos de ressincronização da ovulação. O tratamento com hCG resultou no aumento da área de CL e da produção de P4, porém, este efeito não favoreceu a taxa de prenhez da primeira IATF. Exp.2 184 novilhas Brangus com idade entre 24 a 26 meses e peso corporal médio de 361±29,2 kg foram submetidas a dois programas de IATF. O protocolo de sincronização para a primeira IATF foi o mesmo utilizado no Exp.1. Vinte e quatro dias após a primeira IATF (D24), as novilhas foram aleatoriamente divididas conforme os hormônios utilizados para ressincronização, formando os seguintes grupos experimentais: BE (n = 83, 1 mg de BE, i.m.) e hCG (n = 101, 1000 UI de hCG, i.m.). Novilhas dos grupos BE e hCG receberam um novo dispositivo intravaginal contendo 750mg de progesterona no D24. No D31, os implantes foram removidos e o diagnóstico de gestação por ultrassonografia foi realizado. As taxas de prenhez da primeira IATF no D31 foram de 63,9% (53/83) e 64,9% (65/101), respectivamente, para os grupos BE e hCG. Novilhas diagnosticadas como não gestantes (n=66) receberam 150 μg de PGF, i.m., e 1 mg de CE, im; a segunda IATF foi realizada no D33. Trinta dias após a segunda IATF (D63), foi realizado o segundo diagnóstico de gestação. As perdas gestacionais entre o D31 e D63, das novilhas prenhes da primeira IATF foram 9,4% (5/53) e 6,2% (4/65) respectivamente para os grupos BE e hCG. As taxas de prenhez da segunda IATF foram 40,0% (12/30) e 22,2% (8/36), respectivamente, para os grupos BE e hCG. As taxas de prenhez acumulada para os grupos BE e hCG foram, respectivamente, 72,3% (60/83) e 68,3% (69/101). O uso do BE e hCG para ressincronização da ovulação 24 dias após a primeira inseminação não afetou a taxa de prenhez da primeira IATF. As taxas de prenhez obtidas na segunda IATF foram inferiores às expectativas, considerando a resposta da primeira IATF. Entretanto, as taxas de prenhez acumulada foram similares e satisfatórias para os primeiros 33 dias da estação de acasalamento. / The aim of this study was to evaluate the effect of resynchronization of ovulation, which began 24 days after the first TAI, on the corpus luteum area (CL), plasma progesterone production (P4) and pregnancy rates. Exp.1 526 Brangus heifers between 24 and 26 months of age were submitted to a TAI program at the beginning of the breeding season. The protocol synchronization for the first TAI started with the insertion of an intravaginal implant containing 750 mg progesterone (P4) and the administration of 2 mg of estradiol benzoate (EB) intramuscular (i.m.) on day -9 (D-9). After seven days (D-2), P4 implants were removed, and 150 μg D-cloprostenol (PGF), and 1 mg estradiol cypionate (EC), were administered, i.m. The TAI was carried out between 48 and 54 hours after removal of the P4 implant (D0). Twenty-four days after the first TAI (D24), heifers were divided randomly into the following groups: control (n = 167, untreated), EB (n = 208, 1 mg EB, i.m.) and hCG (n = 151, 1000 IU hCG, i.m.). Heifers of the EB and hCG groups received a new intravaginal implant containing 750 mg of P4 on D24. On day 31 (D31), P4 implants were removed and the pregnancy diagnosis was performed by ultrasonography. Pregnancy rates for the first TAI, on D31, were 58.7% (98/167), 53.4% (111/208) and 52.9% (80/151), respectively, for the control, EB and hCG groups. Non-pregnant heifers received 150 μg PGF, i.m., and 1 mg EC, i.m., and the second TAI was performed 48 to 54 hours after removal of the P4 implant (D33). On D31, subgroups of pregnant cows from each experimental groups were randomly divided to determine the surface area of the CL by ultrasound and blood samples were collected to determine P4 concentrations: control (n = 13), BE (n = 26), and hCG (n = 24). The surface area of the CL was significantly higher (P<0.05) in the hCG group (3.42±0.76 cm2) compared to the EB (2.44±0.57 cm2) and control (2.61±0.61 cm2) groups. Also, P4 concentrations were significantly higher (P<0.05) in the hCG group (12.43±3.48 ng/mL) compared to the EB groups (6.92±3.04 ng/mL) and control (7.29±2.45 ng/mL). The use of EB and hCG in ovulation resynchronization programs 24 days after TAI did not affect the pregnancy rates of the first TAI. It is likely that EB mechanism of action does not affect the activity of the CL and P4 production, consequently having no negative effect on the maintenance of pregnancy. Nevertheless, the hCG treatment on D24 increased the area of CL and P4 plasma levels, but this effect neither improves nor compromised pregnancy rate of the first TAI. Exp.2 184 aged 24-26 months Brangus heifers old with mean body weight of 361±29.2 kg were submitted to two consecutive TAI programs. The synchronization protocol to the first TAI was the same as in Exp.1. Twenty-four days after the first TAI (D24), heifers were randomly divided according to the hormones used for resynchronization, according to the following groups: BE (n = 83, 1 mg EB, i.m.) and hCG (n = 101, hCG 1000 IU, i.m.). Heifers of the EB and hCG groups received a new intravaginal device containing 750 mg of progesterone on D24. On D31, P4 implants were removed and pregnancy diagnosis was performed by ultrasonography. The first TAI pregnancy rates on D31 were 63.9% (53/83) and 64.9% (65/101), respectively, for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Heifers diagnosed as open received 150 μg PGF, i.m., and 1 mg EC, i.m.; the second TAI was performed on D33. Thirty days after the second TAI (D63), the second pregnancy diagnosis was performed. Pregnancy loss rates from D31 to D63 were 9.4% (5/53) and 6.2% (4/65) respectively for the EB and hCG groups. Pregnancy rates for the second TAI were 40.0% (12/30) and 22.2% (8/36), for the EB and hCG groups respectively. The cumulative pregnancy rates for EB and hCG groups were, respectively, 72.3% (60/83) and 68.3% (69/101). The use of hCG and EB for resynchronization of ovulation 24 days after the first insemination did not affect pregnancy rates of the first TAI. Pregnancy rates obtained in the second TAI were below expected values, considering the first TAI response. However, cumulative pregnancy rates were similar and satisfactory for the first 33 days of the breeding season.

Made available in DSpace on 2014-07-29T15:07:30Z (GMT). No. of bitstreams: 1 Ubirajara_Bilego.pdf: 525711 bytes, checksum: 92b1baad77eca0e7758c3f7bfe166098 (MD5) Previous issue date: 2009-02-26 / The aim of this study was to describe the physiological responses to the Ovsynch protocol in Girolando heifers breeding in extensive model on Centro-Oeste of Brazil region and to determine relationships with environments factors in two stations, dry and rain. This responses were describe through the characterization of ovarian structures how: (NTL) total number of follicles; (DFOLIPR) mean of follicular diameter on protocol s begin; (DFOLOV) ovulatory diameter follicles; (DCL) corpus luteum ovulatory diameter; (SCL) ovulatory corpus luteum brightness score; pregnancy rates and ovulation rate on day 9 obtained in two year stations. Were utilized 40 heifers 18-24 months age, split in two groups of 20 animals being G1 group of dry station and G2 group of rain station. During the evaluations were found differences for the total number of follicles 8,07±0,25 e 8,83±0,26 in two stations applied. The mean of follicular diameter on protocol s begin was not show differences, but the measure of the ovulatory follicle diameter, have differences for the year stations (P<0,01), being the diameter for the dry station 11,88 ± 0,4mm whereas the rain station was 10,13 ± 0,36mm. Have in this study, higher frequency of follicles that ovulate with 11 to 12 mm on dry station and 10 to 11mm on rain station. The mean of the corpus luteum diameter on the present study not differ among the stations (P>0,05), with10,46±0,41mm for the dry station and 10,49±0,34mm for the rain station, being the higher frequency of CL 11 to 12mm in both stages. The CL brightness score not have differ(P>0,05), being 2,6 ± 0,16 for the dry and 2,31 ± 0,11 for the rain. The pregnancy rate have differences on two stages (P<0,01) with values 45% and 11% on station dry and rain respectively. The THI values differ among the stations dry and rain (P<0,01). The ovulation rate on D9 do not differ (P>0,05) although to have numeric differences. It was possible to conclude that in two stations evaluated, dry and rain, have differences on follicular morfometry and pregnancy rates. / Objetivou-se com este estudo descrever a resposta fisiológica do protocolo Ovsynch em novilhas Girolando, criadas extensivamente na região Centro-Oeste do Brasil em duas estações do ano, seca e águas. Tais respostas foram descritas através da caracterização das estruturas ovarianas como: (NTF) Número total de folículos; (DFOLIPR) Diâmetro folicular médio no início do protocolo; (DFOLOV) Diâmetro do folículo ovulatório; (DCL) Diâmetro do corpo lúteo; (SCL) Escore de ecogenicidade do corpo lúteo; Taxa de gestação e Taxa de ovulação no D9 obtidas em duas estações do ano. Foram utilizadas 40 novilhas girolando com idades entre 18-24 meses, divididas em dois grupos de 20 animais, sendo (G1) de estação seca e (G2) grupo de estação de águas. Durante as avaliações foram encontradas diferenças quanto ao número total de folículos 8,07±0,25 e 8,83±0,26 nas duas estações avaliadas. O diâmetro folicular médio no início do protocolo não mostrou diferenças, porém, houve diferença entre as estações do ano (P< 0,01), quanto ao diâmetro folicular ovulatório sendo o diâmetro médio para a estação seca 11,88 ± 0,4mm enquanto para a estação das águas foi 10,13 ± 0,36mm. Houve nesse estudo maior freqüência de folículos que ovularam em torno de 11 a 12mm na estação seca e 10 a 11mm na estação das águas. O diâmetro médio do corpo lúteo no presente estudo não diferiu entre as estações (P>0,05), com 10,46±0,41mm para a estação seca e 10,49±0,34mm, com maior freqüência de CLs de 11 a 12mm em ambas as estações. Os escores de corpo lúteo também não mostraram diferenças (P>0,05) sendo 2,6 ± 0,16 para seca e 2,31 ± 0,11 para águas. A taxa de gestação também mostrou diferença entre as estações com valores de 45% e 11% nas estações seca e águas respectivamente. Taxa de ovulação no D9 não diferiu (P>0,05) entre as estações apesar de ser numericamente diferente entre os tratamentos. Os valores de THI diferiram entre as estações seca e águas (P<0,01). Foi possível concluir que as duas estações avaliadas, seca e águas, mostraram diferenças quanto morfometria folicular e taxas de gestação.

Corpus luteum (CL), a transient endocrine structure formed from the ruptured ovarian follicle after ovulation, secretes progesterone (P4) that is essential for establishment and maintenance of pregnancy in mammals. The biosynthesis and secretion of P4 from CL depends, in general, on trophic hormones of the anterior pituitary gland and on hormones or factors originating from ovary, uterus, embryo and placenta. The structure and function of CL tissue is regulated by intricate interplay between two types of factors, namely, the luteotrophic factors, which stimulate CL growth and function, i.e., P4 secretion, and the luteolytic factors, which inhibit CL function and lead to luteal regression. In monoovulatory species such as higher primates and bovines, a striking diversity in the regulation of CL function exists not only between species, but also within the species during different stages of the luteal phase. In higher primates, unlike other species, one of the important characteristics of CL regulation is that, during non-fertile cycle, circulating LH appears to be the sole trophic factor responsible for maintenance of its function, and during fertile cycle, chorionic gonadotropin (CG), an LH analogue, originating from placenta maintains CL function. In higher primates, the role/involvement of luteolytic factors during luteolysis remains elusive. On the other hand, in the bovine species, the role/involvement of luteolytic factor, prostaglandin (PG) F2α during luteolysis is well established. It should be pointed out that in both the species, the mechanism of luteolysis is still poorly understood and the work presented in this thesis attempts to address these lacunae. Further, in bovines, studies have been carried out to examine potential trophic factor(s) responsible for the maintenance of CL function. Chapter I provides an extensive review of literature on CL structure and function with emphasis on factors that influence its growth, development, function and demise in primates and bovines. In Chapter II, employing bonnet monkey (Macaca radiata) as the representative animal model for higher primates, various studies have been conducted to examine the role of molecular modulators involved in regulation of CL function, particularly during spontaneous luteolysis. Although, it is well established that LH is essential for the maintenance of CL function in higher primates, the mechanism(s) responsible for the decline in serum P4 levels at the end of non-fertile cycles, without a concomitant change in circulating LH milieu, remains to be addressed. Several experiments have been conducted to examine the component(s) of luteotrophic (LH/CG) signaling that is/are modulated during luteolysis in the bonnet monkey CL. To understand the relative lack of responsiveness of CL to the circulating LH during the late luteal phase, LH/CG receptor (R) dynamics (expression of LH/CGR and its various transcript variants) was examined throughout the luteal phase and during different functional states of the monkey CL. The results indicated presence of LH/CGR mRNA, its transcript variants and functional LH/CGR protein in the monkey CL on day 1 of menses. Moreover, the functionality of receptors was tested by confirming the biological response of the CL to bolus administration of exogenous LH preparations, which eventually suggested factor(s) downstream of LH/CGR activation to account for the decline in CL function observed during non-fertile cycle. Studies have been conducted to identify molecular modulators that would selectively exploit intraluteal processes to regulate trophic signaling pathways that are critical to the control of luteal function. Immunoblot and qPCR analyses were carried out to examine presence and activation of Src family of kinases (SFKs) and cAMP-phosphodiesterases (PDEs) during various functional states of CL. The results revealed an increased activation of Src (phosphorylated at Tyr 416) during spontaneous and PGF2α/CET-induced luteolysis that may participate in the regulation of cAMP levels in part by increasing the cAMP-PDE activity observed during spontaneous luteolysis. This observation raised the question on the possible mechanism by which CG, an analog of pituitary LH, rescues CL function during early pregnancy. Thus, subsequent experiments involving LH/hCG administration in CET-treated animals as well as simulated early pregnancy animal model were conducted and the results revealed that, a bolus of LH/hCG decreased Src activation and cAMP-PDE activity accompanying a momentous increase in cAMP levels in both these models that further led to a concomitant increase in P4 secretion. Although the mechanisms of action of LH/CG involve modulation of a number of signaling pathways in the CL, by far, the results from various experiments suggested that it leads to activation of Src kinase and cAMP-PDE, thus causing inhibition of various elements of the primary signaling cascade- AC/cAMP/PKA/CREB during spontaneous luteolysis. One of the consequences of activation of Src kinase and cAMP-PDE was the regulation of expression of genes associated with steroidogenesis and it was observed that expression of SR-B1, a membrane receptor associated with trafficking of HDL-CE into the luteal cells, was lower in the regressed CL. The results taken together suggest that the decrease in responsiveness of CL to LH milieu during non-fertile cycles is not associated with changes in LH/CGR dynamics, but, is instead coupled to the activation of Src kinase and cAMP-PDE, inhibition of molecules downstream of LH signaling, and a decrease in the SR-B1 expression that regulates cholesterol economy of the luteal cell, and in turn, P4 secretion. The control of primate CL function appears to be dominated by the luteotrophic factors (LH/CG) over the luteolytic factors, since the process of luteal regression was overcome by administration of LH/CG. Further, in the primate CL, the molecular modulators of LH/CG signaling (Src kinase and PDE) are maintained in the repressed state by the luteotrophic factor LH/CG for maximum steroidogenic function. In contrast, in non-primate species, without invoking a role for the luteotrophic factor, essentially the synthesis and secretion of luteolytic factor, PGF2α, from the uterus is kept in check during pregnancy by the trophoblast derived IFN- and thus allowing CL to continue to function that is essential for maintenance of pregnancy. In the bovine species, the mechanism of PGF2α-induced luteolysis that involves a change in expression of genes associated with various processes of cellular function is poorly understood. Experiments were conducted utilizing buffalo cows (Bubalus bubalis) as a model system, to determine temporal changes in the global gene expression profile of the CL in response to PGF2α treatment. For this purpose, CL tissues were collected on day 11 of estrous cycle without treatment (designated as 0 h) and at 3, 6 and 18 h post PGF2α treatment for various analyses. Global changes in gene expression pattern in the CL were investigated employing Affymetrix GeneChip bovine genome array and the results are presented in Chapter III. The hybridization intensity values obtained by microarray analysis were subjected to R/Bioconductor tool. Following the application of highly stringent statistical filters to eliminate false positives, a set of differentially expressed genes were identified. The differentially expressed genes were further classified based on a fold change cut-off filter of ≥2, and the analysis revealed 127 genes to be differentially expressed within 3 h of PGF2α administration, of these 64 and 63 genes were up-regulated and down-regulated, respectively. Analysis of microarray data at 6 h post PGF2α administration revealed 774 genes to be differentially expressed, of which 544 genes were up-regulated, while 230 genes were down-regulated. The microarray analysis performed on CL tissues collected at 18 h post PGF2α administration showed that out of the total 939 differentially expressed genes, 571 genes were up-regulated, while 368 genes were down-regulated. Analysis of the ontology report for the biological processes category showed that initially in response to PGF2α administration, genes regulating steroidogenesis, cell survival and transcription were differentially regulated in the CL, but at later time points, differential expression of genes involved in apoptosis, PGF2α metabolism, tissue remodeling and angiogenesis was observed. Further, involvement of molecules downstream of LH/IGF-1 activation was investigated and the results obtained indicated that PGF2α interfered with the LH/IGF-1 signaling since the expression of LH/CGR, GHR and pAkt were down-regulated following PGF2αadministration. Furthermore, the functional luteolysis observed post PGF2αadministration appeared to be due to an interruption in cholesterol trafficking to inner mitochondrial membrane, since StAR expression was inhibited. The results obtained also demonstrated that the expression of AGTR1, VEGFR2 and R3 were down-regulated following PGF 2α administration. Further, the data obtained also suggested modulation of expression of pro- and anti-angiogenic factors upon PGF2α-treatment indicative of an involvement of other autocrine or paracrine factor(s) in the regression of bovine CL. This was an interesting finding as it suggests a novel and potential functional relationship between angiogenesis and the luteolytic response of CL to PGF2α administration. In bovines, despite extensive research being carried out to examine factors involved in the regulation of development and function of the CL, the trophic factor(s) required for maintenance of CL function, especially, P4 biosynthesis and secretion are not well characterized. It was hypothesized that the function of the CL during its finite lifespan must be responsive to LH as well as to various growth factors. Thus, experiments were conducted to examine the effects of increased LH and GH/IGF-I on the maintenance of CL function during mid luteal phase and post PGF2α administration and the results of these studies are presented in Chapter IV. To elucidate the role of LH as a trophic factor in the regulation of CL function, effects of increased endogenous LH through GnRH administration and exogenous hCG injections were examined. The results indicated an absence of noticeable effect of various hCG/GnRH treatments on circulating P4 levels. On the other hand, administration of GH resulted in increased serum IGF-1 and P4 levels. It was further observed that the administration of a combination of hCG and GH increased serum P4 levels better than treatment with GH alone. Further experiments were carried out to examine the complex reciprocal relationship between LH/GH and PGF2α on expression of genes involved in the regulation of luteal structure and function. In buffalo cows, administration of exogenous hCG and/or GH following inhibition of CL function by PGF2α administration did not prevent the PGF2α-induced decline in serum P4 levels, but PGF2-mediated decrease in expression of LH/CGR and GHR genes was prevented upon GH administration. However, the decrease in StAR expression was not restored by hCG and GH treatments, thereby indicating that PGF2 action was not prevented by hCG and/or GH treatments. Taken together, the results of studies carried out in buffalo cows employing various experimental model systems suggest essential role for LH and GH/IGF-1, however, these factors were unable to reverse PGF2α-induced luteolysis. Further, our crucial findings of the effects of increased endogenous LH and IGF-1, in addition to their relationship with luteolytic agents such as PGF2α will open new avenues for studying the mechanisms involved in the regulation of structural and functional properties of the buffalo CL. It is well known that a large number of buffalo cows experience loss of pregnancy and infertility due to inadequate luteal function and/or failure of timely insemination. Results from our studies suggest that the incorporation of PGF2α and hCG or GH/IGF-1 protocols in buffalo cows to be beneficial for improving their breeding efficiency as these protocols are likely to increase luteal function with defined luteolysis. To summarize, the results of studies described in the present thesis provide new insights into the physiological and molecular mechanisms involved in the regulation of CL function during luteolysis in the monoovulatory species. The results suggest that the maintenance of CL function appears to be dependent on both luteotrophic and luteolytic factors, but with a varied degree of dominance between the two species examined. Further, the results indicate that while the luteotrophic factors (LH/CG) dominate the CL regulation in primates, the regulation of CL function in bovines is dominated by the actions of luteolytic factor (PGF2α). In monoovulatory species, the luteotrophic and luteolytic factors following binding to their specific plasma membrane receptors on the luteal cells, would counteract each other and modulate activation of various downstream signaling molecules subsequently leading to regulation of gene expression and P4 secretion (Fig.5.1). LH: luteinizing hormone; CG: chorionic gonadotropin; LH/CGR: LH/CG receptor; Gαs: stimulatory α-subunit of trimeric G-protein; AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; p: phosphorylation: CREB: cAMP response element binding protein; SR-B1: scavenger receptor class B, type I; SF-1: steroidogenic factor 1; LRH-1: liver receptor homologue 1; P4; progesterone; Src; sarcoma; PDE4D: cAMP phosphodiesterase 4D; StAR, steroidogenic acute regulatory protein; PGF2α: prostaglandin F2α; PTGFR: PGF2α receptor; PLC: phospholipase C; CYP19A1: cytochrome P450 aromatase; PTGR1: Prostaglandin reductase 1; AREG: Amphiregulin; RTK: receptor tyrosine kinase; Akt: protein kinase B; FKHR: forkhead transcription factor; DAPL1: death associated protein like 1; ARG2: Arginase, type II Growth factor LH/CGR RR AC Gαs ? Gα TT P? Gα K PKP src cAMP ? P Akt PDE4D P PFKHR FKHR CREB P LRH-1CREB P SF-1 Genes associated with Genes associated with apoptosis ? CYP19A1, apoptosis SR-B1 PTGR1 DAPL1 SF-1, LRH-1 AREG ARG 2 P4 biosynthesis Apoptosis? P4 biosynthesis Apoptosis MONKEY BUFFALO COW Shown here is the diagram depicting intracellular signaling pathways regulated by luteotrophic factor (LH) and luteolytic factor (PGF2α) and their cross talk to counteract changes in the expressions of genes associated with the biosynthesis and secretion of P4 and apoptosis in the CL. In primates, LH/CG activates a multitude of intracellular signaling cascades, primarily Gαs/AC/cAMP/PKA/CREB leading to changes in gene expression. LH during early and mid luteal phase and CG during pregnancy maintain the activation of Src and PDE in an inhibitory state. However, during the late luteal phase of non-fertile cycle, results in present study suggests that activated Src levels and PDE activity increase, with accompanying decrease in cAMP and pCREB levels leading to concomitant decrease in SR-B1 expression, and in turn, P4 secretion. Surprisingly, regulation of apoptotic gene expression and CL regression are still unclear. In bovines, PGF2α of uterine origin mediates changes in luteal gene expression and results in decreased P4 secretion, principally by reduction in StAR level. The present study suggests that during luteolysis PGF2α affects the genes regulated by LH, by interfering with LH (and perhaps IGF-1) signaling leading to alteration in the expression of genes crucial for CL structure and function. (Pl refer the abstract file for figures)