C. C. R. Quintão

Efficient delivery of DNA into bovine preimplantation embryos by multiwall carbon nanotubes.

The pellucid zone (PZ) is a protective embryonic cells barrier against chemical, physical or biological substances. This put, usual transfection methods are not efficient for mammal oocytes and embryos as they are exclusively for somatic cells. Carbon nanotubes have emerged as a new method for gene delivery, and they can be an alternative for embryos transfection, however its ability to cross the PZ and mediated gene transfer is unknown. Our data confirm that multiwall carbon nanotubes (MWNTs) can cross the PZ and delivery of pDNA into in vitro-fertilized bovine embryos. The degeneration rate and the expression of genes associated to cell viability were not affected in embryos exposed to MWNTs. Those embryos, however, had lower cell number and higher apoptotic cell index, but this did not impair the embryonic development. This study shows the potential utility of the MWNT for the development of new method for delivery of DNA into bovine embryos.

Biocompatibility assessment of fibrous nanomaterials in mammalian embryos.

Currently there is a growing interest in the use of nanotechnology in reproductive medicine and reproductive biology. However, their toxic effects on mammalian embryos remain poorly understood. In this work, we evaluate the biocompatibility of two fibrous nanomaterials (NMs): cotton cellulose nanofibers (CNF) and carboxylated multiwalled carbon nanotubes (MWCNT-COOH), by performing an investigation of the embryonic development, gene expression (biomarkers focused on cell stress, apoptosis and totipotency) and in situ apoptosis in bovine embryos. Exposure to NMs did not interfere in preimplantation development or in the incidence of apoptosis in the bovine embryo, but they did affect the gene expression. The results presented are important for an understanding of the toxicity of cotton CNF and MWCNT-COOH on mammalian embryos. To our knowledge, we report the first evaluation of biocompatibility between these NMs on preimplantation embryos, which may open a new window for reproductive biomedical applications.

184 EFFECT OF HISTONE DEACETYLASE INHIBITOR ON DEVELOPMENT OF EMBRYOS DERIVED FROM HEAT-SHOCKED BOVINE OOCYTES

Heat shock affects the oocyte developmental competence and embryonic gene expression. We found that the chromatin organisation of embryos derived from heat-shocked oocytes can also be affected (Camargo et al. 2015 Reprod. Fert. Dev. 27, 132). This study aimed to evaluate whether Scriptaid, a histone deacetylase inhibitor able to modulate the chromatin structure, could influence the development of embryos derived from heat-shocked oocytes. Bovine oocytes were in vitro-matured under conventional temperature (38.5°C) for 24h (non-heat-shock; NHS group) or under 41.5°C for 12h followed by 38.5°C for 12h (heat-shock; HS group). In vitro fertilization was performed under 38.5°C with 5% CO2 in air for 20h. Right after the end of fertilization the presumptive zygotes from the NHS or HS groups were denuded and randomly exposed to 500 nM Scriptaid for 0, 12, or 24h, comprising 6 treatments as followa: NHS-0h (NHS without Scriptaid, n=185); NHS-12h (NHS plus Scriptaid for 12h, n=178); NHS-24h (NHS plus Scriptaid for 24h, n=177); HS-0h (HS without Scriptaid, n=187); HS-12h (HS plus Scriptaid for 12h, n=180); and HS-24h (HS plus Scriptaid for 24h, n=183). After Scriptaid exposure, zygotes were cultured in CR2aa plus 2.5% FCS at 38.5°C with 5% CO2, 5% O2, and 90% N2. Cleavage rate was calculated on Day 2 (44h post-fertilization) and blastocyst rates on Day 7 and 8 post-fertilization. Six replicates were carried out and data was analysed by logistic regression (Proc Logistic, SAS 9.2, SAS Institute Inc., Cary, NC). Significant data were interpreted as odd ratios considering the 95% confidence interval. Values are shown as mean±standard error of the mean. There was no difference (P>0.05) among NHS treatments (NHS-0h, NHS-12h, and NHS-24h) as well as among HS treatments (HS-0h, HS-12h, and HS-24h) for cleavage and blastocyst rates at Day 7. At Day 8, however, the blastocyst rate in the NHS group decreased (P<0.05) as the time of zygote exposure to Scriptaid increased to 24h (33.9±2.8 and 24.2±1.6% for NHS-0h and NHS-24h, respectively), whereas no difference (P>0.05) was found in the HS group (20.7±1.5, 21.2±1.6, and 20.5±2.4% for HS-0h, HS-12h, and HS-24h, respectively). Comparison between NHS and HS treatments showed that cleavage and blastocyst rates at Day 7 and 8 of NHS-0h (88.7±2.8, 30.1±1.5, and 33.9±2.8%, respectively) were superior (P<0.05) to HS-0h (79.3±3.2, 16.9±1.0, and 20.7±1.5%, respectively). Differences (P<0.05) between NHS-12h and HS-12h on blastocyst rates at Day 7 (32.8±3.8 v. 20.6±1.7%, respectively) and at Day 8 (31.7±2.7 v. 21.2±1.6%, respectively) were also found. However, no difference (P>0.05) between NHS-24h and HS-24h was found. We showed that Scriptaid for 24h right after IVF has a negative impact on further development of zygotes derived from oocytes matured under conventional temperature (NHS group), in contrast to zygotes derived from oocytes matured under high temperature (HS group). We concluded that the effect of Scriptaid on embryo development is influenced by the temperature during oocyte maturation.

Post implantation development reveals that biopsy procedure can segregate “healthy” from “unhealthy” bovine embryos and prevent miscarriages

Embryo biopsy has been performed in bovine in vivo produced embryos for the last twenty years, but little could be done with few embryonic cells in the past. Recently, advances in single cell analysis enabled a wide range of applications using embryo biopsy, from morphology to genetics analysis and different omics-techniques, which are promising for in vitro-fertilized (IVF) embryos. The aim of this study was to address if biopsy procedure would affect post implantation development of IVF blastocyts. Here we show that blastocyst stage do not affect re-expansion of biopsied embryos (regular blastocyst: 73.7%; expanded blastocyst: 73.1%), but affects (p<0.05) implantation (regular blastocyst: 37.8%, expanded blastocyst: 61.0%), so ideally biopsy should be performed in expanded blastocysts. No detrimental effect of biopsy procedure was detected for post-implantation development (calving rates, Biopsy: 47.1%, Control: 41.9%), and normal calves were born (Birth weight, Biopsy: 32.10±7.20kg; Control: 30.95±5.43kg). Surprisingly, we found interesting results suggesting embryo survival can be increased with aggressive procedures (such as embryo biopsy), and this is highly associated with early pregnancy loss (Biopsy: 0%, Control: 17.4%). This finding also suggests morphological classification of day 7 blastocysts is far from ideal, and supposedly, unhealthy embryos can implant but are bound to miscarriage during the first trimester (non-biopsied embryos). Our results show biopsy procedure is safe for bovine IVF embryos, and shed new light into the importance of conceptus in early pregnancy loss in cattle.