Transcriptomic analyses of buffalo oocytes and blastocysts using RNAseq

The analysis of the whole transcripts of the gametes and embryos can generate information about metabolism, cellsignalling pathways and cell cycle regulation (Graf et al., PNAS, 2014, 11:4139) and these information can be useful to improve in vitro embryo production protocols. Here we show partial results of the first whole mRNA transcriptome of mature oocytes and blastocysts of buffalo using RNAseq approach. Buffalo oocytes obtained from abattoir-derived ovaries were in vitro matured in TCM199 media supplemented with 0.5µg/ml FSH, 50µg/ml LH, 50µg/ml gentamycin, 10mg/mL pyruvate and 10% BFS, for 19h at 38.5°C in a 5% CO2 incubator with humidified air. For in vitro fertilization, frozen sperm from one bull was thawed, processed by Percoll gradient and 2x106 sperm/mL of spermatozoa were incubated in 80µL droplets containing 20 oocytes for 24 hours. After fertilization, presumptive zygotes were cultivated in 100µL droplets of modified synthetic oviduct fluid medium supplemented with 5 mg/mL BSA, 10% FBS, 0.2 mM pyruvate, and 50 mg/mL gentamycin for 6 days in a 38.5°C and 5% CO2 incubator with humidified air. Matured oocytes (showing first polar body) and embryos (blastocysts) were frozen in RNAlater® solution and kept at -80°C until mRNA extraction, which was performed using Dynabeads© mRNA Direct Micro Kit. The mRNA libraries were construct using 35 buffalo embryos and 205 in vitro matured oocytes. The resulting libraries were amplified and then quantified on Qubit® 2.0. Barcoded libraries were sequence on an Ion Proton™ system. Sequencing reads of low quality (Phred < 20) were filtered using ‘fastQC’ tool. Just filtered reads were mapped to both Btau_4.6.1 (NCBI annotation) and UMD3.1 (Ensemble annotation) assembly using CLC Genomics Workbench 4.7.2 software. As result, 13ng and 17ng of amplified mRNA were used to construct the oocyte and blastocyst sequencing libraries, respectively. After sequencing, a total of 7.677.937 and 21.626.473 reads were generate from buffalo mature oocytes and blastocysts, respectively. The UMD3.1 genome reference resulted in higher proportion of mapping reads in comparison to the BTAU_4.6.1 for both oocytes (97.9 versus 94.9%, respectively) and blastocysts (97.6 versus 94.7%, respectively). That find was expected and, according literature, it was possibly related to the different proportions of embryonically expressed genes in these annotations (Chitwood et al., BMC Genomics, 2013, 14:350). Considering the UMD3.1 mapping, we were able to find among protein coding genes the percentage of “exon / exon-exon junction” and “intron / intergenic regions” mapped reads in oocytes (66.1 versus 33.9%, respectively) and embryos (53.2 versus 46.8%, respectively). Proportionally, oocytes presented higher percentage of “exon / exon-exon junction” than embryos, we think that it can be explained by the fact that oocytes storages many mature transcripts in cytoplasm to support embryo development and embryos needs to undergo mitosis and for that transcription machinery is very active producing many immature transcripts. This project is currently in development and further results are expected to be publish soon.