Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2. © 2013 Society for Reproduction and Fertility.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells. © CSIRO 2013.

Phenotypic and metabolic aspects of prostatic epithelial cells in aged gerbils after antisteroidal therapy: Turnover in the state of chromatin condensation and androgen-independent cell replacement

The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. © 2013 Elsevier GmbH. All rights reserved.

Adaptabilidade e estabilidade de clones de cana-de-açúcar no Estado de São Paulo

Pós-graduação em Agronomia - FEIS

Growth curve of female collared peccaries (Pecari tajacu) raised in captivity in the Brazilian Amazon Region

Com o objetivo de ajustar modelos não-lineares, foram utilizados registros mensais do peso de 10 fêmeas de cateto (Pecari tajacu) coletados durante dois anos, no criatório do campo experimental Álvaro Adolfo da Embrapa Amazônia Oriental, Belém, PA. Utilizaram-se os modelos de Von Bertalanffy, Brody, Gompertz e Logístico. Os parâmetros foram estimados usando o procedimento NLIN do aplicativo SAS. Os critérios utilizados para verificar o ajuste dos modelos foram: desvio padrão assintótico (ASD); coeficiente de determinação (R²); desvio médio absoluto dos resíduos (ARD) e o índice assintótico (AR). Os modelos Brody e Logístico estimaram, respectivamente, o maior (19,44kg) e o menor (19,18kg) peso assintótico (A), caracterizando a menor (0,0064kg/dia) e a maior (0,0113kg/dia) taxa de maturação (K), haja vista a natureza antagônica entre estes parâmetros, comprovada pela correlação fenotípica variando entre -0,75 à -0,47. O modelo Brody estimou o menor valor para o ARD, fator limitante para caracterizar o menor valor para o AR por este modelo. Considerando o AR, o modelo Brody apresentou o melhor ajuste, contudo, pelos valores encontrados, os demais modelos também apresentaram ajuste adequando aos dados ponderais da referida espécie/sexo. Com base no AR adotado neste trabalho, recomenda-se o modelo Brody para ajustar a curva de crescimento de fêmeas de cateto (Pecari tajacu). Em razão dos valores estimados, sobretudo, para a K, essa característica pode ser incluída em um índice de seleção. Contudo, estudos com grupos mais representativos e criados em outras condições se faz oportuno.

Mandibular growth and dentoalveolar development in the treatment of Class II, division 1, malocclusion using Balters Bionator according to the skeletal maturation

OBJECTIVE: The purpose of the study was to evaluate the influence of the skeletal maturation in the mandibular and dentoalveolar growth and development during the Class II, division 1, malocclusion correction with Balters bionator. METHODS: Three groups of children with Class II, division 1, malocclusion were evaluated. Two of them were treated for one year with the bionator of Balters appliance in different skeletal ages (Group 1: 6 children, 7 to 8 years old and Group 2: 10 children, 9 to 10 years old) and the other one was followed without treatment (Control Group: 7 children, 8 to 9 years old). Lateral 45 degree cephalometric radiographs were used for the evaluation of the mandibular growth and dentoalveolar development. Tantalum metallic implants were used as fixed and stable references for radiograph superimposition and data acquisition. Student's t test was used in the statistical analysis of the displacement of the points in the condyle, ramus, mandibular base and dental points. Analysis of variance one-fixed criteria was used to evaluate group differences (95% of level of significance). RESULTS: The intragroup evaluation showed that all groups present significant skeletal growth for all points analyzed (1.2 to 3.7 mm), but in an intergroup comparison, the increment of the mandibular growth in the condyle, ramus and mandibular base were not statically different. For the dentoalveolar modifications, the less mature children showed greater labial inclination of the lower incisors (1.86 mm) and the most mature children showed greater first permanent molar extrusion (4.8 mm).

Prevalence and phenotypic characterization of Enterococcus spp. isolated from food in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anatomical evaluation of the cervical vertebrae of Wistar rats by means of digital radiographs and its correlation with the maturation stages of human cervical vertebrae

INTRODUCTION: Biological age is an important parameter for growth and development assessment. It can be evaluated through the observation of radiographic changes in skeletal maturation of cervical vertebrae. OBJECTIVE: This study aims to: a) verify if there is correlation between growth curve and the stages of bone age of animals used in laboratories, by evaluating radiographs of the cervical vertebrae; b) correlate these stages with their correspondents in humans. METHODS: 35 Wistar rats were evaluated for a period of 160 days, starting at day 22nd (weaning), with cross sections for periodic weighing, length measurement and digital radiography. Radiographs of the cervical vertebrae (C2 and C3) were measured by means of a computer program (Radio IMP). Data were submitted to statistical analysis (ANOVA) and Pearson correlation. RESULTS: Growth spurt was characterized by fast increasing in weight and length. Through ANOVA, differences were observed in the cervical measurements between days 22, 97, 127, 157, 187 and 217 (p <0.001). A high correlation was found between increasing in body length and weight, as well as in cervical vertebrae height (r = 0.86). Increments in concavities of vertebrae were also observed, similar to humans. CONCLUSIONS: There is correlation between body growth and maturation of cervical vertebrae in rats. Despite the continuous development of concavities, it was not possible to clearly identify the 5/6 stages as in studies of cervical vertebrae maturation in humans.

Dynamics of ovarian maturation during the reproductive cycle of Metynnis maculatus, a reservoir invasive fish species (Teleostei: Characiformes)

Neste estudo, avaliamos a dinâmica da maturação ovariana a desova durante o ciclo reprodutivo de Metynnis maculatus. Fêmeas adultas (n = 36) foram coletadas bimestralmente entre abril de 2010 e março de 2011. O índice gonadossomático (IGS) foi calculado e amostras de ovário e de sangue foram submetidas à avaliação morfométrica e das concentrações plasmáticas dos esteroides por ELISA, respectivamente. A espécie apresenta desenvolvimento ovariano assincrônico, com múltiplas desovas. Neste estudo revelamos que mesmo sendo de desova parcelada, os ovários do M. maculatus mostraram um padrão de desenvolvimento com predomínio de atividade vitelogênica entre abril a agosto e intensificação da desova em setembro. Em outubro houve uma diminuição nos valores médios de IGS, bem como registramos as maiores frequências de folículos pós-ovulatórios (FPOs). Observamos uma correlação positiva entre a frequência de FPOs e a concentração plasmática de 17 α-OHP. O M. maculatus tem potencial para ser usado como fonte para uso de hipófise para preparo de extrato bruto para indução hormonal, sendo o período teórico para coleta de hipófises de setembro a outubro, mas estudos específicos para esta finalidade ainda precisam ser desenvolvidos.

Effects of the Temperature and Maturation Stages on the Germination of Roystonea regia Seeds

Roystonea regia (Kunth) O.F. Cook is largely used as ornamental. The propagation is done almost exclusively by seeds; however, there is a great variation in the germination process influenced by many factors. The objective of this work was to study the effects of the temperature and maturation stages on the germination of R. regia seeds. The experimental design was entirely randomized in a factorial arrangement 6x3 (six temperatures: constant at 20, 25, 30 and 35 degrees C and alternated at 20-30 and 25-35 degrees C, with a photoperiod of 12 hours; and three fruit maturation stages: brown, yellow and black), with four replications of 25 disseminules (seed with stucked endocarp) each. The disseminules had their mesocarp and exocarp were removed and shade dried. Their moisture content was determined, and then they were placed in plastic boxes (gerbox type) containing vermiculite. The disseminules, with the germinative intumescence, were daily noted until germination was steady. The germination rate and the germination speed index were calculated, and the data were submitted to the variance analysis. The means were compared by the Tukey test. It was concluded that the highest germination rate (99.7%) and germination speed were obtained by seeds from mature (black) fruits at the temperature of 35 degrees C.

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation

The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and a parts per thousand yenaEuro parts per thousand 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 +/- 14.11 % and 82.93 +/- 5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 +/- 10.40 % and 79.64 +/- 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 +/- 12.01 % and 81.19 +/- 10.15 %.In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.

Ultrastructural Morphology and Nuclear Maturation Rates of Immature Equine Oocytes Vitrified with Different Solutions and Exposure Times

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Midpalatal suture maturation: Classification method for individual assessment before rapid maxillary expansion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Disentangling the Phylogenetic and Ecological Components of Spider Phenotypic Variation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)