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Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-17Bitstream added on 2014-06-13T20:59:32Z : No. of bitstreams: 1 trevisol_e_me_botfmvz.pdf: 451549 bytes, checksum: 24926e449f40ad04e47e00cb8e719536 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O objetivo do presente estudo foi estudar as alterações da concentração plasmática de progesterona (P4), perfusão sanguínea e volume luteais após administração de sub-dose de cloprostenol sódico (análogo da PGF2α) no sexto dia do ciclo estral em bovinos. Foram utilizadas 18 vacas da raça Caracu adultas, e em todas foram inicialmente sincronizadas as ovulações antes de serem iniciados os três módulo experimentais. Em cada módulos as vacas foram divididas aleatoriamente entre os grupos 1 (desafiadas com 2 mL de solução fisiológica 0,9%; IM, n=6), 2 (duas doses de 500 μg de cloprostenol sódico com intervalo de 2 horas, n=6) e 3 (83,33 μg de cloprostenol sódico IM, n=7). As coletas de dados foram iniciadas com avaliações ultrassonográficas modo B e Power-Doppler, seguida de coleta de sangue para dosagem de P4, realizadas nos momentos 0, 8, 16, 24, 32, 40 e 48 horas após o tratamento. Os dados foram submetidos a analise de variância e para diferenças entre as médias foi aplicado o teste TUKEY com 5% de probabilidade. Os tratamentos influenciaram as concentrações plasmáticas de P4, perfusão sanguínea e volume luteal. No tratamento 1 não foi observado alterações na P4, perfusão sanguínea e volume luteal, porém no tratamento 2 a progesterona diminuiu no momento 16 e nos momentos seguintes foram semelhas ao observados inicialmente. A perfusão sanguínea e volume luteal diminuíram 48 horas após o tratamento 2. No tratamento 3 a P4 diminuiu até a hora 16 e voltou a aumentar, igualando a valores iniciais.Porém a perfusão sanguínea e volume não alteraram. No grupo luteólise quando excluído um animal que não apresentou luteólise, a P4 manteve-se baixa a partir da hora 16, e no grupo luteólise parcial quando excluído o animal que apresentou luteólise total, a P4 48 horas após tratamento foi maior que na hora 16... / The objective of this study was to evaluate progesterone plasmatic concentration, blood flow and luteal volume after cloprostenol (PGF2α analogue) sub-doses on sixth day of bovine estrus cycles. Initially all animals has the ovulations synchronize to initiate every stage. In tree firsts stage on day 6 the animals receive treatment and in every stage it were randomly allocate between group 1 (2 mL Physiologic solution 0,9%; IM, n=6 ), 2 (two doses 500 μg cloprostenol 2 hours interval, n=6) and 3 (83,33 μg cloprostenol IM on D6, n=7). Data collection were initiate with mode B ultrasonography and Power-Doppler evaluations follow by blood collection for progesterone (P4) measure at moments 0, 8, 16, 24, 32, 40 e 48 hours after treatment. Data was submitted to variance analyze between mean the TUKEY test with 5% probability was applied. Treatments influenced P4 concentration, blood flow and luteal volume. On treatment 1 there was no P4, blood perfusion and luteal volume changes, but on treatment 2 P4 decrease on moment 16 the follow moments was similar to the initial.Blood flow and luteal volume decreased 48 hours after treatment 2. In treatment 3 was observed a decrease in P4 concentrations from moment 0 to 16 with a increase returning to initial values after this moment. Although this, blood flow and luteal volume did not change. On luteolysis group when excluded a animal that had partial luteolysis P4 concentrations remained low after hour 16. On partial luteolysis group when a animal that had luteolysis was excluded P4 at 48 hours after treatment was higher than 16 hour. Is possible to conclude that 83,33 μg cloprostenol causes partial luteolysis and that is characterized by an initial P4 decrease 16 hours after treatment follow by luteal function rescue on the follow moments

Orientador: João Carlos Pinheiro Ferreira / Banca: Mario Binelli / Banca: Roberto Sartori Filho / Resumo: O objetivo do presente estudo foi estudar as alterações da concentração plasmática de progesterona (P4), perfusão sanguínea e volume luteais após administração de sub-dose de cloprostenol sódico (análogo da PGF2α) no sexto dia do ciclo estral em bovinos. Foram utilizadas 18 vacas da raça Caracu adultas, e em todas foram inicialmente sincronizadas as ovulações antes de serem iniciados os três módulo experimentais. Em cada módulos as vacas foram divididas aleatoriamente entre os grupos 1 (desafiadas com 2 mL de solução fisiológica 0,9%; IM, n=6), 2 (duas doses de 500 μg de cloprostenol sódico com intervalo de 2 horas, n=6) e 3 (83,33 μg de cloprostenol sódico IM, n=7). As coletas de dados foram iniciadas com avaliações ultrassonográficas modo B e Power-Doppler, seguida de coleta de sangue para dosagem de P4, realizadas nos momentos 0, 8, 16, 24, 32, 40 e 48 horas após o tratamento. Os dados foram submetidos a analise de variância e para diferenças entre as médias foi aplicado o teste TUKEY com 5% de probabilidade. Os tratamentos influenciaram as concentrações plasmáticas de P4, perfusão sanguínea e volume luteal. No tratamento 1 não foi observado alterações na P4, perfusão sanguínea e volume luteal, porém no tratamento 2 a progesterona diminuiu no momento 16 e nos momentos seguintes foram semelhas ao observados inicialmente. A perfusão sanguínea e volume luteal diminuíram 48 horas após o tratamento 2. No tratamento 3 a P4 diminuiu até a hora 16 e voltou a aumentar, igualando a valores iniciais.Porém a perfusão sanguínea e volume não alteraram. No grupo luteólise quando excluído um animal que não apresentou luteólise, a P4 manteve-se baixa a partir da hora 16, e no grupo luteólise parcial quando excluído o animal que apresentou luteólise total, a P4 48 horas após tratamento foi maior que na hora 16... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate progesterone plasmatic concentration, blood flow and luteal volume after cloprostenol (PGF2α analogue) sub-doses on sixth day of bovine estrus cycles. Initially all animals has the ovulations synchronize to initiate every stage. In tree firsts stage on day 6 the animals receive treatment and in every stage it were randomly allocate between group 1 (2 mL Physiologic solution 0,9%; IM, n=6 ), 2 (two doses 500 μg cloprostenol 2 hours interval, n=6) and 3 (83,33 μg cloprostenol IM on D6, n=7). Data collection were initiate with mode B ultrasonography and Power-Doppler evaluations follow by blood collection for progesterone (P4) measure at moments 0, 8, 16, 24, 32, 40 e 48 hours after treatment. Data was submitted to variance analyze between mean the TUKEY test with 5% probability was applied. Treatments influenced P4 concentration, blood flow and luteal volume. On treatment 1 there was no P4, blood perfusion and luteal volume changes, but on treatment 2 P4 decrease on moment 16 the follow moments was similar to the initial.Blood flow and luteal volume decreased 48 hours after treatment 2. In treatment 3 was observed a decrease in P4 concentrations from moment 0 to 16 with a increase returning to initial values after this moment. Although this, blood flow and luteal volume did not change. On luteolysis group when excluded a animal that had partial luteolysis P4 concentrations remained low after hour 16. On partial luteolysis group when a animal that had luteolysis was excluded P4 at 48 hours after treatment was higher than 16 hour. Is possible to conclude that 83,33 μg cloprostenol causes partial luteolysis and that is characterized by an initial P4 decrease 16 hours after treatment follow by luteal function rescue on the follow moments / Mestre

Orientador: José Luiz Moraes Vasconcelos / Banca: Rui Machado / Banca: Heraldo César Gonçalves / Resumo: Foram realizados quatro experimentos com o objetivo de avaliar possíveis mecanismos relacionados ao aumento da prenhez com a utilização de ácidos graxos poliinsaturados (PF). No exp. 1 foram utilizadas 51 vacas multíparas Nelore não lactantes, ovuladas, para avaliar se PF alteram a produção de progesterona (P4) e o momento da luteólise. No exp. 2 foram utilizadas 43 vacas multíparas Nelore não lactantes, ovuladas, para avaliar se PF alteram a sensibilidade do corpo lúteo de seis dias a aplicação de prostaglandina (i.m. 12,5mg de dinoprost trometamina, Lutalyse). No exp. 3 foram utilizadas 27 vacas multíparas Nelore, ovuladas, com 30 a 40 dias pós parto para avaliar se PF diminuem a incidência de ciclo curto. Os tratamentos utilizados foram: grupo controle (100g mineral + 100g milho + 100g caolin vaca dia); grupo SF (100g mineral + 100g milho + 100g/vaca/dia de Megalac (7-9% C18:2; Arm&Hammer a Church&Dwight Company, EUA); grupo PF (100g mineral + 100g milho + 100g/vaca/dia de Megalac-E QGN, Brasil: 40-42% de C18:2; 2-3% de C18:3). No exp. 4 (1457 vacas multíparas Nelore) foi avaliado se a suplementação com PF pós IATF por diferentes períodos altera a taxa de prenhez. Nos experimentos 1, 2 e 3 não foi detectado efeito de PF nas concentrações de P4 durante o período avaliado, no momento da luteólise e na incidência de ciclo curto (P>0,1). Ao se agrupar os dados do Exp 1 e 2, a concentração de P4 (P=0,01) no dia 6 foi maior nos animais suplementados com PF em relação ao SF e controle (4,45; 3,25; 3,48 ng/ml, respectivamente; EPM=0,278). No experimento 4 vacas recebendo PF por 21 e 28 dias após a IATF tiveram maior (P<0,05) taxa de prenhez (50,38%) quando comparadas com os outros tratamentos agrupados (42,38%). Não foi detectado diferença entre os tratamentos PF21 (50,99%) e PF28 (49,81). Os resultados em conjunto mostram que apesar de suplementação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Four experiments were designed to evaluate the possible mechanisms related to the increased pregnancy rates in cows supplemented with PF. In Experiment 1, 51 ovulated non-lactating Nelore multiparous cows were used to evaluate if PF supplementation affects circulating progesterone (P4) concentrations and timing of luteolysis. In Experiment 2, 43 ovulated non-lactating Nelore multiparous cows were used to evaluate if PF supplementation alters the sensitivity of a 6-d corpus luteum to exogenous prostaglandin treatment. In Experiment 3, 27 ovulated postpartum Nelore cows were used to evaluate if PF supplementation affects the incidence of premature luteolysis. Beginning at the d of estrus, the daily treatments in these 3 experiments were: Control (0.1 Kg Mineral + 0.1 Kg corn + 0.1 Kg kaolin); SF (0.1 Kg Mineral + 0.1 Kg Megalac-S® [7-9% linoleic acid] + 0.1 Kg corn), this group was used just in experimental 1 and 2; PF (0.1 Kg Mineral + 0.1 Kg Megalac-E® [40-42% linoleic acid; 2-3% linolenic acid] + 0.1 Kg corn). In Experiment 4, we evaluated if the length of PF supplementation in different times after timed-AI alters the pregnancy rate. No effect was detected on P4 concentration, luteolysis or short cycle (P>0.1), but when the cows of exp. 1 and 2 were grouped had higher (P=0.01) concentration of P4) on cows that were supplemented with PF compared with Sf or control (4.45; 3.25; 3.48 ng/ml, respectively; SEM=0,278. Cows supplemented with PF during 21 (PF21,) or 28 d post- AI (PF28) had higher pregnancy rates (50.38%; P < 0.05) than cows from other treatments (42.38%). There was no difference between PF21 (50,99%) and PF28 (49,81%) treatments. These experiments indicated that the possible mechanism for greater conception with PF supplementation post-AI may be due to effects on embryo development, animals supplemented for more than 21d had greater pregnancy rates. / Mestre

Prostaglandin F2α (PGF2α) is commonly given to female cattle intramuscularly (IM) for the synchronization of estrus. A novel site for administration of PGF2α that improves beef quality assurance is the ischiorectal fossa (IRF). The objective of this study was to determine whether administration of PGF2α in the IRF results in a similar physiologic response to administration of PGF2α given IM. Yearling angus-cross heifers (n=112) were blocked by weight and randomly assigned within blocks to be injected with 5 mL PGF2α either IM in the neck or in the IRF. Blood samples were taken at 0, 8, 16, 24, 36, and 48 h post-injection. Serum samples were analyzed for progesterone concentration using a radioimmunoassay. Progesterone concentration curves for each heifer were plotted to determine luteolysis. The median times to luteolysis for neck and IRF injections were 18.1 hrs and 20.0 hrs, respectively (p=0.06). Angus cross commercial beef cows (n=1471) at least 30 days post-partum were blocked by age and randomly assigned to be injected with 5 mL PGF2α either IM in the neck muscle or in IRF as part of a 7-Day CO-Synch + CIDR ovulation protocol. Pregnancy diagnosis was performed via ultrasound at 60 days post insemination. Results were analyzed with Proc Glimmix (SAS). Pregnancy rates for neck and IRF injections were 52.6% and 57.2%, respectively (p=0.06). In summary, injection of PGF2α in the IRF for estrus synchronization and lysis of the corpus luteum did not differ from injection in the neck muscle. Utilizing the ischiorectal fossa as an injection site for PGF2α may be considered as an alternative that more closely aligns with beef quality assurance objectives. / Master of Science

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)

O 17&#946;-E2 estimula a expressão de receptores endometriais, ER e OXTR. A ativação de OXTR induz a ativação da cadeia de síntese de PGF2&#945;. A hipótese do presente estudo é que as enzimas de síntese de PGF2&#945; são reguladas pelo 17&#946;-E2. Objetivou-se determinar os efeitos do 17&#946;-E2 na expressão gênica e proteica, assim como na localização de proteínas envolvidas na síntese de PGF2&#945;. Vacas Nelore multíparas, solteiras e cíclicas foram sincronizadas por aplicação de BE e inserção de dispositivo de P4. Após 8 dias realizou-se a remoção do dispositivo de P4 e aplicação de PGF2&#945;, seguido por 4 dias de observação de estro (D0=dia do estro). Entre D14 e D27 foram realizadas avaliações diárias da área do CL (cm2), fluxo sanguíneo (%) e concentração plasmática de progesterona (P4). No D15 as vacas foram divididas em três grupos: Controle (C; não tratado;N= 10), Placebo (P; 6mL de etanol 50%; IV; N= 21) e Estradiol (E; 3mg 17&#946;-E2; 6mL de etanol 50%; IV;N=21). Subsequente aos tratamentos, biópsias uterinas foram coletadas nos tempos 0h (C; N=10); 4h (E4h, N=11 e P4h; N=10) ou 7h (E7h, N=10 e P7h, N=11). Amostras de sangue foram obtidas nos tempos 0h a 7h, para mensuração das concentrações PGFM no D15. O grupo E apresentou acentuada diminuição da área do CL, fluxo sanguíneo e concentração de P4 (P&lt;0,05), comparado ao grupo P. Comparado ao grupo P, as vacas do grupo E anteciparam o dia da luteólise funcional e estrutural em 2 e 3 dias, respectivamente. O grupo E apresentou maior concentração de PGFM nos tempos 4h, 6h e 7h (P&lt;0,05), comparado ao grupo P. A quantificação dos transcritos realizada por qPCR (N=6/grupo). Na hora 4, a abundância dos genes ESR1, ESR2, PRKC&#945;, PRKC&#946;, PLA2G4, AKR1B1 e AKR1C4 foi menor nas amostras E4h, enquanto OXTR foi maior nas mesmas amostras comparando-se com as amostras P4h (P&lt;0,05). A expressão gênica de PTGS2 não diferiu entre os grupos E4h e P4h (P&gt;0,05). Na hora 7, as amostras E7h também apresentaram menor abundância de ESR1, PRKC&#945;, PRKC&#946;, AKR1B1 e AKR1C4 (P&lt;0,05) e houve tendência para menor expressão de ESR2, comparado às amostras P7h. Contudo, não houve diferença na abundância de OXTR, PLA2G4 e PTGS2 entre as amostras E7h e P7h (P&gt;0,05). A abundância da enzima PKC&#945; analisada por Western Blotting (N=3/grupo) foi diminuída tanto nas amostras E4h como nas E7h, em relação às amostras P4h e P7h, respectivamente. Na avaliação por imunohistoquímica (N=5/grupo), o grupo E4h apresentou maior imunomarcação de PGR no epitélio glandular (P&lt; 0,05) e houve tendência para maior imunomarcação de PKC&#978; no epitélio luminal, comparado ao grupo P4h (P=0,08). Houve tendência para menor imunomarcação de ER&#945; no epitélio glandular do grupo E4h comparado ao grupo E7h (P=0,1). Concluí-se que a aplicação do 17&#946;-E2 levou a redução da maioria dos transcritos das moléculas de síntese de PGF2&#945;, assim como da abundância de PKC&#945;. O possível mecanismo para a estimulação de PGFM por 17&#946;-E2 pode incluir o aumento da ativação de enzimas que participam na cascata de síntese de PGF2&#945;. / 17&#946;-E2 stimulates the expression of endometrial receptors, ER and OXTR. Activation of OXTR induces the activation of the synthesis of PGF2&#945; pathway. The central hypothesis is that the enzymes involved in PGF2 synthesis are reguleted by 17&#946;-E2. The objective of this study was to determine the effects of 17&#946;-E2 on gene and protein expression and localization of the enzymes involved in PGF2&#945; synthesis. Multiparous, non-lactating and cyclic Nelore cows were synchronized by BE application and P4 device insertion. After 8 days the P4 device was removed and a single dose of PGF2&#945; applied, followed by 4 days of estrus detection (D0 = day of estrus). Daily measurements of CL area (cm2), blood flow (%), and plasma progesterone concentration (P4) were performed between D14 and D27. On D15 cows were divided into three groups: Control (C, untreated, N = 10), Placebo (P; 6mL of ethanol 50%, IV; N = 21) and Estradiol (E; 3mg 17&#946;-E2; Ethanol 50%, IR: N = 21). After the treatments administration, uterine biopsies were collected at times 0h (C; N = 10); 4h (E4h, N = 11 and P4h, N = 10) or 7h (E7h, N = 10 and P7h, N = 11). Blood samples were obtained from time 0h to 7h for the measurement of the PGFM concentrations on D15. Group E showed a marked decrease in CL area, blood flow, and P4 concentration (P &lt;0.05) compared to group P. Also, when compared to group P, cows from group E anticipated the day of functional and structural luteolysis in 2 and 3 days, respectively. Group E presented higher concentration of PGFM at 4h, 6h and 7h (P &lt;0.05), compared to group P. The transcripts abundance was performed by qPCR (N = 6 / group). The transcripts abundance of ESR1, ESR2, PRKC&#945;, PRKC&#946;, PLA2G4, AKR1B1, and AKR1C4 genes was lower in the E4h samples, while OXTR was higher in the same samples compared to the P4h (P &lt;0.05) samples in the time 4h. The gene expression of PTGS2 did not differ between groups E4h and P4h (P&gt; 0.05). At time 7h, samples E7h also had lower abundance of ESR1, PRKC&#945;, PRKC&#946;, AKR1B1 and AKR1C4 (P &lt;0.05) and there was a tendency for lower ESR2 expression, compared to samples P7h. Nevertheless, there was no difference in the abundance of OXTR, PLA2G4, and PTGS2 between samples E7h and P7h (P&gt; 0.05). The abundance of the PKC&#945; enzyme analyzed by Western Blotting (N = 3 / group) was decreased in both the E4h and E7h samples, relative to the samples P4h and P7h, respectively. In the evaluation by immunohistochemistry (N = 5 / group), the E4h group presented greater PGR immunostaining in the glandular epithelium (P &lt;0.05) and there was a tendency for a greater immunostaining of PKC&#978; in the luminal epithelium, compared to the P4h group (P = 0,08). There was a tendency for lower ER&#945; immunostaining in the glandular epithelium of the E4h group compared to the E7h group (P = 0.1). It was concluded that the application of 17&#946;-E2 led to the reduction of most of the transcripts of the PGF2&#945; synthesis molecules, as well as the abundance of PKC&#945;. The possible mechanism for stimulation of PGFM by 17&#946;-E2 may include increased activation of enzymes that participate in the cascade of PGF2&#945; synthesis.

Orientador: Claudia Maria Bertan Membrive / Banca: Flávia Simone Munin / Banca: Fábio Ermínio Mingatto / Resumo: Em fêmeas bovinas, a liberação de prostaglandina F2a (PGF2a) pode ser induzida in vivo pelo estradiol (E2), entretanto, o papel do E2 na síntese de PGF2a ainda não foi esclarecido. Considerando que as concentrações plasmáticas de PGF2a aumentam 3,5 horas após a aplicação de E2 in vivo, acredita-se que o E2 ative não somente enzimas, mas também estimule a síntese de proteínas essenciais para a produção de PGF2a, como a PKC e PLA2. O presente estudo objetivou avaliar o efeito do E2 no incremento da concentração das proteínas totais no endométrio e na modificação da composição protéica de explantes endometriais de fêmeas bovinas tratadas com E2 no 17º dia do ciclo estral. A hipótese é que a administração de E2 em fêmeas bovinas no 17º dia do ciclo estral incrementa a concentração de proteínas no endométrio e modifica a composição protéica nos explantes endometriais. Para tanto, fêmeas bovinas mestiças (Bos taurus taurus vs Bos taurus indicus) foram tratadas no 17° dia do ciclo estral, via intravenosa, com 0mg (Grupo Controle; n=6) ou 3mg de E2 (Grupo E2; n=6) e abatidas duas horas após o tratamento. Explantes endometriais foram isolados e submetidos à extração de proteínas totais. As amostras de proteínas, referentes aos explantes de cada animal, foram avaliadas por eletroforese unidimensional em gel de poliacrilamida 10% SDS-PAGE e coradas com Coomasie Blue ou Nitrato de Prata. A concentração de proteínas totais não diferiu entre os grupos (p=0,1158) e foi de 6296,10 + 439,90μg/mL para o Grupo Controle e de 8426,56 + 1156,00μg/mL para o Grupo E2. Nos géis corados com Coomasie Blue foram identificadas 18 bandas protéicas e não foi observada diferença significativa (p>0,05) no perfil protéico dos explantes endometriais. Na coloração com Nitrato de Prata, foram identificadas 12 bandas protéicas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In cows the release of prostaglandin F2a (PGF2a) can be induced in vivo by estradiol (E2), however, the role of E2 in the synthesis of PGF2a has not yet been clarified. Considering that plasma concentrations of PGF2a increased 3.5 hours after application of E2 in vivo, it is believed that E2 not only active enzymes, but also stimulates the synthesis of essential proteins for the production of PGF2a, as PKC and PLA2 . This study aimed to evaluate the effect of E2 in increasing the concentration of total protein in the endometrium and changes in protein composition of endometrial explants from cows treated with E2 at the 17th day of estrous cycle. The hypothesis is that the administration of E2 in cows on the 17th day of the estrous cycle increases the concentration of protein in the endometrium and changes the composition of protein in endometrial explants. Therefore, days of the estrous cycle, crossbred cows were treated at 17th day of estrous cycle intravenously with 0 mg (Control Group; n = 6) or 3 mg of E2 (E2 Group; n = 6) and killed two hours after treatment. Endometrial explants were isolated and subjected to extraction of total protein. Samples of proteins related to the explants from each animal were analyzed by one-dimensional electrophoresis on polyacrylamide gel 10% SDSPAGE and stained with Coomasie Blue or silver nitrate. The concentration of total protein did not differ between groups (p = 0.1158) and was 6296.10 + 439.90μg/mL for the Control Group and of 8426.56 + 1156.00μg/mL for E2 Group. In gels stained with Coomasie Blue were identified 18 protein bands and there was no significant difference (p>0.05) in the protein profile of endometrial explants. Staining with Silver Nitrate, was identified 12 protein bands and was found in E2 Group greater relative percentage of the bands for the molecular weight of... (Complete abstract, click electronic access below) / Mestre

O 17&#946;-E2 estimula a expressão de receptores endometriais, ER e OXTR. A ativação de OXTR induz a ativação da cadeia de síntese de PGF2&#945;. A hipótese do presente estudo é que as enzimas de síntese de PGF2&#945; são reguladas pelo 17&#946;-E2. Objetivou-se determinar os efeitos do 17&#946;-E2 na expressão gênica e proteica, assim como na localização de proteínas envolvidas na síntese de PGF2&#945;. Vacas Nelore multíparas, solteiras e cíclicas foram sincronizadas por aplicação de BE e inserção de dispositivo de P4. Após 8 dias realizou-se a remoção do dispositivo de P4 e aplicação de PGF2&#945;, seguido por 4 dias de observação de estro (D0=dia do estro). Entre D14 e D27 foram realizadas avaliações diárias da área do CL (cm2), fluxo sanguíneo (%) e concentração plasmática de progesterona (P4). No D15 as vacas foram divididas em três grupos: Controle (C; não tratado;N= 10), Placebo (P; 6mL de etanol 50%; IV; N= 21) e Estradiol (E; 3mg 17&#946;-E2; 6mL de etanol 50%; IV;N=21). Subsequente aos tratamentos, biópsias uterinas foram coletadas nos tempos 0h (C; N=10); 4h (E4h, N=11 e P4h; N=10) ou 7h (E7h, N=10 e P7h, N=11). Amostras de sangue foram obtidas nos tempos 0h a 7h, para mensuração das concentrações PGFM no D15. O grupo E apresentou acentuada diminuição da área do CL, fluxo sanguíneo e concentração de P4 (P&lt;0,05), comparado ao grupo P. Comparado ao grupo P, as vacas do grupo E anteciparam o dia da luteólise funcional e estrutural em 2 e 3 dias, respectivamente. O grupo E apresentou maior concentração de PGFM nos tempos 4h, 6h e 7h (P&lt;0,05), comparado ao grupo P. A quantificação dos transcritos realizada por qPCR (N=6/grupo). Na hora 4, a abundância dos genes ESR1, ESR2, PRKC&#945;, PRKC&#946;, PLA2G4, AKR1B1 e AKR1C4 foi menor nas amostras E4h, enquanto OXTR foi maior nas mesmas amostras comparando-se com as amostras P4h (P&lt;0,05). A expressão gênica de PTGS2 não diferiu entre os grupos E4h e P4h (P&gt;0,05). Na hora 7, as amostras E7h também apresentaram menor abundância de ESR1, PRKC&#945;, PRKC&#946;, AKR1B1 e AKR1C4 (P&lt;0,05) e houve tendência para menor expressão de ESR2, comparado às amostras P7h. Contudo, não houve diferença na abundância de OXTR, PLA2G4 e PTGS2 entre as amostras E7h e P7h (P&gt;0,05). A abundância da enzima PKC&#945; analisada por Western Blotting (N=3/grupo) foi diminuída tanto nas amostras E4h como nas E7h, em relação às amostras P4h e P7h, respectivamente. Na avaliação por imunohistoquímica (N=5/grupo), o grupo E4h apresentou maior imunomarcação de PGR no epitélio glandular (P&lt; 0,05) e houve tendência para maior imunomarcação de PKC&#978; no epitélio luminal, comparado ao grupo P4h (P=0,08). Houve tendência para menor imunomarcação de ER&#945; no epitélio glandular do grupo E4h comparado ao grupo E7h (P=0,1). Concluí-se que a aplicação do 17&#946;-E2 levou a redução da maioria dos transcritos das moléculas de síntese de PGF2&#945;, assim como da abundância de PKC&#945;. O possível mecanismo para a estimulação de PGFM por 17&#946;-E2 pode incluir o aumento da ativação de enzimas que participam na cascata de síntese de PGF2&#945;. / 17&#946;-E2 stimulates the expression of endometrial receptors, ER and OXTR. Activation of OXTR induces the activation of the synthesis of PGF2&#945; pathway. The central hypothesis is that the enzymes involved in PGF2 synthesis are reguleted by 17&#946;-E2. The objective of this study was to determine the effects of 17&#946;-E2 on gene and protein expression and localization of the enzymes involved in PGF2&#945; synthesis. Multiparous, non-lactating and cyclic Nelore cows were synchronized by BE application and P4 device insertion. After 8 days the P4 device was removed and a single dose of PGF2&#945; applied, followed by 4 days of estrus detection (D0 = day of estrus). Daily measurements of CL area (cm2), blood flow (%), and plasma progesterone concentration (P4) were performed between D14 and D27. On D15 cows were divided into three groups: Control (C, untreated, N = 10), Placebo (P; 6mL of ethanol 50%, IV; N = 21) and Estradiol (E; 3mg 17&#946;-E2; Ethanol 50%, IR: N = 21). After the treatments administration, uterine biopsies were collected at times 0h (C; N = 10); 4h (E4h, N = 11 and P4h, N = 10) or 7h (E7h, N = 10 and P7h, N = 11). Blood samples were obtained from time 0h to 7h for the measurement of the PGFM concentrations on D15. Group E showed a marked decrease in CL area, blood flow, and P4 concentration (P &lt;0.05) compared to group P. Also, when compared to group P, cows from group E anticipated the day of functional and structural luteolysis in 2 and 3 days, respectively. Group E presented higher concentration of PGFM at 4h, 6h and 7h (P &lt;0.05), compared to group P. The transcripts abundance was performed by qPCR (N = 6 / group). The transcripts abundance of ESR1, ESR2, PRKC&#945;, PRKC&#946;, PLA2G4, AKR1B1, and AKR1C4 genes was lower in the E4h samples, while OXTR was higher in the same samples compared to the P4h (P &lt;0.05) samples in the time 4h. The gene expression of PTGS2 did not differ between groups E4h and P4h (P&gt; 0.05). At time 7h, samples E7h also had lower abundance of ESR1, PRKC&#945;, PRKC&#946;, AKR1B1 and AKR1C4 (P &lt;0.05) and there was a tendency for lower ESR2 expression, compared to samples P7h. Nevertheless, there was no difference in the abundance of OXTR, PLA2G4, and PTGS2 between samples E7h and P7h (P&gt; 0.05). The abundance of the PKC&#945; enzyme analyzed by Western Blotting (N = 3 / group) was decreased in both the E4h and E7h samples, relative to the samples P4h and P7h, respectively. In the evaluation by immunohistochemistry (N = 5 / group), the E4h group presented greater PGR immunostaining in the glandular epithelium (P &lt;0.05) and there was a tendency for a greater immunostaining of PKC&#978; in the luminal epithelium, compared to the P4h group (P = 0,08). There was a tendency for lower ER&#945; immunostaining in the glandular epithelium of the E4h group compared to the E7h group (P = 0.1). It was concluded that the application of 17&#946;-E2 led to the reduction of most of the transcripts of the PGF2&#945; synthesis molecules, as well as the abundance of PKC&#945;. The possible mechanism for stimulation of PGFM by 17&#946;-E2 may include increased activation of enzymes that participate in the cascade of PGF2&#945; synthesis.