Made available in DSpace on 2016-05-02T13:55:47Z (GMT). No. of bitstreams: 1 Humberto Luis Del Hoyo NeriDissertacao.pdf: 1155571 bytes, checksum: 3a98af31a9aa3f9225c0359f68b2db8e (MD5) Previous issue date: 2013-06-27 / Progesterone (P4) is an important steroid hormone in FTAI programs. Hormonal protocols used in FTAI programs involve considerable financial values, in which P4 represents 43% of the total cost. The P4 release process from vaginal implants occurs by passive diffusion, i.e., the drug release is driven by concentration gradient and enhanced by the contact area between the implant and vaginal epithelium. Given the importance of P4 in the protocols and the significance of this steroid in the treatment costs, several studies described the reutilization of P4 vaginal implants as an alternative to make this technology feasible. However, the results are controversial and the pattern of P4 releasing from vaginal implants used in cows with different luteal activity (amount of endogenous P4 synthesis) is not yet described. The aim of the present study was to evaluate the P4 profile in cows with different luteal activity treated with a new vaginal implant (1g of P4) for 8 days; evaluate and compare to each other and to a new vaginal implant the P4 releasing from implant previously used in females with different endogenous progesterone conditions; and correlate progesterone releasing from new and used (second use) implants with follicular dynamics. For this purpose, two experiments were performed. Experiment 1: Group 1(G1a) with corpus luteum during all treatment period; Group 2 (G2a) with corpus luteum during half of treatment period; Group 3 (G3a) without corpus luteum. At the beginning of treatment (D0), G1a and G2a animals had a functional corpus luteum which was formed eight days prior to implant insertion. Three day after implant insertion (D3), 0.15 mg of D-cloprostenol were administered in G2a animals. The G3a animals (n=10) started the treatment without ovarian luteal activity. Blood samples were collected on D0 in the morning and afternoon, D3, D5 and D8. P4 concentrations were determined by radioimmunoassay (RIA). Average P4 concentrations in each collection were compared by Tukey s test. G1a and G2a was different from G3a on D0 (5,3+3,1a; 5,3+1,4a and 0,6+0,3b ng/mL, respectively (p<0,05)) and on D3 (5,7+2,6a; 5,4+1,95a and 3,6+0,8b ng/mL, respectively (p<0,05)). On D5, 36 hours after PGF administration, P4 concentration of G2a became similar to G3a and both were different from G1a (G1a=3,3+1,6a, G2a=2,4+0,9b and G3a=2,1+0,7b ng/mL (p<0,05)). On D8, the groups maintained the same characteristics (G1a=3,1+1,3a, G2a=1,8+0,8b and G3a=1,6+0,6b ng/mL (p<0,05)). Furthermore, the difference in the P4 serum concentration between D3 and D0 were lower in G1a and G2a than in G3a (G1a=0,4+1,8a; G2=0,2+1,4a e G3=2,8+0,9b ng/mL (p<0,05)). In experiment 2, the same animals were reallocated into 4 groups without CL. Group 1(G1b) implants from G1a of Exp1; Group 2 (G2b) implants from G2a of Exp1; Group 3 (G3b) implants from G3a of Exp1; and Group 4 (G4b) new implants. There is no difference in the number of animals with P4 lower than 1 ng/mL (G1b = 16.7%, G2b = 66%, G3b = 50% and, G4b = 0; P=0,14) and in the number of times that this occurred in relation to the amount of samples evaluated (G1b = 12.5 %, G2b = 22.9 %, G3b = 12.5 % and, G4b = 0; P=0,07), i.e., there were no P4 releasing pattern from used implants and only new implants properly maintained the concentration of this hormone. The average follicular growth rate (G1b = 1,0+0,5; G2b = 1,0+0,3; G3b = 0,7+0,5 e G4b = 0,8+0,3) mm/day) and the diameter of the largest follicle on D8 (G1b = 13,0+3,3; G2b = 12,0+2,3; G3b = 10,5+2,9 e G4b = 11,4+0,6 mm) were also not affected by the releasing pattern of the implants. In conclusion, animals with CL and endogenous P4 during FTAI protocols consumed less P4 from the implants when compare to animals without CL; new P4 vaginal implants maintained adequate levels of P4 for TFAI, regardless the physiological condition of the treated animal; implants previously used by animals with different ovarian activity did not maintain the P4 profile similar to that observed with new implants; follicular dynamics was not changed by the lack of P4 releasing pattern from the used implants when compared to new implants; despite the greater progesterone release, did not affect follicular development pattern in heifers. / A progesterona (P4) é imprescindível para a aplicação da técnica de Inseminação Artificial em Tempo Fixo (IATF). Os protocolos hormonais utilizados para a IATF movimentam importante valor financeiro, em que a P4 representa 43 % do custo total. A liberação da P4 pelos dispositivos vaginais ocorre por difusão passiva, ou seja, a droga é liberada obedecendo gradiente de concentração, potencializado pela área de superfície de contato entre o dispositivo e o epitélio vaginal. Dada a importância desse esteroide nos protocolos, vários estudos descrevem a reutilização dos dispositivos como uma alternativa de viabilizar a técnica. No entanto, os resultados são controversos e não há uma descrição sobre o padrão de liberação da P4 de implantes utilizados em vacas em diferentes etapas do ciclo estral. Os objetivos foram: 1) avaliar o perfil de P4, de implantes novos contendo 1g de P4, utilizados por 8 dias, em fêmeas com diferentes condições de atividade ovariana luteal; 2) avaliar e comparar entre si e em relação a um dispositivo novo, a liberação de P4 de implantes previamente usados em fêmeas com diferentes condições de progesterona endógena e 3) correlacionar a liberação de progesterona de implantes de 1º e 2º uso com a dinâmica de desenvolvimento folicular. Para isso, foram realizados dois experimentos. Exp. 1: Grupo 1 (G1a): com corpo lúteo durante todo o tratamento; Grupo 2 (G2a): com corpo lúteo a metade do tratamento e Grupo 3 (G3a): sem corpo lúteo. Os animais dos Grupos G1a e G2a iniciaram o tratamento (D0) com um corpo lúteo funcional, formado oito dias antes da inserção do implante. No G2a, foi aplicada , três dias após a inserção do implante, D3, (0,15 mg de D-cloprostenol) visando luteólise. Os 10 animais do G3a iniciaram o tratamento sem atividade ovariana luteal. Duas amostras de sangue foram coletadas no D0, pela manhã e à tarde, e no D3, no D5 e no D8, à tarde. O nível de P4 foi obtido por radioimunoensaio (RIA). As médias de P4 das amostras foram comparadas pelo teste de tukey. G1a e G2a diferenciaram de G3a no D0 (5,3+3,1a; 5,3+1,4a e 0,6+0,3b ng/mL, respec. (p<0,05)) e no D3 (5,7+2,6a; 5,4+1,95a e 3,6+0,8b ng/mL, respec. (p<0,05)). Em D5, 36 horas após a PGF do G2a, este passou a níveis semelhantes a G3a e ambos se diferenciaram de G1a (G1a=3,3+1,6a, G2a=2,4+0,9b e G3a=2,1+0,7b ng/mL (p<0,05)) e no D8 os grupos mantiveram as mesmas características (G1a=3,1+1,3a, G2a=1,8+0,8b e G3a=1,6+0,6b ng/mL (p<0,05)). Além disso, a diferença entre o nível sérico de D3 e D0 também foi diferente de G1a e G2a quando comparadas com G3a (G1a=0,4+1,8a; G2=0,2+1,4a e G3=2,8+0,9b ng/mL (p<0,05)). Exp. 2: Os mesmos animais foram redistribuídos e, desta vez, divididos em quatro grupos sem a presença de CL. Grupo 1 (G1b): implantes do G1a, Exp. 01; Grupo 2 (G2b): implantes do G2a, Exp. 01; Grupo 3(G3b): dispositivos de G3a, Exp. 1 e Grupo 4(G4b): implantes novos. Não houve diferença no percentual de animais que apresentaram P4 abaixo de 1 ng/mL durante o tratamento (G1b = 16,7%; G2b = 66%; G3b = 50% e G4b = 0; (p=0,14)) e no número de vezes que isso ocorreu em relação à quantidade de amostras avaliadas (G1b = 12,5 %; G2b = 22,9 %; G3b = 12,5 % e G4b = 0; (p=0,07)), ou seja, não houve padrão na liberação de P4 de implantes reutilizados e somente os implantes novos mantiveram níveis adequados desse hormônio. A média das taxas de crescimento folicular (mm/dia) (G1b = 1,0+0,5; G2b = 1,0+0,3; G3b = 0,7+0,5 e G4b = 0,8+0,3) e o diâmetro (mm) do maior folículo em D8 (G1b = 13,0+3,3; G2b = 12,0+2,3; G3b = 10,5+2,9 e G4b = 11,4+0,6) também não foram alterada pelo perfil de liberação a partir dos dispositivos. Diante desses resultados, conclui-se que animais com a presença de CL e P4 endógena durante o protocolo de IATF consomem menor quantidade de P4 dos implantes que animais sem CL; implantes de P4 novos mantêm níveis satisfatórios de P4 para a IATF, independentemente da condição fisiológica da fêmea tratada; dispositivos reutilizados oriundos de animais em diferentes condições de ciclicidade não mantêm perfis de progesterona semelhantes àqueles do uso prévio (1º uso); a dinâmica folicular não foi alterada em função da ausência de padrão na liberação de P4 dos implantes reutilizados quando comparados a implantes novos e que Implantes novos, a despeito da maior liberação de progesterona, não interferem no padrão de desenvolvimento folicular em novilhas.

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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. / 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.

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.

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.