Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation.

Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.

Energy balance and hippo effector activity in endometrium and corpus luteum of early pregnant ewes.

CONTEXT: The establishment of pregnancy in cows requires uterine activity regulation of the main Hippo signalling effector yes-associated protein 1 (YAP). It remains unknown (1) how YAP activity at the corpus luteum (CL) correlates with early pregnancy-related events in ruminants; and (2) if YAP activity in the uterus and CL can be affected by metabolic disorders that may lead to pregnancy failure in ruminants. AIMS AND METHODS: To determine the effect of early pregnancy on total and phospho-YAP expression and its transcriptional activity in the CL, we compared non-pregnant vs pregnant ewes. To understand the YAP activity dysregulation with disorders that may result in pregnancy loss, we induced negative energy balance in pregnant ewes. KEY RESULTS AND CONCLUSIONS: Our main results indicate that early pregnancy alters the expression and activity patterns of YAP in the ovine CL but not in the endometrium. In addition, while our NEB-induced model fails to alter YAP activity at the endometrium level, we found that fasting during the first but not second week of pregnancy affects YAP activity in the CL of pregnant ewes. IMPLICATIONS: The data presented herein provide considerable insight into the activity of a signalling pathway that may be a key player in pregnancy recognition and establishment in ewes.

Trabecular Bone is Increased in a Rat Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) in an intricate disorder characterized by reproductive and metabolic abnormalities that may affect bone quality and strength along with the lifespan. The present study analysed the impact of postnatal androgenization (of a single dose of testosterone propionate 1.25 mg subcutaneously at day 5 of life) on bone development and markers of bone metabolism in adult female Wistar rats. Compared with healthy controls, the results of measurements of micro-computed tomography (microCT) of the distal femur of androgenized rats indicated an increased cortical bone volume voxel bone volume to total volume (VOX BV/TV) and higher trabecular number (Tb.n) with reduced trabecular separation (Tb.sp). A large magnitude effect size was observed in the levels of circulating bone formation Procollagen I N-terminal propeptide (P1NP) at day 60 of life; reabsorption cross-linked C-telopeptide of type I collagen (CTX) markers were similar between the androgenized and control rats at days 60 and 110 of life. The analysis of gene expression in bone indicated elements for an increased bone mass such as the reduction of the Dickkopf-1 factor (Dkk1) a negative regulator of osteoblast differentiation (bone formation) and the reduction of Interleukin 1-b (Il1b), an activator of osteoclast differentiation (bone reabsorption). Results from this study highlight the possible role of the developmental programming on bone microarchitecture with reference to young women with PCOS.

Virgin Coconut Oil Associated with High-Fat Diet Induces Metabolic Dysfunctions, Adipose Inflammation, and Hepatic Lipid Accumulation.

Obesity reaches an epidemic level worldwide, and this condition is associated with chronic low-grade inflammation and secondary comorbidities, largely driven by global changes in lifestyle and diet. Various dietary approaches are proposed for the obesity treatment and its associated metabolic disorders. Good taste, antioxidant functions, and vitamins have been attributed to virgin coconut oil (VCO). However, VCO contains a large amount of saturated fatty acids, and the consumption of this fat is associated with a number of secondary diseases. We evaluate the effects of VCO supplementation on biochemical, inflammatory, and oxidative stress parameters in rats fed with high-fat diet (HFD). After feeding with HFD for 12 weeks, the animals were supplemented with VCO for 30 days. HFD+VCO group increased in diet intake, weight gain, low-density lipoprotein cholesterol level, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These findings were accompanied by increased in hepatic lipid profile and fat deposition in the liver. Adipocyte hypertrophy was observed in the HFD+VCO group, which was associated with elevated expression of tumor necrosis factor alpha (TNF-α) in adipose tissue. These results revealed that VCO associated with HFD induced important metabolic alterations, adipose inflammation, and hepatic lipid accumulation in rats.

1α, 25-Dihydroxyvitamin D3 alters ectonucleotidase expression and activity in human cutaneous melanoma cells.

PURPOSE: We hypothesized that vitamin D decreases rates of adenosine formation in human cutaneous melanoma cells through the inhibition of extracellular adenosine 5'-triphosphate breakdown, thereby affecting tumor cell viability. Therefore, the objective of this study was to explore the mechanisms of action of 1α, 25-dihydroxyvitamin D3 (1,25(OH)2 D3) on the activity and expression of ectonucleotidases in cutaneous melanoma cells. METHODS: A human melanoma cell line, SK-Mel-28, was treated with 1 to 50 nM of the active vitamin D metabolite (1,25(OH)2 D3) over 24 hours, followed by determination of NTPDase1/CD39 and ecto-5'-nucleotidase/CD73 activity and expression rates of the purinergic system-related NTPDASE1, NT5E and adenosine deaminase and vitamin D receptor. An 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay was used to evaluate cellular viability. RESULTS: 1,25(OH)2 D3 decreased adenosine monophosphate hydrolysis via ecto-5'-nucleotidase/CD73 and expression of CD73, but did not change NTPDase1/CD39 activity; it increased the CD39 expression. We also observed an increase of cell viability at 1 nM, but this viability decreased as the concentrations of vitamin D active metabolite increased to 50 nM. There were no differences in gene expression levels. CONCLUSION: To the best of our knowledge, we showed for the first time a mechanism of control of adenosine production via modulation of the purinergic system in cutaneous melanoma cells treated with the active metabolite of vitamin D. This study provides original information regarding mechanisms, in which vitamin D plays a key role in preventing tumor progression in human melanoma cells.

Prenatal Androgenization of Ewes as a Model of Hirsutism in Polycystic Ovary Syndrome.

The main clinical feature associated with hyperandrogenism in polycystic ovary syndrome (PCOS) in humans is hirsutism, where hair increases its length, pigmentation, and particularly its diameter. Currently, it is not known whether PCOS animal models also exhibit changes in the hair. Therefore, the aim of this study was to explore the wool characteristics in sheep prenatally androgenized (PA) with testosterone propionate. After 4 and 13 months of life, wool was collected from the top of the shoulder of both females and males (both androgenized and controls). The offspring sheep were followed for up to 19 months of life to evaluate testosterone and androstenedione serum levels by ultra-high-performance liquid chromatography-tandem mass spectrometry, determine insulin and glucose response to intravenous glucose tolerance test, and address estrus cyclicity during the second breeding season. PA male animals showed a reduction in wool fiber diameter at 4 months of age compared with controls (P = 0.02) but not at 13 months, whereas PA females showed increased hair diameter at 13 months (P = 0.002), with no difference at 4 months. No substantial changes in other hair parameters (length, color, and medullation) were identified. In addition, increased levels of serum testosterone were observed in PA female sheep compared with controls at 12 months (P = 0.03). Our results indicate for the first time, to our knowledge, that changes in wool fiber diameter observed in PA ewes replicate, at the translational level, the increase in hair diameter in hirsute women with PCOS.

Effects of dietary microencapsulated Cymbopogon flexuosus essential oil on reproductive-related parameters in male Rhamdia quelen.

In aquaculture, nutrition and supplemented diets have been shown to affect broodstock reproductive performance. In this study, we investigated the effects of dietary supplementation with Cymbopogon flexuosus essential oil (CFEO) microcapsules on reproductive-related parameters in silver catfish (Rhamdia quelen) male broodfish. Adult male broodstocks were separated into three groups according to the concentrations of supplemented CFEO (0.0 = control; 1.0 or 3.0 mL per kg of diet). After 20 days under experimental conditions, the animals were euthanized and the gonads were harvested for gonadosomatic index, sperm analysis, oxidative stress, and histopathology; testosterone levels were measured in the plasma; gene expression of prl, smtl, pomca, and pomcb was assessed in the pituitary gland by real-time PCR. The results showed no alterations on reproductive parameters in R. quelen males treated with Cymbopogon flexuosus essential oil compared to the control-diet animals. In conclusion, CFEO microcapsules supplied for 20 days in the concentrations of 1.00 or 3.00 mL per kilogram of diet did not affect the reproduction criteria evaluated in this study in male silver catfish.

Systemic inflammatory and stress markers in cattle and sheep submitted to different reproductive procedures / Marcadores sistêmicos de inflamação e estresse em bovinos e ovinos submetidos a diferentes procedimentos reprodutivos

ABSTRACT: Previous studies have evaluated the effects of different reproductive procedures on discomfort markers in sheep and cattle. Such studies may help stimulate the adoption of techniques that are more beneficial for animal welfare. However, markers that are commonly used to evaluate discomfort are highly influenced by external factors. To overcome this, several systemic markers can be evaluated to more precisely identify stress, pain, and inflammation. Such markers include cortisol, acute phase proteins, bradykinin, and substance P. We aimed to review the potential markers of stress, pain, and inflammation, and discuss how and when they are regulated after different stimuli related to reproductive procedures in cattle and sheep. Furthermore, we aimed to review how reproductive procedures with different degrees of invasiveness cause stress and provide information that may help develop strategies to limit animal discomfort.

RESUMO: Estudos anteriores avaliaram o efeito de diferentes procedimentos reprodutivos sobre marcadores de desconforto em bovinos e ovinos. Tais estudos podem estimular a adoção de técnicas que preservem o bem-estar animal. Entretanto, os marcadores comumente utilizados apresentam alta influência de fatores externos. Para contornar isso, a avaliação conjunta de diferentes parâmetros sistêmicos pode ser utilizada para determinar com maior precisão a presença de estresse, dor ou inflamação, como cortisol, proteínas de fase aguda, a bradicinina e a substância P. O objetivo desta revisão é relacionar potenciais marcadores de inflamação e estresse, discutindo como e quando são regulados frente aos estímulos em bovinos e ovinos. Ainda, pretende-se revisar de que forma procedimentos reprodutivos com diferentes graus de invasividade acarretam em desconforto, fornecendo informações para a elaboração de estratégias que possibilitem minimizá-lo.

Prepartum progestagen supplementation in swine: a strategy to facilitate piglet care and prevent early parturition / Suplementação de progestágenos no pré-parto em suínos: estratégia para facilitar assistência aos leitões e evitar partos precoces

ABSTRACT: Gestation length in swine has a considerable amplitude and both early and delayed parturition are common. This variation increases the occurrence of unassisted farrowing and could lead to a wide-ranging age at weaning for piglets born from one batch. Supervision of sow parturition is crucial to reduce mortality of neonate piglets. To facilitate assistance, induction of farrowing using prostaglandin F2α (PGF) has been widely used in batch farrowing systems, whereby synchronization would concentrate the time of farrowing, allowing for better organization of employees. However, a viable alternative method that can be implemented to manage farrowing is to sustain high progestagen levels in the final days of gestation and, consequently, prevent early parturition. Efficient techniques to delay farrowing such as using oral progestagen supplementation have been previously described, but are only recently being considered for commercial use. The present manuscript reviews publications regarding delaying parturition and discusses the use of intravaginal devices (IVDs) containing progestagen. There is limited data addressing the effect of progestagen treatment during gestation on productive and reproductive performance. Therefore, future studies should focus on improving synchronization protocols following progestagen supplementation and evaluating piglet viability and sow fertility, before widely using progestagen supplementation to manipulate parturition.

RESUMO: Como a duração da gestação de suínos pode ter ampla variação, é comum a ocorrência de partos antecipados ou gestações prolongadas. Isso aumenta as chances de partos sem assistência e leva a uma grande variação de idade dos leitões dentro do lote de produção. Portanto, a supervisão do parto é indispensável para reduzir as perdas neonatais. Para facilitar o auxílio aos leitões, a indução do parto com prostaglandina F2α (PGF) é eficaz e amplamente utilizada, sendo indicada para concentrar os partos em momentos mais adequados, preferencialmente durante o horário com maior disponibilidade de colaboradores. Uma alternativa viável é manipular o momento do parto, através da manutenção de níveis plasmáticos elevados de progestágeno durante o final da gestação, a fim de evitar partos antecipados. Formas eficientes de evitar o parto através de suplementação oral de progestágenos foram descritas há décadas, mas apenas recentemente tem sido cogitada a utilização comercial. A presente revisão aborda estudos disponíveis na literatura relacionados ao protelamento do parto, incluindo a utilização de dispositivos intravaginais (DIVs) impregnados com progestágeno. São poucos os dados disponíveis relacionados ao uso de progestágenos na gestação com índices produtivos e reprodutivos. Portanto, alguns pontos ainda devem ser melhor avaliados, especialmente com relação à determinação da sincronia dos partos após o fim da suplementação com progestágenos, à viabilidade dos neonatos e à fertilidade subsequente das fêmeas antes da ampla adoção desta técnica.

Reversible meiotic arrest of bovine oocytes by EGFR inhibition and follicular hemisections.

The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 µM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 µM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.

Uncaria tomentosa extract alters the catabolism of adenine nucleotides and expression of ecto-5'-nucleotidase/CD73 and P2X7 and A1 receptors in the MDA-MB-231 cell line.

ETHOPHARMACOLOGICAL RELEVANCE: Uncaria tomentosa (Willd.) DC. (Rubiaceae) (Ut), also known as cat's claw, is a woody liana widely spread throughout the Amazon rainforest of Central and South America, containing many chemical constituents such as oxindole alkaloids, which are responsible for various biological activities. Since ancient times, the indigenous people of Peru have used it as a bark infusion for the treatment of a wide range of health problems gastric ulcers, arthritis and rheumatism. Recently, Ut is distributed worldwide and used as an immunomodulatory and anti-inflammatory herbal remedy. Additionally, U. tomentosa also has antitumural activity. However, little is known about the action of U. tomentosa on the purinergic system mechanisms, which is involved in tumor progression. AIM OF THE STUDY: Considering the pharmacological properties of U. tomentosa, we sought to evaluate the hydroalcoholic extract U tomentosa is able to influence the purinergic system in breast cancer cells, MDA-MB-231. Through the activity and expression of ectonucleotidases (NTPDase - CD39; Ecto-5'-nucleotidase - CD73) and purinergic repceptores (P2X7 and A1). MATERIALS AND METHODS: A hydroalcoholic extract was prepared in two concentrations, 250 and 500µg/mL. (Ut250; Ut500). The effect of these concentrations on the activity and expression of ectonucleotidases, as well as on the density of purinergic receptors were investigated in MDA-MB-231 breast cancer cells. Cells were treated with the hydroalcoholic extract of Uncaria tomentosa and/or doxorubicin (Doxo 1µM; Ut250+Doxo; Ut500+Doxo) for 24h. RESULTS: Although the results were not significant for the hydrolysis of the ATP, they presented an increase in the ADP hydrolysis in the Ut500+Doxo group when compared to the control group. Additionally, the activity of 5'-nucleotidase was inhibited in all groups when compared with the untreated group of cells. Inhibition of the enzyme was more evident in groups with U. tomentosa per se. The expression of CD39 was increased in the Ut250 and Ut250+Doxo groups when compared to the control group. No changes were found in the CD73 expression. Furthermore, a reduction in the density of the P2X7 receptor in all treated groups was detected. On the other hand, the density of the A1 receptor increased in all groups compared to the control group, with the exception of the Ut500+Doxo group. CONCLUSION: Therefore, we conclude that hydroalcoholic extract of U. tomentosa may be responsible for the reduction of adenosine levels in the extracellular medium, which accelerates tumor progression. Interestingly, the dysregulation of A1 and P2X7 receptors in the MDA-MB-231 cells exacerbate the proliferation of this cells and U. tomentosa treatment may be stimulate the antitumor activity of adenosine A1 receptor and control the P2X7 effects. Our study demonstrates the significant participation of purinergic pathway in the regulation of MDA-MB-231 progression; additionally, U. tomentosa treatment alone or combined with chemotherapy may favor the action of doxorubicin.

DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (P<0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200µM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P<0.05). Only the oocytes' cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.

Evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep / Avaliação de um kit de ensaio imunoenzimático na detecção de glicoproteínas associadas à prenhez bovina para o diagnóstico sorológico de gestação em ovinos

ABSTRACT: Pregnancy diagnosis is an important tool for farm management. Ultrasonography is the main technique used for pregnancy diagnosis in ewes. As an alternative, radioimmunoassay (RIA) allows accurate and early detection of pregnancy-associated glycoproteins (PAGs) in sheep blood. However, radioactive-based techniques, as RIA, have been increasingly inadvisable due to environmental risk. Homology between ovine and bovine PAGs is high, and ELISA kits used for PAGs detection in cattle are safer than RIA. Thus, this study aimed to evaluate the feasibility of PAGs detection for pregnancy diagnosis in sheep serum samples using an ELISA kit produced for cattle. The sensitivity and specificity of the ELISA kit were 93.5% and 98.9%, respectively, whereas positive and negative predictive values were 99.0% and 93.1%, respectively, in comparison to ultrasonography diagnostic (control). PAGs reached consistently detectable concentrations in ovine serum around 33 days after mating. Accuracy of the ELISA test was 96.1% from 33 days of pregnancy until lambing. After parturition, PAGs were still detectable seven days post-lambing. However, from 21 days post-parturition, PAGs from the previous pregnancy were no longer detected in serum samples. In conclusion, the bovine ELISA kit can accurately detect pregnancy in sheep 33 days following mating, while PAGs levels from the previous gestation are no longer detected from 21 days post-partum. The evaluated ELISA test is a reliable tool for pregnancy diagnosis in sheep at random stages and as a complementary exam at early gestation.

RESUMO: O diagnóstico de gestação é uma ferramenta importante no manejo da propriedade. A ultrassonografia é a principal técnica utilizada no diagnóstico de prenhez em ovelhas. Como alternativa, o radiomunoensaio (RIA) permite acurácia e a detecção precoce de proteínas associadas à prenhez (PAGs) no sangue. No entanto, as técnicas radioativas, como o RIA, têm sido cada vez mais desaconselháveis, devido ao risco ambiental. A homologia entre PAGs de ovinos e bovinos é alta, e os kits de ELISA utilizados para a detecção de PAGs em bovinos são mais seguros do que RIA. Portanto, este estudo teve como objetivo avaliar a viabilidade de detecção de PAGs no soro ovino para diagnóstico de gestação em soro ovino, utilizando um kit de ELISA produzido para bovinos. A sensibilidade e a especificidade do kit de ELISA foram de 93,5% e 98,9%, respectivamente, enquanto que os valores preditivos positivo e negativo foram de 99,0% e 93,1%, respectivamente, em comparação com a ultrassonografia (utilizada como referência). As PAGs atingiram concentrações consistentemente detectáveis no soro ovino em torno de 33 dias após o acasalamento. A acurácia do teste de ELISA foi de 96,1% a partir de 33 dias de gestação até o parto. As PAGs ainda eram detectáveis sete dias pós-parto. No entanto, a partir de 21 dias após o parto, as PAGs da prenhez anterior já não eram detectadas no soro. Em conclusão, o kit de ELISA bovino pode detectar a prenhez com precisão em ovelhas a partir de 33 dias após o acasalamento, e os níveis de PAGs da gestação anterior não são detectados a partir de 21 dias pós-parto. O teste de ELISA avaliado é uma ferramenta confiável para o diagnóstico de gestação em ovelhas em estágios aleatórios e como exame complementar no início da gestação.

Guaraná a Caffeine-Rich Food Increases Oxaliplatin Sensitivity of Colorectal HT-29 Cells by Apoptosis Pathway Modulation.

We investigated the in vitro effects of guaraná and its main metabolites (caffeine, theobromine and catechin) on cytotoxicity and cell proliferation on colorectal cancer (CRC) line HT-29 cells and on oxaliplatin sensitivity. The cells were exposed to different concentrations of guaraná extract with and without oxaliplatin. The concentrations of bioactive molecules were also estimated considering their potential proportion on guaraná hydro-alcoholic extract. Apoptosis effect was analyzed by annexin V quantification using flow cytometry, while apoptosis pathway gene modulation (p53, Bax/Bcl-2 genes ratio, caspases 8 and 3) was determined by qRT-PCR analysis. Cells exposed to guaraná at a concentration of 100 µg/mL presented a similar cytotoxic effect as HT-29 cells treated with oxaliplatin and did not affect the sensitivity of the drug. Guaraná presented cell anti-proliferative effect and increased anti-proliferative oxaliplatin sensitivity at all concentrations tested here. Guaraná was able to induce apoptosis and up-regulate the p53 and Bax/Bcl-2 genes.

Lack of FSH support enhances LIF-STAT3 signaling in granulosa cells of atretic follicles in cattle.

Subordinate follicles (SFs) of bovine follicular waves undergo atresia due to declining FSH concentrations; however, the signalling mechanisms have not been fully deciphered. We used an FSH-induced co-dominance model to determine the effect of FSH on signalling pathways in granulosa cells of the second-largest follicles (SF in control cows and co-dominant follicle (co-DF2) in FSH-treated cows). The SF was smaller than DF in control cows while diameters of co-DF1 and co-DF2 in FSH-treated cows were similar. The presence of cleaved CASP3 protein confirmed that granulosa cells of SFs, but not of DFs and co-DFs, were apoptotic. To determine the effect of FSH on molecular characteristics of the second-largest follicles, we generated relative variables for the second largest follicle in each cow. For this, variables of SF or co-DF2 were divided by the variables of the largest follicle DF or co-DF1 in each cow. There was higher transcript abundance of MAPK1/3 and AKT1/2/3 but lower abundance of phosphorylated MAPK3/1 in SF than co-DF2 granulosa cells. Abundance of mRNA and phosphorylated protein of STAT3 was higher in granulosa cells of control SF than FSH-treated co-DF2. SF granulosa cells had higher levels of LIFR and IL6ST transcripts, the two receptors involved in STAT3 activation. Further, lower transcript abundance of interleukin 6 receptor (IL6R), another receptor involved in STAT3 activation, indicated that STAT3 activation in SF granulosa cells could be mainly due to leukemia inhibitory factor (LIF) signalling. These results indicate that atresia due to lack of FSH is associated with activated LIF-STAT3 signalling in SF granulosa cells, as FSH treatment reversed such activation.

Crioprotetor para sêmen de carneiro a base de plasma de gema mantém membrana acrossomal intacta após a descongelação / Ram semen cryoprotector based on egg yolk plasma maintain the viability of acrosomal membrane

O presente estudo teve como objetivo avaliar a capacidade crioprotetora das lipoproteínas de baixa densidade (LDL) presentes no plasma de gema de ovo, adicionado ao trihidroxiaminometano (TRIS) para congelar sêmen ovino. Trinta e seis ejaculados foram coletados para formar 12 "pool". Cada alíquota de sêmen foi diluída em TRIS-gema de ovo (TRISG) ou TRIS- plasma de gema de ovo (TRISP) antes de congelar o sêmen. Para a obtenção do plasma da gema de ovo, foi utilizado o método de ultracentrifugação. Após o descongelamento, não houve diferença entre os dois extensores em relação aos parâmetros seminais (motilidade, viabilidade, membrana acrossômica e plasma). No entanto, no Teste de Termo Resistência Lenta (TTRL - 4h/38°C), o sêmen congelado com TRISP resultou no aumento do número de espermatozoides com acrossoma intacto (P<0,032). Conclui-se que o diluente TRISP é uma alternativa para criopreservação do sêmen de carneiros, pois permite uma melhora da característica seminal em relação ao diluente TRIS gema, mas sem a necessidade do processo de purificação das LDL.

The present study aimed to evaluate the cryoprotectant low-density lipoprotein (LDL) present in the plasma of egg yolk added to the extender trihidroxiaminometano (TRIS) for freezing ram semen. Thirty-six ejaculates were collected to form 12 pool. Each aliquot of semen was diluted in TRIS-egg yolk (TRISG) or TRIS-egg yolk plasma (TRISP) before freezing the semen. The plasma of egg yolk was obtained by ultracentrifugation. After thawing, no difference was detected between the two extenders in relation to seminal parameters (motility, viability, plasma membrane and acrosome). However, in the thermal resistance slow test (4h in a water bath at 38°C), the semen frozen with TRISP resulted in higher number of sperm with intact acrosome than those with TRISG (P<0.032). It was concluded that the TRISP extender is an alternative for cryopreservation of ram semen, once it improves the semen characteristics in comparison to TRISG, avoiding the purification process of LDL.

Grb10 characterization in bovine cumulus oocyte complexes from different follicle sizes / Caracterização do Grb10 em complexos cumulus-oócito oriundos de folículos bovino de diferentes tamanhos

The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs) from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and >8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and >8mm; P>0.05), had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P>0.05). After it was performed an immunofluorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and >8mm) to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100). Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500). Positive fluorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.

O objetivo deste estudo foi investigar a expressão de RNAm e a localização da proteína Grb10 em complexos cumulusoócito (CCO), oriundos de folículos bovinos em diferentes fases de desenvolvimento. Primeiramente, foi investigada a expressão de RNAm e relacionada com as taxas de maturação. CCOs oriundos de folículos com diâmetro de 1-3, 4-6, 6-8 e >8mm foram utilizados para avaliar a expressão de RNAm por PCR em tempo real e para analisar os estádios de maturação nuclear. Foi observado que os oócitos mais competentes para a progressão meiótica (oriundos de folículos com diâmetro de 6-8 e >8mm; P<0.05) tiveram menor expressão de RNAm para Grb10, quando comparados aos CCOs oriundos de folículos com 1-3 e 4-6mm (P<0.05). Posteriormente, foi realizada a técnica de imunofluorescência em CCOs oriundos de folículos de diferentes tamanhos (1-3, 4-6, 6-8 e >8mm) para investigar a localização da proteína Grb10. As amostras foram incubadas com o anticorpo primário: anti-Grb10 policlonal, produzido em coelho (1:100). O anticorpo primário foi detectado utilizando um anticorpo secundário, IgG anti-coelho, produzido em cabra, conjugado com Alexa Fluor 488 (1:500). A fluorescência foi detectada em todas as amostras analisadas, porém, menos evidente nos CCOs oriundos dos folículos maiores. Os resultados apresentados neste estudo caracterizam o gene Grb10 em CCOs de bovinos e fornecem evidências do seu envolvimento na maturação molecular do oócito.

Nested-PCR multiplex test with increased sensitivity for detection of allogeneic cells transplanted from male to female mice / Nested-PCR multiplex com aumento da sensibilidade de detecção de células alogênicas transplantadas de camundongos machos para fêmeas

Cell therapy has shown encouraging perspectives for human and veterinary medicine. Experimentally, genetic manipulation allows to mark and locate allogeneic cells. However, this makes their genotype/phenotype different from non-marked cells used clinically. Alternatively, the presence of the Y-chromosome enables male donor cells detection in female organisms. However, the concentration of engrafted cells may be minimal in tissues, due to systemic distribution. In this study, a nested-PCR multiplex test was developed, aiming to increase the sensitivity of the presence/absence diagnosis of male mice adipose-derived (ADSC-Y) and bone marrow mononuclear (BMNC-Y) cells in samples of blood and lungs from females, after endovenous transplantation. Four females received placebos; four females received ADSC-Y from two males; and four females received BMNC-Y from two males. The PCR first-step included two primer sets (multiplex): one for amplification of a Y-chromosome fragment (SRYout; 300bp); the other for amplification of an X-chromosome (DXNds3 gene) fragment. In the PCR second-step, one primer set (SRYinn) was used for amplification of a 110bp fragment, restrained in the SRYout amplification product. The PCR internal control (DXNds3 gene) was detected in all DNA samples, whereas the SRY gene external fragment (300bp) was detected exclusively in ADSC-Y and BMNC-Y pure DNA samples. The SRY gene internal fragment (110bp) was detected in 100% of the blood and lung samples from the ADSC-Y and BMNC-Y female recipients. The nested-PCR technique increased sensitivity and reliability for molecular diagnostic of presence or absence of male mice cells in body fluids and tissues of female recipients after endovenous transplantation.

A terapia celular traz perspectivas encorajadoras à medicina humana e veterinária. Experimentalmente, a manipulação genética permite a marcação e a localização de células alogênicas. Porém, isso torna seu genótipo/fenótipo diferente daquelas usadas clinicamente, sem marcação. Alternativamente, a presença do cromossomo Y possibilita detectar células de doadores machos no organismo de fêmeas. Todavia, a concentração de células transplantadas pode ser mínima em certos tecidos, pela distribuição sistêmica. Neste estudo, foi desenvolvida uma nested-PCR multiplex, visando a aumentar a sensibilidade do diagnóstico de presença/ausência de células derivadas do tecido adiposo (CDTA-Y) e derivadas da fração mononuclear da medula óssea (CFMO-Y) de camundongos machos, em amostras de sangue e de pulmões de camundongos fêmeas, após transplante endovenoso. Quatro fêmeas receberam placebo; quatro fêmeas receberam CDTA-Y de dois machos; e quatro fêmeas receberam CFMO-Y de dois machos. A primeira fase da PCR teve dois pares de primers (multiplex): um para amplificação de fragmento do cromossomo Y (SRYout; 300pb); outro para amplificação de fragmento do cromossomo X (gene DXNds3). Na segunda fase da PCR, foi usado um par de primers para amplificação de fragmento de 110pb (SRYinn) interno ao produto amplificado pelo SRYout. O controle interno da reação (gene DXNds3) foi detectado em todas as amostras de DNA testadas, enquanto que o fragmento externo do gene SRY (300pb) foi detectado apenas nas amostras puras de DNA de CDTA-Y e CFMO-Y. O fragmento interno do gene SRY (110pb) foi detectado no sangue e nos pulmões de 100% das receptoras de CDTA-Y e CFMO-Y. A técnica de nested-PCR aumentou a sensibilidade e a segurança do diagnóstico molecular de presença ou ausência de células de camundongos machos em fluidos e tecidos de receptoras fêmeas após transplante endovenoso.

Steroidogenic enzymes mRNA expression profile and steroids production in bovine theca cells cultured in vitro and stimulated with angiotensin II / Perfil de expressão de RNAm de enzimas esteroidogênicas e produção de esteroides a partir de células da teca bovina cultivadas in vitro e estimuladas por Angiotensina II

The main objective of this study was to detect the steroidogenic effects of Ang II in bovine theca cells in vitro. Bovine theca cells were obtained from follicles (larger than 10mm of diameter) collected from a local abattoir and submitted to different treatments in a sequence of experiments. In experiment 1, CYP17A1 mRNA profile was evaluated in LH- (10ng ml-1) and Ang II-treated (0.1µM) theca cells. In experiment 2, a dose-response effect of Ang II (0.001; 0.1 e 10µM) plus insulin (100ng ml-1) and LH (100ng ml-1) was evaluated on steroidogenesis of bovine theca cells. Experiment 3 explored the effects of saralasin (an antagonist of Ang II receptors) on steroid production and steroidogenic enzymes regulation in theca cells. After 24 hours, culture media from experiments 2 and 3 was collected to evaluate testosterone and androstenedione levels by High-Performance Liquid Chromatography. In parallel, mRNA levels of key steroidogenic enzymes (HSD3B2, CYP11A1, CYP17A1) and STAR were assessed by RT-PCR. There was no difference in testosterone and androstenedione production between treated and controls groups, as well as in mRNA levels of the evaluated genes. In conclusion, the results suggest that Ang II does not regulate steroidogenesis in bovine theca cells.

O objetivo deste trabalho foi verificar o efeito da Angiotensina II (Ang II) sobre a esteroidogenese nas células da teca bovina, cultivadas in vitro. Para isso, células da teca bovina foram obtidas de folículos maiores que 10 mm de diâmetro de ovários oriundos de abatedouro e submetidas a diferentes tratamentos em uma sequência de experimentos. No experimento 1, o perfil de expressão do RNAm de CYP17A1 foi avaliado nas células da teca em resposta ao LH (10ng ml-1) e/ou Ang II (0,1µM) em diferentes momentos de tratamento. No experimento 2, foi investigado o efeito dose-resposta de Ang II (0,001; 0,1 e 10µM), acrescido de insulina (100ng ml-1) e LH (100ng ̸ml) sobre a esteroidogênese nas células da teca bovina. O experimento 3 explorou os possíveis efeitos da Ang II por meio do tratamento de células da teca com saralasina (antagonista dos receptores da Ang II). Após 24 horas, nos experimentos 2 e 3, o meio de cultura foi coletado e avaliado quanto aos níveis de testosterona e androstenediona pela técnica de HPLC. Em paralelo, a expressão gênica de enzimas-chave da esteroidogênese (HSD3B2, CYP11A1, CYP17A1) e STAR foi avaliada por qRT-PCR. Não se observou diferença na produção de testosterona e androstenediona entre controle e grupos tratados, bem como, na expressão do RNAm para os genes estudados. Em conclusão, nossos resultados não demonstraram um papel da Ang II sobre a esteroidogenese nas células da teca bovina.