180 HEAT STRESS INDUCES ALTERATION IN EXPRESSION OF GENES RELATED TO COMPETENCE AND IMPLANTATION IN NELORE BOVINE IN VITRO-PRODUCED EMBRYOS

Abstract

Several factors affect early embryonic development in cattle, including heat stress. These factors can contribute to high early embryonic loss, probably altering gene expression. Studies using microarray-profiled genome-wide RNA expression for in vitro-produced blastocysts have compared embryos resulting in calf delivery or no pregnancy, and they have identified genes with potential roles in pregnancy and embryo competence. The aim of the present work was to compare the expression of some genes (PLAC8, HSF1, COX-2, and CDX-2) related to embryo competence and embryonic implantation between in vitro-produced embryos from the Nelore breed (Bos indicus), submitted or not submitted to heat stress. Oocytes from Nelore cows were aspirated by ovum pickup and matured for 22 h (TCM-199 with bicarbonate, supplemented with 10% FCS, 2 μL mL^–1 of pyruvate, 75 μg mL^–1 of amicacin, 20 μg mL^–1 of FSH, and 2 IU mL^–1 of hCG) at 38.5°C with 5% CO₂ in air. The fertilization (Day 0) was performed with semen from Nellore bulls. After a 12-h fertilization period, in Tyrode’s lactate stock medium supplemented with 6 mg mL^–1 of BSA, 2 mL mL^–1 of pyruvate, 75 mg mL^–1 of amicacin, 11 mg mL^–1 of heparin, and 44 mL mL^–1 of phenylalanine solution, presumptive zygotes were denuded and randomly divided in 2 groups: nonstressed and stressed. The culture medium was SOFaaci supplemented with sodium pyruvate (0, 2%), 5 mg mL^–1 of BSA and 5% FCS. Embryo culture was performed at 38.5°C, 90% N₂, 5% CO₂, and 5% O₂. In the stressed group, 96 h after fertilization, the embryos were subjected to heat stress of 41°C for 6 consecutive hours and then returned to a temperature of 38.5°C. On Day 7, pools of 5 blastocysts (nonstressed, n = 9; stressed, n = 7) were submitted to total RNA extraction (RNeasy, Qiagen, Valencia, CA). The gene expression of target genes was measured by real-time RT-PCR with oligo-dT in the reverse transcription and bovine-specific primers in the PCR. Expression of cyclophlin A was used as an internal control. The means of mRNA levels of target genes between the groups were compared by t-test. The PLAC8 mRNA levels were higher in nonstressed blastocysts in comparison with the stressed group. The HSF1 and CDX2 mRNA was detectable only in nonstressed embryos. The COX2 mRNA levels did not differ between groups. The higher levels of PLAC8 and the CDX2 expression on nonstressed embryos indicate better competence of embryos not submitted to heat stress. Furthermore, the absence of HSF1 mRNA in the stressed embryos does not reflect the lack of biological activity of this protein. In conclusion, the data indicate that heat stress alters the gene expression pattern of in vitro-produced embryos in the Nelore breed.