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- Dynamics of notch pathway expression during mouse testis post-natal development and along the spermatogenic cyclePublication . Murta, Daniel; Batista, Marta; Silva, Elisabete; Trindade, Alexandre; Henrique, Domingos; Duarte, António; Lopes da Costa, LuísThe transcription and expression patterns of Notch pathway components (Notch 1–3, Delta1 and 4, Jagged1) and effectors (Hes1, Hes2, Hes5 and Nrarp) were evaluated (through RT-PCR and IHC) in the mouse testis at key moments of post-natal development, and along the adult spermatogenic cycle. Notch pathway components and effectors are transcribed in the testis and expressed in germ, Sertoli and Leydig cells, and each Notch component shows a specific cell-type and timewindow expression pattern. This expression at key testis developmental events prompt for a role of Notch signaling in prepubertal spermatogonia quiescence, onset of spermatogenesis, and regulation of the spermatogenic cycle.
- Cytokines and angiogenesis in the corpus luteumPublication . Galvão, António M.; Ferreira-Dias, Graça; Skarzynski, Dariusz J.In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum(CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant fromthe balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase.Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.
- Role of tumor necrosis factor-α, interferon-γ and Fas-ligand on in vitro nitric oxide activity in the corpus luteumPublication . Galvão, A.M.; Szóstek, Anna Z.; Skarzynski, Dariusz J.; Ferreira-Dias, Graça M.Normal reproductive function involves the expression of inflammatory mediators. Regarding the corpus luteum (CL), cytokines promote the cross-talk between immune, vascular and steroidogenic cells, among others. Moreover, TNF, IFNG and FASL were shown to regulate equine CL establishment and regression. We hypothesized that cytokines action on equine CL may be mediated by nitric oxide (NO), through the regulation of endothelial NO synthase (eNOS) expression. TNF increased eNOS mRNA level and NO metabolite (nitrite) production during CL growth. Cytokines combined action (TNF + IFNG + FASL) promoted eNOS protein upregulation in mid-CL and nitrite production in mid and late-CL. However, in late-CL, TNF alone decreased nitrite secretion. These results indicate that in equine CL, cytokines TNF, IFNG and FASL regulate NO activity, via eNOS expression modulation.
- Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen rich plants : potent endocrine disruptors inducing infertilityPublication . Ferreira-Dias, G.; Botelho, M.; Zagrajczuk, A.; Rebordão, M.R.; Galvão, A.M.; Bravo, P. Pinto; Piotrowska-Tomala, K.; Szóstek, A.Z.; Wiczkowski, W.; Piskula, M.; Fradinho, M.J.; Skarzynski, D.J.Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-β estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E2), progesterone (P4), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.
- Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia modelsPublication . Cristofaro, Brunella; Yu Shi; Faria, Marcella; Suchting, Steven; Leroyer, Aurelie S.; Trindade, Alexandre; Duarte, Antonio; Zovein, Ann C.; Iruela-Arispe, M. Luisa; Nih, Lina R.; Kubis, Nathalie; Henrion, Daniel; Loufrani, Laurent; Todiras, Mihail; Schleifenbaum, Johanna; Gollasch, Maik; Zhuang, Zhen W.; Simons, Michael; Eichmann, Anne; Le Noble, FerdinandArteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Deltalike 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic lossand gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4+/− mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4+/− mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.
- Carcass fat partitioning and meat quality of Alentejana and Barrosã young bulls fed high or low maize silage dietsPublication . Costa, Ana S. H.; Costa, Paulo; Bessa, Rui J. B.; Lemos, José P. C.; Simões, Jorge A.; Silva, José Santos; Fontes, Carlos M. G. A.; Prates, José A. M.This study assessed the effect of breed and diet on carcass composition, particularly fat partitioning, and meat quality in young bulls. An experiment with forty young bulls from two phylogenetically distant Portuguese bovine breeds, Alentejana and Barrosã, fed two diets with different maize silage to concentrate ratios, but isoenergetic and isonitrogenous, was carried out until the animals reached 18 months of age. In the longissimus lumborum muscle, Barrosã bulls fed the low silage diet had the highest intramuscular fat (IMF) content. Bulls fed the low silage diet also had the highest IMF content in the semitendinosus muscle. Diet determined the proportions of total visceral fat and individual fat depots. Under these experimental conditions, it was shown that the genetic background is a major determinant of carcass composition and meat quality, and that the dietary differences studied had limited effect on carcass composition.
- Expression of genes controlling fat deposition in two genetically diverse beef cattle breeds fed high or low silage dietsPublication . Costa, Ana Sofia Henriques da; Pires, Virgínia Maria Rico; Fontes, Carlos Mendes Godinho Andrade; Prates, José António MestreBackground: Both genetic background and finishing system can alter fat deposition, thus indicating their influence on adipogenic and lipogenic factors. However, the molecular mechanisms underlying fat deposition and fatty acid composition in beef cattle are not fully understood. This study aimed to assess the effect of breed and dietary silage level on the expression patterns of key genes controlling lipid metabolism in subcutaneous adipose tissue (SAT) and longissimus lumborum (LL) muscle of cattle. To that purpose, forty bulls from two genetically diverse Portuguese bovine breeds with distinct maturity rates, Alentejana and Barrosã, were selected and fed either low (30% maize silage/70% concentrate) or high silage (70% maize silage/30% concentrate) diets. Results: The results suggested that enhanced deposition of fatty acids in the SAT from Barrosã bulls, when compared to Alentejana, could be due to higher expression levels of lipogenesis (SCD and LPL) and β-oxidation (CRAT) related genes. Our results also indicated that SREBF1 expression in the SAT is increased by feeding the low silage diet. Together, these results point out to a higher lipid turnover in the SAT of Barrosã bulls when compared to Alentejana. In turn, lipid deposition in the LL muscle is related to the expression of adipogenic (PPARG and FABP4) and lipogenic (ACACA and SCD) genes. The positive correlation between ACACA expression levels and total lipids, as well trans fatty acids, points to ACACA as a major player in intramuscular deposition in ruminants. Moreover, results reinforce the role of FABP4 in intramuscular fat development and the SAT as the major site for lipid metabolism in ruminants. Conclusions: Overall, the results showed that SAT and LL muscle fatty acid composition are mostly dependent on the genetic background. In addition, dietary silage level impacted on muscle lipid metabolism to a greater extent than on that of SAT, as evaluated by gene expression levels of adipogenic and lipogenic factors. Moreover, the response to diet composition evaluated through mRNA levels and fatty acid composition showed interesting differences between Alentejana and Barrosã bulls. These findings provide evidence that the genetic background should be taken into account while devising diet-based strategies to manipulate fatty acid composition of beef cattle tissues.
- Genetic background and diet impact beef fatty acid composition and stearoyl-CoA desaturase mRNA expressionPublication . Costa, Ana S. H.; Silva, Marta P.; Alfaia, Cristina P. M.; Pires, Virgínia M. R.; Fontes, Carlos M. G. A.; Bessa, Rui J. B.; Prates, José A. M.The intramuscular fat composition of ruminant meats influences the quality of the final product, which explains the increasing interest in assessing the fatty acid profile of meat from different production systems. In this study, it was hypothesized that there are breed- and diet induced variations on lipid metabolism in the muscle, which may be, at least partially, modulated by the stearoyl-CoA desaturase (SCD) gene expression levels. Forty purebred young bulls from two phylogenetically distant autochthonous cattle breeds, Alentejana and Barrosã(n = 20 for each breed), were assigned to two different diets (low vs. high silage) and slaughtered at 18 months of age. Meat fatty acid composition, including the detailed conjugated linoleic acid (CLA) isomeric profile, was determined along with the SCD mRNA levels. Meat from Barrosã bulls fed the low silage diet was richer in monounsaturated fatty acids, CLA and trans fatty acids, when compared to that from Alentejana bulls. The meat content in polyunsaturated fatty acids was similar across experimental groups. Moderate positive correlations between the SCD mRNA levels and the products of this enzyme activity were found, although they were not reflected on the calculated desaturase indices. Overall, these findings highlight the importance of taking into account the genetic background while devising feeding strategies to manipulate beef fatty acid composition.
- Effect of pig breed and dietary protein level on selected fatty acids and stearoyl-coenzyme A desaturase protein expression in longissimus muscle and subcutaneous fatPublication . Bessa, R. J. B.; Hughes, R. A.; Jeronimo, E.; Moreira, O. C.; Prates, J. A. M.; Doran, O.The aims of the study were 1) to investigate effects of a low protein diet on fatty acids content and composition of the LM and subcutaneous adipose tissue in 3 genetically diverse breeds, Large White × Landrace, Alentejano, and Bizaro, and 2) to determine whether the effect of the low protein diet of fatty acid composition is associated with dietary modulation of stearoyl-CoA desaturase (SCD) protein expression. The experiments were conducted on 12 Large White × Landrace, 12 Alentejano, and 10 Bízaro female and male pigs. The average animal BW at the beginning of experiments was 40.8, 40.7, and 38.3 kg for Large White × Landrace, Alentejano, and Bízaro, respectively, and the BW of animals at slaughter was 90 kg. The diets contained 202 or 169 g/kg DM of CP (high and low protein diets, respectively) and were balanced in essential AA. The diets were fed until the animals reached 90 kg BW (approximately 73 d). It was established that Large White × Landrace pigs had a less (P = 0.001) total fatty acid content in subcutaneous adipose tissue when compared with Alentejano and Bízaro and less (P < 0.001) intramuscular fat (IMF) content when compared with Alentejano. There was a positive relationship between SCD protein expression in the LM and MUFA content (r = 0.627, P = 0.029) and SCD protein expression and total muscle fatty acids content (r = 0.725, P = 0.008) in Large White × Landrace but not in Alentejano and Bizaro breeds. It has been suggested that SCD protein expression is associated with regulation of fat deposition only in breeds with genetic predisposition to a low IMF content.
- Nutritional status of Lusitano broodmares on extensive feeding systems: body condition, live weight and metabolic indicatorsPublication . Fradinho, Maria J.; Bessa, Rui J. B.; Martin-Rosset, William; Ferreira-Dias, Graça; Caldeira, Rui M.The present research aimed to evaluate the effects of foaling season and feeding management in extensive systems on the nutritional status of Lusitano broodmares throughout the gestation/lactation cycle, by assessment of body condition (BC), body weight (BW), and some blood metabolic indicators. Four groups of Lusitano broodmares (A, B, C, D) were monitored during four years, in a total of 119 gestation/lactation cycles. All mares were kept on pasture, and A and B mares were daily supplemented. Monthly, mares were weighed and BC evaluated. Suckling foals from these mares were also monitored for BW and withers height. Glucose, non-esterified fatty acids, urea and albumin concentrations were determined in blood. BW changes were influenced by reproductive stage and foaling season (P<0.001), reflecting also pasture availability. Changes on BC were observed (P<0.05), although with small amplitudes within each group. Higher scores were reached at the end of spring, decreasing 0.25 point until late summer. Early foaling had also a marked effect, hindering the recovery of BC along the cycle. Glucose values decreased from late gestation to early lactation (P<0.05) and lower levels were recorded during the summer months. Uremia was mainly influenced by the reproductive stage (P<0.05). Under nutrition was not detected. Foals born in February-March had higher average daily gain than those born in April-May (P<0.05), probably reflecting differences in milk production of the mares. BC and BW changes and, particularly, blood indicators showed an overall balanced nutritional status, reflecting an adaptation to feed availability and climate.