The effects of different dietary energy levels [100 and 170% for maintenance (M) and high energy (1.7M), respectively] on metabolic, endocrine, and reproductive parameters were evaluated in nonlactating Bos indicus (Gir; n=14) and Bos taurus (Holstein; n=14) cows submitted to ultrasound-guided ovum pick-up followed by in vitro embryo production. The oocyte donor cows were housed in a tiestall system and fed twice daily (0800 and 1600 h). Twenty-one days before the beginning of the experiment, the animals were fed with a maintenance diet for adaptation followed by the experimental diets (M and 1.7M), and each cow underwent 9 ovum pick-up procedures 14 d apart. The recovered oocytes were cultured in vitro for 7 d. We measured glucose and insulin concentrations and performed glucose tolerance tests and the relative quantification of transcripts (PRDX1, HSP70.1, GLUT1, GLUT5, IGF1R, and IGF2R) from the oocytes recovered at the end of the experimental period. No interactions were observed between the effects of genetic groups and dietary energy level on the qualitative (viable oocytes, quality grade, and oocyte quality index) and quantitative (oocytes recovered) oocyte variables. There were no effects of dietary energy level on the qualitative and quantitative oocyte variables. However, Bos indicus cows had greater numbers of recovered structures, viable oocytes, and A and B oocyte grades as well as better oocyte quality index scores and lower DNA fragmentation rates compared with Bos taurus donors. In vitro embryo production (cleavage and blastocyst rates and number of embryos) was similar between diets, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Moreover, Bos indicus cows on the 1.7M diet showed lower transcript abundance for the HSP70.1, GLUT1, IGF1R, and IGF2R genes. All cows fed 1.7M diets had greater glucose and insulin concentrations and greater insulin resistance according to the glucose tolerance test. In conclusion, increasing dietary energy did not interfere with oocyte numbers and quality, but the 1.7M diet reduced in vitro embryo production in Bos indicus cows after 60 d of treatment. Finally, Bos indicus cows had greater oocyte quality, greater numbers of viable oocytes and greater in vitro embryo yield than Bos taurus.

Apesar da velhice não ocupar um espaço central na temática cinematográfica, são inúmeros os filmes que geram, em luz e sombra, múltiplas imagens do envelhecimento humano, propiciando uma possibilidade a mais de entender a velhice e de compreender sua influência cultural. O presente artigo consiste em uma reflexão sobre a utilização de filmes como forma de análise crítica sobre o processo de viver o envelhecimento. Tem como objetivo ressaltar as possibilidades do filme Copacabana (Brasil, 2001) como recurso no processo de ensino em Gerontologia, na perspectiva de que a exposição do aluno à multiplicidade de imagens e situações de convivência de idosos acabe por favorecê-lo na compreensão de aspectos particulares da velhice. Concluímos que, por meio da pedagogia crítica da mídia podemos cultivar a cidadania e, dessa forma, construir uma sociedade, na qual os idosos sejam respeitados e exerçam novos papéis sociais com plenitude.

No abstract

Trichostatin A is a histone deacetylase inhibitor that improves histone acetylation and chromatin remodeling of somatic cell nuclear-transferred embryos (Iager et al. 2008 Cloning Stem Cells 10, 371–379; Maalouf et al. 2009 BMC Dev. Biol. 9, 11). We have previously observed that it also improves quality of bovine cloned embryos, which may increase pregnancy rates. This study aimed to evaluate the effect of trichostatin A treatment of zygotes on relative abundance of 9 transcripts in bovine nuclear-transferred blastocysts. In vitro matured oocytes were enucleated, fused to somatic cells and activated with ionomycin (Camargo et al. 2011 Reprod. Fertil. Dev. 23, 122). After activation, putative zygotes were randomly separated into 2 groups: NT-TRICHO, zygotes were cultured for 4 h in 6-DMAP followed by 7 h in CR2 aa medium plus with 2.5% fetal calf serum (FCS; Nutricell, Campinas, Brazil), both supplemented with 50 nM trichostatin A (Sigma); NT-CONT, zygotes were cultured in the same described conditions without thichostatin A supplementation. In vitro-fertilized embryos (IVF group) were used as a calibrator for relative transcript quantification. Embryos from the 3 groups were cultured in CR2 aa supplemented with 2.5% FCS under 5% CO2, 5% O2 and 90% N2 at 38.5°C. At 168 h postactivation, the embryos were rapidly frozen in liquid nitrogen. Pools of 10 blastocysts for each group were subject to RNA extraction and reverse transcription, in which cDNA was amplified by real-time PCR using the β-actin and GAPDH genes as endogenous references. The transcripts analysed encode high mobility group N1 (HMGN1), peroxiredoxin 1 (PRDX1), octamer-binding protein 4 (OCT4), insulin-like growth factor 1 and 2 receptors (IGF1r and IGF2r), glucose transporter 1 and 5 (GLUT1 and GLUT5), histone acetyltransferase (HAT) and heat shock protein 70.1 (HSP70) genes. Results were analysed by a pair-wise fixed reallocation randomization test using the REST software v.2. Data from NT-TRICHO and NT-CONT were compared with the IVF group and between themselves. The relative abundance of HSP70, PRDX1, IGF2r and HMGN1 transcripts was higher (P < 0.05) in NT-TRICHO compared with the IVF group and no difference was detected for the other transcripts. In the NT-CONT group, the relative abundance of IGF2r and HAT was higher (P < 0.05), whereas IGF1r and OCT4 were lower (P < 0.05) compared with IVF embryos. When data from NT-TRICHO and NT-CONT were compared, a higher amount (P < 0.05) of stress-associated transcripts (HSP70 and PRDX1) were found in NT-TRICO blastocysts. These results suggest that although trichostatin A may improve chromatin remodeling, alterations on gene expression still persist in bovine somatic cell nuclear-transferred blastocysts in comparison with IVF embryos. Financial support: Embrapa Project 01.07.01.002, CNPq 403019/2008–7 and Fapemig.

Oocyte competence is associated with the amount of transcripts stored in the ooplasm and oocyte ability to extrude polar bodies (PB). To our knowledge, however, no data comparing mRNA levels between bovine oocytes maturated in vitro with or without PB are available. The aim of the present study was to compare the relative abundance of transcripts of glucose transporter 1 (GLUT1), insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R), growth differentiation factor-9 (GDF9) and aquaporin 3 (AQP3) genes between oocytes with and without PB (PB and NPB groups, respectively) following in vitro maturation. Immature bovine oocytes were obtained by follicular aspiration and matured in TCM-199 (Gibco Life Technologies, New York, NY, USA) containing 10% of oestrus cow serum and 20 μg mL–1 of FSH (Pluset, Serono, Italy) for 24 h under 5% CO2 in air at 38.5°C. Subsequently, oocytes were visually classified according to the presence or absence of PB and then denuded and rapidly frozen in liquid nitrogen. Three pools of 10 oocytes for each group were subjected to total RNA extraction using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions and treated with DNase. Reverse transcription and cDNA amplification were performed using the TransPlex Complete Whole Transcriptome Amplification Kit (WTA2, Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Relative abundance of the target transcripts was performed by quantitative RT-PCR (Applied Biosystems Prism 7300 Sequence Detection Systems, Foster City, CA, USA) using a mixture of SYBR® Green PCR Master Mix (Applied Biosystems), 200 ng of cDNA, nuclease-free water and specific primers for each reaction. Expression of the β-actin gene was used as an endogenous reference. Relative gene expression analysis was performed using the software REST© 2005 using the Pair Wise Fixed Reallocation Randomization Test©. The relative expression values are presented as mean ± standard error. The relative abundance of GLUT1 (0.81 ± 0.07), IGF2R (0.72 ± 0.07) and GDF9 (0.82 ± 0.10) genes was lower (P < 0.05) for NPB oocytes. There was no difference (P > 0.05) in relative abundance between PB and NPB groups for the other genes. The results suggest that the amount of some transcripts stored in the matured ooplasm is associated with the presence of PB. The authors acknowledge FAPEMIG, CNPq and Innovation Network Project on Animal Reproduction (01.07.01.002).

One requirement for somatic cell nuclear transfer (NT) is the coordination between donor cell cycle and recipient cytoplasm. As an alternative to commercially available substances to synchronize the cell cycle in G0/G1, we tested 2 extracts, aqueous and hexane obtained from the plant Azadirachta indica A. Juss (popularly called Neem). Extracts from this plant have shown antiviral, antibacterial and anticancer activities, widely described in the literature (Kumar et al. 2009 Invest. New Drugs 27, 246–252). The hexane extract was prepared in the Soxhlet apparatus until total collapse and then submitted to rotary evaporation. The aqueous extract was prepared by dynamic maceration and was subsequently lyophilized. Bovine fibroblasts collected from Gyr cows were cultured in DMEM (Sigma) supplemented with 10% fetal cow serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. After obtaining 70% of cell confluence, the extract was added to cells at the following concentrations: 0 μg mL–1 (negative control), 50 μg mL–1, 100 μg mL–1, 200 μg mL–1 and 300 μg mL–1, for 12 and 24 h. Simultaneously, a group with serum starvation (positive control; cells cultured in DMEM plus 0.5% FBS for 3 days) was prepared. Three repetitions were performed in triplicate for each concentration and control groups. Cell cycle readings were performed by flow cytometry (Facs Callibur, Becton Dickinson, San Jose, CA, USA) and DNA histograms were analysed by WinMDI software to determine the percentage of cells in G0/G1 phase, S and G2 cell cycle, so that 10 000 cells were analysed in each reading on a flow cytometer. Data were analysed by analysis of variance and means were compared by Student-Newman-Keuls test. The percentages of cells at G0/G1 phase for aqueous extracts were lower (P < 0.05), regardless of the concentration and exposure time, than the 0 μg mL–1 (83.73 ± 1.14%) and serum starvation (86.64 ± 1.44%). In contrast, the percentages of cells synchronized at G0/G1 with 50 μg mL–1 for 12 h (84.23 ± 0.56%), 50 μg mL–1 for 24 h (85.66 ± 0.57%), 100 μg mL–1 for 12 h (87.85 ± 0.51%) and 200 μg mL–1 for 12 h (85.87 ± 0.45%) using hexane extracts were higher (P < 0.05) than with 0 μg mL–1 (81.44 ± 0.29%), but lower (P < 0.05) than the serum starvation (91.33 ± 0.31%). In conclusion, hexane extracts from the plant Azadirachta indica A. Juss can synchronize mammalian cell cycle at G0/G1 despite the low proportion when compared with serum starvation. Studies to evaluate efficiency of cell cycle resuming and viability after somatic cell nuclear transfer are ongoing. Financial support: FAPEMIG, CNPq and Project 01.07.01.002.

The 90-kDa heat shock protein (HSP90) is a chaperone that is important for maintaing protein homeostasis under stress conditions. HSP90 seems also to be required for maturation of Xenopus oocytes (Fisher et al. 2000 EMBO J. 19, 1516) and first cleavage of mouse zygotes (Audouard et al. 2011 PloS One 6, e17109). This study aimed to evaluate the effect of inhibition of HSP90 by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma St. Louis, MO, USA) during in vitro maturation (IVM) on bovine oocyte developmental competence. Immature cumulus–oocyte complexes (COC) were randomly allocated in 3 treatments during IVM: T0 (control; n = 240), no HSP90 inhibitor; T1: 2 μM HSP90 inhibitor (17AAG; n = 250) for the first 12 h of IVM; and T2: 2 μM HSP90 inhibitor (n = 188) for 24 h of IVM. In vitro maturation was performed in Nunc plates containing 400 μL of TCM-199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2, 95% humidity, and 38.5°C for 24 h. Oocytes were in vitro fertilized for 20 h and incubated under the same IVM conditions. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) an IVF performed with 2 × 106 spermatozoa mL–1. Presumptive zygotes were completely denuded in a PBS solution with hyaluronidase and then cultured in wells with 500 μL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h post-fertilization and blastocyst rates were evaluated at Day 7 and Day 8. Data from 6 repetitions were analysed by generalized linear model procedure of SAS software (version 9.1; SAS Institute Inc., Cary, NC, USA), and means were compared by Student-Newman-Keuls test. Values are shown as mean ± s.e.m. There was a tendency (P = 0.08) for a lower cleavage rate in T2 (52.6 ± 5.8%) than in T0 (control; 74.2 ± 4.1%). Inhibition of HSP90 by 17AAG for 12 h and 24 h of IVM (T1 and T2, respectively) decreased blastocyst rates at Day 7 (20.4 ± 3.0% and 14.3 ± 2.6%, respectively; P < 0.01) and Day 8 (22.6 ± 4.1% and 16.9 ± 2.7%, respectively; P < 0.05) when compared with control (T0 = 31.8 ± 2.5% and 34.1 ± 2.9% for Day 7 and Day 8, respectively). In addition, the inhibition of HSP90 for 24 h decreased (P < 0.05) the proportion of hatched blastocysts at Day 8 (9.5 ± 5.0% for T2, respectively) when compared with control (T0 = 35.8 ± 3.9%), indicating a reduction on embryo quality. In conclusion, inhibition of HSP90 by 17AAG during IVM results in lower developmental competence, suggesting that this protein is also important for bovine oocytes. Further studies are required to investigate if the role of HSP90 on developmental competence of bovine oocyte is affected when under stress conditions. The authors acknowledge CNPq 473484/2011-0, FAPEMIG and FAPES for financial support.

Heat stress has been a challenge for bovine reproduction in tropical and subtropical environments. Although the role of the oocyte in thermotolerance has been studied, little attention has been paid to the contributions of sperm to embryo resistance to heat shock. The current study aimed to evaluate the development of fertilized and nonfertilized (parthenogenetic) bovine embryos undergoing heat stress during the pre-implantation stage. Cumulus–oocyte complexes obtained from ovaries collected from Bos indicus × Bos taurus crossbred cows at slaughter were in vitro matured with TCM-199 supplemented with 20 μg mL–1 of FSH, under 5% CO2 at 38.5°C for 24 h. Afterward, oocytes were randomly allocated into 2 groups: 1) IVF and 2) PART (chemical activation for parthenogenesis induction). In vitro-fertilized oocytes were cultured with 2.0 × 106 Holstein sperm mL–1 in Fert-TALP medium supplemented with heparin, for 20 h. For chemical activation, oocytes were activated with calcium ionomycin for 4 min, followed by 6-DMAP for 4 h, both in CR2aa medium supplemented with 0.1% BSA. Presumptive IVF (n = 1 262) or PART (n = 1 206) zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% of FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. At 44 h post-insemination or chemical activation, embryos were exposed to 38.5 or 41°C for 12 h in an atmosphere of 5% CO2, 5% O2, and 90% N2. After that, embryos were cultured at 38.5°C under 5% CO2, 5% O2, and 90% N2 until Day 8 post-insemination. Blastocyst rates were evaluated at Day 7 and Day 8 post-insemination and were calculated based on the total number of presumptive zygotes. Blastocysts at 192 h post-insemination or activation were fixed and permeabilized for TUNEL assay (DeadEndTM Florimetric TUNEL System, Promega, Madison, WI) according to the manufacturer’s instructions. The effect of heat stress was compared within groups (IVF or PART) and the data were analysed by ANOVA. As expected, heat stress reduced the blastocyst rate of IVF embryos at Day 7 (24.3 ± 2.0% and 17.4 ± 2.2% for nonstressed and stressed IVF embryos; P < 0.05) and at Day 8 (32.4 ± 1.9% and 23.0 ± 2.1% for nonstressed and stressed IVF embryos; P < 0.01). However, the effect of heat stress on blastocyst rate of PART embryos was observed only at Day 8 post-insemination (30.0 ± 1.7% and 22.6 ± 2.0% for nonstressed and stressed PART embryos; P < 0.05), with no difference in blastocyst rate at Day 7 (21.6 ± 1.5% and 18.2 ± 1.8% for nonstressed and stressed PART embryos; P > 0.05). There was no difference in total cell numbers between nonstressed and stressed IVF or PART embryos. Apoptosis cell numbers and the apoptotic cell index were higher (P < 0.05) for stressed IVF (18.45 ± 1.24 and 0.16 ± 0.00) and PART (16.40 ± 5.20 and 0.17 ± 0.00) embryos than for nonstressed IVF (13.70 ± 0.75 and 0.13 ± 0.00) and PART (14.15 ± 0.86 and 0.13 ± 0.00) embryos. In conclusion, heat stress can induce apoptosis in both IVF and PART embryos, but its effect on pre-implantation development may occur at earlier stages in IVF embryos when compared with PART embryos. Financial support from Fapemig and CNPq.