In vivo and in vitro matured bovine oocytes present a distinct pattern of single-cell gene expression.

Oocyte gene expression is a well controlled event that promotes gamete competence to undergo maturation, fertilization, and to support early embryo development, directly affecting reproductive outcomes. Considering that in vivo controlled ovarian stimulation or in vitro maturation (IVM) for the acquisition of mature oocytes has distinct implications for gene expression, we sought to evaluate the effects of these procedures on the expression of competence-related genes in single-cell oocytes. Healthy Nelore cows of reproductive age were synchronized to harvest in vivo matured oocytes; ovaries from slaughtered animals were used to obtain cumulus-oocyte complexes that were in vitro matured. Single-cell gene expression was performed using TaqMan Low-Density Arrays and 42 genes were evaluated. In silico analysis of protein interactions and Gene Ontology (GO) analysis was performed. Reduced gene expression was observed for 24 targets in IVM oocytes when compared with those of in vivo matured oocytes (P < 0.05). Differences ranged from 1.5-fold to 4.8-fold higher in in vivo oocytes and the BMP15 (5.28), GDF9 (6.23), NOBOX (7.25), HSPA8 (7.85) and MSX1 (11.00) showed the greatest fold increases. The strongest score of functional interactions was observed between the CDC20 and CKS2, with the differentially expressed gene CDC20 being the main marker behind GO enrichment. IVM negatively affected the expression of important genes related to oocyte competency, and showed higher expression levels in in vivo matured oocytes. In vivo controlled ovarian stimulation may be a better strategy to achieve proper oocyte competence and increase the success of assisted reproductive technologies.

Small extracellular vesicles derived from in vivo- or in vitro-produced bovine blastocysts have different miRNAs profiles-Implications for embryo-maternal recognition.

In vivo- and in vitro-produced bovine embryos have different metabolic profiles and differences in gene transcription patterns. These embryos also have a distinct ability to establish and sustain early pregnancies. Small extracellular vesicles (sEVs) are secreted by embryos and carry bioactive molecules, such as miRNAs. We hypothesize that in vivo or in vitro-produced bovine hatched blastocysts on Day 9 and the sEVs secreted by them have different miRNA profiles. To address this hypothesis, embryos of both groups were placed in in vitro culture on Day 7. After 48 h, hatched embryos and hatched embryo-conditioned media (eCM) of both groups were collected. A total of 210 miRNAs were detected in embryos of both groups, of these 6 miRNAs were downregulated, while 7 miRNAs were upregulated in vitro group when compared to in vivo group. sEVs were isolated from eCM to determine miRNA profile. A total of 106 miRNAs were detected in both groups, including 14 miRNAs upregulated in sEVs from in vivo-eCM, and 2 miRNAs upregulated in sEVs from in vitro-eCM. These miRNAs express in embryos and sEVs secreted by them regulate early embryonic developmental and endometrial pathways, which can modify embryo-maternal communication during early pregnancy and consequently affect pregnancy establishment.

A diferença de idade é um fator determinante na modulação do estresse oxidativo muscular induzido pelo exercício agudo

Resumo Este estudo teve por objetivo comparar os efeitos do exercício agudo com carga de trabalho relativa em gastrocnêmio de ratos Wistar com três e 18 meses. Os animais foram divididos em quatro grupos (n = 6): controle de três e 18 meses; exercício de três e 18 meses. Os grupos exercitados foram submetidos a uma única sessão de corrida na esteira com 60 minutos em velocidade de 0,8 km/h e 1,2 km/h. Foram analisados parâmetros do metabolismo (níveis de lactato, conteúdo de glicogênio, níveis de succinato desidrogenase, citocromo c oxidase e de estresse oxidativo (SOD, CAT e GPX). Os resultados mostraram um aumento nos marcadores metabólicos após o exercício agudo independentemente da idade dos animais e similaridade nos danos oxidativos apesar de as enzimas antioxidantes apresentarem modulação diferenciada nos animais mais velhos em comparação com animais jovens.

Abstract The effects of exercise on the generation of reactive oxygen species and the response to muscle oxidative stress determines longevity. This study compares the effects of acute exercise with similar relative workload in rats with 3 and 18 months. The animals were divided into four groups (n = 6): control 3-months; exercise 3-months; control 18-months and exercise 18-months. Exercised groups underwent to a single bout of running with 60 minutes in the speed 0.8 km/h (18 months) and 1.2 km/h (3 months). Metabolic parameters (lactate levels, glycogen content, succinate dehydrogenase levels, cytochrome c oxidase) and oxidative stress (activity of superoxide dismutase, catalase and glutathione peroxidase). The results showed an increase in metabolic markers after acute exercise regardless of the age and similarity in oxidative damage even if the antioxidant enzymes are age-dependent. These results suggest that the effects of acute exercise at moderate intensity, affects the metabolism independent of age, and, that the differential response in enzymatic antioxidant system between groups do not promote protection against oxidative damage.

Este estudio ha tenido como objetivo comparar los efectos del ejercicio agudo con carga de trabajo relativamente similar en el gastrocnemio de ratas Wistar con 3 y 8 meses. Se separó a los animales en cuatro grupos (n = 6): control de 3 y 18 meses, y ejercicio de 3 y 18 meses. Se sometió a los grupos de ejercicio a una sola sesión de carrera en la cinta durante 60 minutos con una velocidad de 0,8 km/h y 1,2 km/h. Se analizaron algunos parámetros del metabolismo (niveles de lactato, contenido de glucógeno, niveles de succionato-deshidrogenasa, citocromo c-oxidasa e de estrés oxidativo [superóxido-dismutasa, catalasa y glutatión-peroxidasa]). Los resultados mostraron un aumento de los marcadores metabólicos después del ejercicio agudo independientemente de la edad de los animales y la similitud de los daños oxidativos aunque las enzimas antioxidantes presentan modulación diferenciada en animales viejos en comparación con animales jóvenes.

Effects of different frequencies of physical training on electron transport chain and oxidative damage in healthy mice

The present study investigated the effect of different frequencies (three and five times a week) on electron transport chain and oxidative stress after 8 weeks of run training. Methods: Eighteen male mice (CF1, 30-35g) were distributed into the following groups (n=6): untrained (UT); trained three-time per week (T3) and trained five- time per week (T5). All training sessions were at the same intensity and duration (45min/day) in a treadmill for small animals. Forty-eight hours after the last training session, the animals were killed by decapitation and quadriceps (red portion) was removed and stored at -70ºC. Succinate dehydrogenase (SDH), complexes I, II, II-III, IV and hydroperoxides were measured. Results: Training sessions for five times per week were more effective in increasing the mitochondrial respiratory chain enzyme activities (SDH, complexes I, II, II-III, IV) as well as in decreasing the formation hydroperoxides than sessions performed for three times training per week (p<0.05). Conclusion: Our findings clearly showed that a higher the frequency of training session promotes a greater activity of the electron transport chain and consequently reduces the oxidative stress in healthy animals.(AU)

A Flow Cytometry Protocol to Estimate DNA Content in the Yellowtail Tetra Astyanax altiparanae.

The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100) at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5%) and sucrose (74 mM) and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at -20°C) and fixation in saturated NaCl solution, acetic methanol (1:3), ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl2.7H20; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 µg mL-1; preservation procedure: somatic cells (dorsal fin samples) frozen at -20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

In vitro maturation impacts cumulus-oocyte complex metabolism and stress in cattle.

The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di- and triacylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress-related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the ß-oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.

Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development.

Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3-6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3-6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3-6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3-6 mm follicles) in embryos. Interestingly, the addition of EVs from 3-6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3-6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.

The role of continuous versus fractionated physical training on muscle oxidative stress parameters and calcium-handling proteins in aged rats.

Age-associated decline in skeletal muscle mass and strength is associated with oxidative stress and Ca(2+) homeostasis disturbance. Exercise should be considered a viable modality to combat aging of skeletal muscle. This study aimed to investigate whether continuous and fractionated training could be useful tools to attenuate oxidative damage and retain calcium-handling proteins. We conducted the study using 24-month-old male Wistar rats, divided into control, continuous, and fractionated groups. Animals ran at 13 m min(-1) for five consecutive days (except weekends) for 6 weeks, for a total period of 42 days. Each session comprised 45 min of exercise, either continuous or divided into three daily sessions of 15 min each. Metabolic and oxidative stress markers, protein levels of mitochondrial transcription factors, and calcium-handling proteins were analyzed. Continuous exercise resulted in reduced ROS production as well as showed a decrease in TBARS levels and carbonyl content. On the other hand, fractionated training increased the antioxidant enzyme activities. The ryanodine receptor and phospholamban protein were regulated by continuous training while sodium calcium exchange protein was increased by the fractionated training. These data suggest that intracellular Ca(2+) can be modulated by various training stimuli. In addition, the modulation of oxidative stress by continuous and fractionated training may play an important regulatory role in the muscular contraction mechanism of aged rats, due to changes in calcium metabolism.

Follicle and corpus luteum size and vascularity as predictors of fertility at the time of artificial insemination and embryo transfer in beef cattle / Tamanho e vascularização do folículo e corpo lúteo como preditores de fertilidade após inseminação artificial e transferência de embriões em gado de corte

Two ultrasound based fertility prediction methods were tested prior to embryo transfer (ET) and artificial insemination (AI) in cattle. Female bovines were submitted to estrous synchronization prior to ET and AI. Animals were scanned immediately before ET and AI procedure to target follicle and corpus luteum (CL) size and vascularity. In addition, inseminated animals were also scanned eleven days after insemination to target CL size and vascularity. All data was compared with fertility by using gestational diagnosis 35 days after ovulation. Prior to ET, CL vascularity showed a positive correlation with fertility, and no pregnancy occurred in animals with less than 40% of CL vascularity. Prior to AI and also eleven days after AI, no relationship with fertility was seen in all parameters analyzed (follicle and CL size and vascularity), and contrary, cows with CL vascularity greater than 70% exhibit lower fertility. In inseminated animals, follicle size and vascularity was positive related with CL size and vascularity, as shown by the presence of greater CL size and vascularity originated from follicle with also greater size and vascularity. This is the first time that ultrasound based fertility prediction methods were tested prior to ET and AI and showed an application in ET, but not in AI programs. Further studies are needed including hormone profile evaluation to improve conclusion.

Duas técnicas de predição de fertilidade, baseadas em ultrassonografia, foram testadas no momento da transferência de embriões (TE) e inseminação artificial (IA) em bovinos. Fêmeas bovinas foram submetidas a protocolos de sincronização de estro para TE e IA. Os animais foram escaneados por ultrassonografia imediatamente antes do procedimento de TE e IA para identificar o tamanho e vascularização do folículo e corpo lúteo (CL). Além disso, os animais inseminados foram escaneados onze dias após a inseminação para identificar o tamanho e vascularização do CL. Todos os dados foram comparados com a fertilidade utilizando-se do diagnóstico gestacional 35 dias após a ovulação. No momento da TE, a vascularização do CL apresentou-se positivamente relacionada com a fertilidade, sendo que animais com menos de 40% de vascularização do CL não ficaram gestantes. No momento da IA, assim como onze dias após a IA, nenhuma relação foi encontrada entre fertilidade e os parâmetros analizados (tamanho e vascularização do folículo e CL), enquanto que contrariamente, houve uma queda na fertilidade em vacas com vascularização do CL acima de 70%. Nos animais inseminados, o tamanho e vascularização do folículo foi positivamente relacionado ao tamanho e vascularização do CL, demonstrado pela presença de um CL maior e mais vascularizado proveniente de um folículo maior e mais vascularizado. O presente estudo é o primeiro a mostrar métodos de predição de fertilidade baseados em ultrassonografia no momento da IA e TE, demonstrando uma aplicabilidade prática no momento da TE. Novos estudos são necessários para suportar os resultados mostrados, incluindo análises hormonais.

Plasma steroid dynamics in late- and near-term naturally and artificially conceived bovine pregnancies as elucidated by multihormone high-resolution LC-MS/MS.

The plasma levels of corticosteroids and sex steroids during pregnancy are key indicators of mammalian placental function and the onset of parturition. Steroid hormones are believed to be disturbed in pregnancies produced using assisted reproductive technologies (ARTs) due to placental dysfunction and the frequently observed lack of parturition signals. To elucidate the plasma steroid dynamics, a liquid chromatography-tandem mass spectrometry method was developed and used to determine the levels of corticosteroids (corticosterone, 11-deoxycortisol, and cortisol) and their direct precursors (progesterone and 17α-OH-progesterone) as well as sex steroids (androstenedione, estrone, estrone sulfate, testosterone, and 17ß-estradiol) in bovine plasma. The levels of these 10 steroids in recipient cows carrying naturally conceived (control), in vitro fertilized (IVF), or cloned (somatic cell nuclear transfer) conceptuses were compared during late-term pregnancy (30 days before parturition), during near-term pregnancy (1 day before parturition), and on the day of parturition (day 0). Significant differences were observed among the corticosteroid levels: higher levels of corticosterone, 11-deoxycortisol, and cortisol were detected in cloned pregnancies at day 30; lower levels of corticosterone were observed in ART-derived pregnancies at days 1 and 0; and estrone and estradiol levels were higher in IVF pregnancies throughout the final development. These results suggested an upregulation of the P450C11 and P450C21 enzymes 30 days before parturition in somatic cell nuclear transfer pregnancies and an overactivation of the aromatase enzyme in IVF pregnancies. Taken together, the monitoring of multiple steroid hormones revealed that the pregnancies obtained using ART exhibited plasma steroid concentration dynamics compatible with the dysregulation of steroidogenic tissues.

Conceptus-induced changes in the gene expression of blood immune cells and the ultrasound-accessed luteal function in beef cattle: how early can we detect pregnancy?

We aimed to identify the functional characteristics of the corpus luteum (CL) by color Doppler ultrasonography and changes in interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs) during early pregnancy in beef cows. We then aimed to use these features to establish earlier pregnancy diagnosis methods. In experiment 1, the CL size and blood flow were accessed by Doppler ultrasonography, and the PBMCs were isolated on Days 8, 12, 15, 18, 20, 22, 25, 30, 45, and 60 post-timed artificial insemination (TAI) from pregnant (n = 10) and nonpregnant cows (n = 12). The abundance of ISG (OAS1, MX1, MX2, and ISG15) transcripts was measured by quantitative PCR. Analyses of OAS1 and MX2 expression in isolated PBMCs (ISG-PBMC method) and Doppler imaging of CL (Doppler-US method) were performed to test the accuracy of these methods for the diagnosis of pregnancy on Day 20 post-TAI (n = 110; experiment 2). In experiment 1, the luteal volume and blood flow were reduced in nonpregnant cows during the first weeks post-TAI, but an evaluation of CL vascularization and size was efficient in identifying nonpregnant cows on Day 20 post-TAI. The expression of ISGs in PBMCs can be stimulated by the presence of a viable conceptus between Days 15 and 22 post-TAI, and the expression of these genes reaches a peak on Day 20. In experiment 2, the Doppler-US and ISG-PBMC methods resulted in similar specificity (85.5 and 87.7%, respectively). However, only the Doppler-US method resulted in 100% sensitivity. In conclusion, the greatest abundance of ISGs in PBMCs and a high detection of luteolysis by Doppler imaging on Day 20 post-TAI can be feasibly used for the earlier detection of nonpregnant cows in reproductive programs. The level of accuracy for our described pregnancy methods is high on Day 20 (80%-91%), but only the Doppler imaging method results in an absence of false-negative diagnoses.

Intratracheal co-administration of antioxidants and ceftriaxone reduces pulmonary injury and mortality rate in an experimental model of sepsis.

BACKGROUND AND OBJECTIVE: Recent studies showed that both sepsis and antibiotic therapy are associated with cell death and linked to reactive oxygen species generation. This study investigated the effects of intratracheal administration of combinations of antioxidants (n-acetyl cysteine (NAC), vitamins C and E) in the treatment of sepsis-induced lung injury. METHODS: Ninety-six male Wistar rats subjected to sepsis were treated with ceftriaxone plus NAC with or without vitamins C and E and compared to appropriate controls. As an index of oxidative damage protein carbonyls, sulfhydryl groups, lipid peroxidation and superoxide anion were measured, as well as superoxide dismutase and catalase. Histopathological alterations and mortality rate were also analyzed. RESULTS: Twenty-four hours after sepsis induction, markers of oxidative stress increased in all lungs examined. Ceftriaxone plus intratracheal combination of NAC, vitamins C and E decreased lung injury in infected animals by reducing superoxide anion production (54%), lipid peroxidation (53%) and protein carbonyl (58%) and restored the redox status (7.5 times). This therapy also reduced the imbalance of antioxidant enzymes activities and attenuated the alveolar architectural disorganization, inflammatory cell infiltration and pulmonary oedema. Survival increased from 66.6% with ceftriaxone to 83.2% with ceftriaxone plus antioxidants. CONCLUSIONS: Ceftriaxone plus intratracheal co-administration of antioxidants provides better protection, by decreasing pulmonary oxidative stress, limiting histophatological alterations and improving survival. Antioxidants should be explored as a co-adjuvant in the treatment of severe lung injury.

Effects of taurine supplementation following eccentric exercise in young adults.

The purpose of the present study was to investigate the effects of taurine supplementation on muscle performance, oxidative stress, and inflammation response after eccentric exercise (EE) in males. Twenty-one participants (mean age, 21 ± 6 years; weight, 78.2 ± 5 kg; height, 176 ± 7 cm) were selected and randomly divided into two groups: placebo (n = 10) and taurine (n = 11). Fourteen days after starting supplementation, subjects performed EE (3 sets until exhaustion, with EE of the elbow flexors on the Scott bench, 80% 1 repetition maximum (RM)). Blood samples were collected and muscle performance was measured on days 1, 14, 16, 18, and 21 after starting the supplements. Then, performance, muscle damage, oxidative stress, and inflammatory markers were analyzed. The taurine supplementation resulted in increased strength levels and thiol total content and decreased muscle soreness, lactate dehydrogenase level, creatine kinase activity, and oxidative damage (xylenol and protein carbonyl). Antioxidant enzymes (superoxide dismutase, catalase, and gluthatione peroxidase) and inflammatory markers (tumor necrosis factor, interleukin-1ß (IL-1ß), and interleukin-10 (IL-10)) were not altered during the recovery period compared with the placebo group. The results suggest that taurine supplementation represents an important factor in improving performance and decreasing muscle damage and oxidative stress but does not decrease the inflammatory response after EE.

Creatine supplementation does not decrease oxidative stress and inflammation in skeletal muscle after eccentric exercise.

Thirty-six male rats were used; divided into 6 groups (n = 6): saline; creatine (Cr); eccentric exercise (EE) plus saline 24 h (saline + 24 h); eccentric exercise plus Cr 24 h (Cr + 24 h); eccentric exercise plus saline 48 h (saline + 48 h); and eccentric exercise plus Cr 48 h (Cr + 48 h). Cr supplementation was administered as a solution of 300 mg · kg body weight(-1) · day(-1) in 1 mL water, for two weeks, before the eccentric exercise. The animals were submitted to one downhill run session at 1.0 km · h(-1) until exhaustion. Twenty-four and forty-eight hours after the exercise, the animals were killed, and the quadriceps were removed. Creatine kinase levels, superoxide production, thiobarbituric acid reactive substances (TBARS) level, carbonyl content, total thiol content, superoxide dismutase, catalase, glutathione peroxidase, interleukin-1b (IL-1ß), nuclear factor kappa B (NF-kb), and tumour necrosis factor (TNF) were analysed. Cr supplementation neither decreases Cr kinase, superoxide production, lipoperoxidation, carbonylation, total thiol, IL-1ß, NF-kb, or TNF nor alters the enzyme activity of superoxide dismutase, catalase, and glutathione peroxides in relation to the saline group, respectively (P < 0.05). There are positive correlations between Cr kinase and TBARS and TNF-α 48 hours after eccentric exercise. The present study suggests that Cr supplementation does not decrease oxidative stress and inflammation after eccentric contraction.

Reproductive stem cell differentiation: extracellular matrix, tissue microenvironment, and growth factors direct the mesenchymal stem cell lineage commitment.

The mesenchymal stem cells (MSCs) have awakened interest in regenerative medicine due to its high capability to proliferate and differentiate in multiple specialized lineages under defined conditions. The reproductive system is considered a valuable source of MSCs, which needs further investigations. Many factors have been reported as critical for these cell lineage specification and determination. In this review, we discuss the main effects of extracellular matrix or tissue environment and growth factors in the cell lineage commitment, including the reproductive stem cells. The MSCs responses to culture medium stimuli or to soluble factors probably occur through several intracellular activation pathways. However, the molecular mechanisms in which the cells respond to these mechanical or chemical perturbations remain elusive. Recent findings suggest a synergic effect of microenvironment and soluble cell culture factors affecting cell differentiation. For future applications in cell therapy, protocols of reproductive MSCs differentiation must be established.

Acute exercise induce endothelial nitric oxide synthase phosphorylation via Akt and AMP-activated protein kinase in aorta of rats: Role of reactive oxygen species.

BACKGROUND: Acute exercise increases reactive oxygen species (ROS) levels, including hydrogen peroxide (H2O2). H2O2 promotes endothelial nitric oxide synthase (eNOS) activation and phosphorylation in endothelial cells. With this in mind, the present study was designed to evaluate ex vivo eNOS phosphorylation in rat aortas incubated with H2O2 and to test this hypothesis in vivo in the aortas of rats submitted to acute exercise. METHODS: For ex vivo studies, six groups of aortic tissue were formed: control, H2O2, N-acetylcysteine (NAC), LY294002, compound C, and LY294002 plus compound C. While incubation with H2O2 increased Akt, AMPK and eNOS phosphorylation, pre-incubation with NAC strongly reduced the phosphorylation of these enzymes. For in vivo studies, male Wistar rats were divided into four groups: control, cont+NAC, exercise, and exer+NAC. After a 3h swimming session, animals were decapitated and aortas were excised for biochemical and immunoblotting analysis. RESULTS: Acute exercise increased superoxide levels and dichlorofluorescein (DCF) concentrations, and this increase was related to phosphorylation of Akt, AMPK and eNOS. On the other hand, use of NAC reduced superoxide levels and DCF concentration. Reduced superoxide levels and DCF in the exer+NAC group were associated with decreased Akt, AMPK and eNOS phosphorylation. These results appear to be connected with vascular function because VASP phosphorylation increased in acute exercise and decreased in exer+NAC. CONCLUSION: Our results indicate that ROS induced by acute exercise play the important role of activating eNOS, a process apparently mediated by Akt and AMPK.

Impact of different resistance training protocols on muscular oxidative stress parameters.

This study analyzes oxidative stress in skeletal muscle using different resisted training protocols. We hypothesize that different types of training produce different specifics. To test our hypothesis, we defined 3 resistance training protocols and investigated the respective biochemical responses in muscle. Twenty-four male Wistar rats were distributed in 4 groups: untrained (UT), muscular resistance training (RT), hypertrophy training (HT), and strength training (ST). After 12 weeks of training on alternate days, the red portion of the brachioradialis was removed and the following parameters were assessed: lactate and glycogen content, superoxide production, antioxidant enzyme content, and activities (superoxide dismutase, SOD; catalase, CAT; GPx, glutathione peroxidase). Thiobarbituric acid-reactive substances (TBARS), carbonyl, and thiol groups were also measured. Results showed increased superoxide production (UT = 5.348 ± 0.889; RT = 5.117 ± 0,651; HT = 8.412 ± 0.431; ST = 6.354 ± 0.552), SOD (UT = 0.078 ± 0.0163; RT = 0.101 ± 0.013; HT = 0.533 ± 0.109; ST = 0.388 ± 0.058), GPx (UT = 0.290 ± 0.023; RT = 0.348 ± 0.014; HT = 0.529 ± 0.049; ST = 0.384 ± 0.038) activities, and content of GPx (HT = 3.8 times; ST = 3.0 times) compared with the UT group. CAT activity was lower (UT = 3.966 ± 0.670; RT = 3.474 ± 0.583; HT = 2.276 ± 0.302; ST = 2.028 ± 0.471) in HT and ST groups. Oxidative damage was observed in the HT group (TBARS = 0.082 ± 0.009; carbonyl = 0.73 ± 0.053; thiol = 12.78 ± 0.917) compared with the UT group. These findings indicate that HT causes an imbalance in oxidative parameters in favor of pro-oxidants, causing oxidative stress in skeletal muscle.

Effects of different physical training protocols on ventricular oxidative stress parameters in infarction-induced rats.

AIM: Physical exercise is important in the prevention and treatment of cardiovascular diseases. Nevertheless, controversy remains around type and intensity of effort required for significant biochemical protective changes. This study investigates two exercise protocols on ventricular oxidative parameters in rats post-infarction. MAIN METHODS: Thirty-six 2-month-old male Wistar rats were divided in two groups (n=18): Sham and acute myocardial infarction (AMI) conducted by blocking the coronary artery. Thirty days after AMI, animals were divided in 6 subgroups (n=6): sham, sham+continuous training (60 min), sham+interval training, AMI, AMI+continuous training, and AMI+interval training. Training was conducted in water (30-32°C) 5 times a week for 6 weeks. Animals were sacrificed 48 h after the last exercise routine. Left ventricles were used for oxidative stress analyses (antioxidant enzyme activity and level, oxidative damage) and HIF1α and cit c oxidase expression. KEY FINDINGS: After AMI, both exercise models decreased superoxide levels significantly. Training routines did not alter SOD expression and activity, though CAT expression increased with continuous training and GPX level diminished in both training groups, which coincided with the increase in GPX activity. Lipid damage decreased only in the continuous training group, while protein damage decreased only in the interval training group. Cytochrome C increased in both groups, while HIF-1 α dropped significantly after both exercise protocols. SIGNIFICANCE: Significant improvement occurred in myocardium redox status in rats challenged with AMI after different training routines. However, continuous training seems to be more efficient in improving the parameters analyzed.

Alterations in muscular oxidative metabolism parameters in incremental treadmill exercise test in untrained rats.

The present study investigates the effects of incremental exercise test on muscular oxidative metabolism. Thirty-six 2-month-old male Wistar rats were distributed in seven groups that performed exercise at different levels: first level (control), second level (0.6 km/h), third level (0.6 and 0.8 km/h), fourth level (0.6, 0.8 and 1.0 km/h), fifth level (0.6, 0.8, 1.0 and 1.2 km/h), sixth level (0.6, 0.8, 1.0, 1.2 and 1.4 km/h), and seventh level (0.6, 0.8, 1.0, 1.2, 1.4 and 1.6 km/h). At the end of the exercise challenge, level of blood lactate (BL), glycogen content (MG), creatine kinase (CK), complexes (CI, CII, CIII, CIV), oxidative damage, succinate dehydrogenase (SDH), cytochrome c oxidase as well as antioxidant enzymes (SOD and CAT) expression were measured. The speed of 1.0 km/h increased BL level, while 1.2 km/h decreased MG and increased serum CK. Increased SDH expression was observed after intensity levels 6 and 7, and cytochrome c oxidase expression increased after levels 5, 6 and 7, in comparison with lower intensity levels, ETC enzyme activities increased when exercise was applied at intensities of 0.8 km/h (CI), 1.0 km/h (CII and CIII), and 1.2 km/h (CIV). The increase in SOD expression did not occur as observed for superoxide production, except for rats that underwent exercise at level 7, but CAT expression increased significantly in all levels, starting from level 3. Our results show interesting alterations in the muscular metabolism parameters, and suggest a differential response of muscle oxidative metabolism when intense exercise is applied at different speeds.