Inhibition of Hsp90 during in vitro maturation under thermoneutral or heat shock conditions compromises the developmental competence of bovine oocytes.

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower (P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower (P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased (P < 0.05) the blastocysts rates. In the third experiment, the association of 2 µM 17AAG with HS for 12 h during IVM resulted in lower (P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

RA33, an analogue of resveratrol, improves the development of in vitro-fertilized bovine embryos.

Oxidative stress is an undesirable effect of in vitro culture, which requires antioxidant supplementation. This study investigated the analogue of resveratrol (RA33) as an alternative to resveratrol, an antioxidant molecule, for the in vitro culture of in vitro-fertilized bovine embryos. The effect of different concentrations of RA33 on embryo development was evaluated and a comparison between RA33 and resveratrol was performed. The cleavage rate was higher (P < 0.05) with 2.5 µM (69.0 ± 4.4%) than at 0, 0.1 or 0.5 µM RA33 (62.1 ± 2.0%, 60.7 ± 5.9% and 56.7 ± 5.8%, respectively). The blastocyst rates on days 7 and 8 post-fertilization with 2.5 µM RA33 (19.4 ± 3.3% and 24.6 ± 3.3%, respectively) were higher (P < 0.05) than for 0 µM (12.4 ± 2.5% and 15.2±2.5%, respectively). When 2.5 µM RA33 was compared with 0.5 µM resveratrol, similar (P > 0.05) cleavage and blastocyst rates were found between them, but the cleavage rate was higher (P < 0.05) in the control (80.8 ± 3.4%) than for the resveratrol treatment (76.4 ± 3.6%). The numbers of apoptotic cells and the apoptotic index were lower (P < 0.05) with RA33 (6.5 ± 0.6 cells and 6.4 ± 0.7%, respectively) and resveratrol (5 ± 0.8 cells and 5.5 ± 1.0%, respectively) than in the control group (9.8 ± 1.2 cells and 8.9 ± 1.1%, respectively). In conclusion, RA33 can enhance the preimplantation development of in vitro-fertilized bovine embryos and be an alternative to resveratrol in embryo culture medium.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice.

The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Challenges in the use of nanostructures as carriers of nucleic acids in clinical practice

ABSTRACT The delivery of nucleic acids to cells is considered a crucial step for the success of genetic modifications aimed at therapeutic purposes or production of genetically modified animals. In this context, nanotechnology is one of the most promising fields of science, with the potential to solve several existing problems. Nanostructures have desirable characteristics to be used as carriers, such as nanometric size, large surface area, cell internalization capacity, prolonged and controlled release, among others. Genetically modified animals can contribute to the production of biopharmaceuticals, through the expression of high-associated-value molecules. The production of these animals, also known as biofactories, further enhances Brazilian agribusiness, since it allows adding value to the final product, and favors the integration between the agricultural market and the pharmaceutical sector. However, there is a growing concern about the safety and possible harmful effects of nanostructures, since data on the safe use of these materials are still insufficient. The objective of this review was to address aspects of the use of nanostructures, mainly carbon nanotubes as nucleic acid carriers, aiming at the production of genetically modified animals, with the certainty that progress in this field of knowledge depends on more information on the mechanisms of interaction between nanostructures, cells and embryos, as well as on its toxicity.

Heat shock during in vitro maturation of bovine oocytes disturbs bta-miR-19b and DROSHA transcripts abundance after in vitro fertilization.

While microRNAs (miRNAs) are a class of non-coding RNAs important for embryo development, the relationship between them and heat stress during oocyte maturation has not yet been established. This study investigated the effect of heat shock during in vitro maturation (IVM) on the abundance of bta-miR-20a, -27b, -103, -21-5p, -19b, -1246 miRNAs and DROSHA and DICER1 mRNAs, previously reported for being involved in oocyte maturation, response to heat stress and miRNA biogenesis. Oocytes were exposed for 12h to heat shock during IVM, fertilized in vitro and the presumptive zygotes cultured for eight days. The relative quantification of miRNAs and mRNAs was performed by real-time PCR in vitro-matured oocytes and 8-cell stage embryos. Progression of meiosis, embryonic development and apoptotic indices was also evaluated. Heat shock compromised (p < .05) oocyte nuclear maturation, cleavage and embryo development, with a higher (p < .05) embryonic apoptotic index than the control group. The abundance of bta-miR-19b increased (p < .05) whereas the abundance of DROSHA transcripts decreased (p < .05) in embryos derived from heat-shocked oocytes. In conclusion, heat shock during IVM influences the abundance of bta-miR-19b and DROSHA in pre-implantation embryos, indicating a persistent effect of heat shock that can be associated with impaired embryo development.

Effects of resveratrol in bull semen extender on post-thaw sperm quality and capacity for fertilization and embryo development.

Resveratrol, a potent antioxidant, can be an alternative semen extender constituent to protect spermatozoa against reactive oxygen species (ROS); however, effects on sperm quality post-thawing and sperm function is not well understood. This study, therefore, was conducted to investigate effects of resveratrol supplementation to semen extender on sperm quality post-thawing. Bull semen was cryopreserved using extenders not supplemented or supplemented with 0.05, 0.1, or 1 mM resveratrol. Supplementation of extender with resveratrol at 0.05 mM resulted in greater (P < 0.05) sperm progressive motility, average path velocity, straight linear velocity, linearity and straightness when compared with no or 1 mM supplementations. Furthermore, effects of 0.05 mM resveratrol supplementations on plasma membrane and acrosome integrity and sperm fertilization capacity using in vitro procedures were investigated. Supplementation of semen extender with resveratrol resulted in a greater (P < 0.05) proportion of frozen-thawed spermatozoa with an intact acrosome and plasma membrane. Results from in vitro fertilization studies indicated there were no differences (P> 0.05) when there was no supplementation or supplementation with 0.05 mM resveratrol on embryo development to the cleavage and blastocyst stages. In conclusion, addition of resveratrol to bull semen extender resulted in greater sperm quality post-thawing in a dose-dependent manner, with values for variables related to sperm quality being greater when there was resveratrol supplementation at the 0.05 mM concentration. Proportion of embryo developing to the cleavage and blastocyst stages after in vitro fertilization was not affected by resveratrol supplementation to semen extenders.

Chromium supplementation improves glucose metabolism and vaginal temperature regulation in Girolando cows under heat stress conditions in a climatic chamber.

This study aimed to evaluate chromium supplementation on productive, reproductive, and metabolic parameters at lactating Girolando cows subjected to heat stress conditions in a climatic chamber. Thirty-six lactating Girolando cows were subjected to two sequential trials. In trial 1 (thermoneutral environment), the effect of chromium supplementation was evaluated (0 vs. 0.50 mg/kg of dry matter). In trial 2, the cows were fed the same diets, but they were divided into three environmental conditions: heat stress conditions in climatic chamber, fed ad libitum (HS); thermoneutral environment, fed ad libitum (TN); and thermoneutral environment, pair-fed (PF). In thermoneutral conditions, chromium supplementation did not affect productive or metabolic parameters, although supplemented cows had lower viability of oocytes (65.11 ± 0.08% vs. 76.86 ± 0.08%). During heat stress, chromium supplementation lowered plasma glucose levels (61.17 ± 1.90 vs. 67.11 ± 1.90 mg/dL), and increased the insulin:glucose ratio (0.39 ± 0.04 vs. 0.27 ± 0.04). Cows fed the control diet in the HS group had higher vaginal temperature values (39.40 ± 0.10 °C) than the cows in the TN group and PF group (38.89 ± 0.10 °C and 38.85 ± 0.11 °C, respectively). However, supplemented cows heat-stressed maintained the same vaginal temperature as cows in thermoneutral conditions. In conclusion, chromium supplementation improved glucose metabolism and prevented body temperature increases under heat stress conditions.

Differential gene expression between in vivo and in vitro maturation: a comparative study with bovine oocytes derived from the same donor pool.

OBJECTIVE: In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors. METHODS: A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo-matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR. RESULTS: Oocytes in the IN VITRO-OPU group had lower (p<0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups (p<0.05). CONCLUSION: In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.