186 TOTAL RNA AND TRANSCRIPT ABUNDANCE IN HEAT-STRESSED BOVINE OOCYTES AND SURROUNDING CUMULUS

Previous efforts of our laboratory revealed heat-induced perturbations in bovine oocytes that were coincident with reduced developmental potential. The objective of this study was to examine the effect of heat stress on total RNA and specific transcripts during oocyte maturation. After cumulus–oocyte complex (COC) collection, a subset at the germinal vesicle (GV) stage was denuded. Oocytes and surrounding cumulus were stored in RNA lysis buffer. Remaining COCs were matured for 24 h at 38.5 or 41�C (first 12 h of IVM followed by 38.5�C). At 12 and 24 h of IVM, subsets of COCs were denuded and stored in lysis buffer. Four to eight-cell embryos and blastocysts (developmental controls) derived from control and heat-stressed oocytes were collected at 40.5 and 192 h after IVF, respectively. Total RNA was isolated (PicoPure™, Molecular Devices Corp., Sunnyvale, CA, USA), quantified (RiboGreen�, Molecular Probes, Inc., Eugene, OR, USA), spiked with GFP cRNA (for normalization), and reverse transcribed with random primers. Real-time PCR was performed in triplicate using 0.1 oocyte or embryo equivalents or 100 pg cumulus RNA for analysis of BMP15, GDF9, HSP70, cyclin B1, poly(A) polymerase (PAP), and 18S and 28S rRNAs. Data were calibrated to GV-stage and analyzed using the ΔΔCt method. The experiment was replicated 9 times. Data were analyzed as a randomized block design using GLIMMIX (SAS; SAS Institute, Inc., Cary, NC, USA). Heat stress for the first 12 h of IVM reduced blastocyst formation after IVF (20.4%v. 30.5%; SEM = 1.9; P < 0.001), but had no effect on total RNA in oocytes (1.9 to 2.2 ng per oocyte; SEM = 0.7; P > 0.7) or in 4- to 8-cell embryos derived from heat-stressed oocytes (2.4 and 2.9 ng per embryo for 38.5 and 41�C, respectively; SEM = 0.7; P > 0.5). Total RNA was higher in blastocysts derived from heat-stressed oocytes (3.7 v. 5.4 ng; SEM = 0.7; P < 0.03). Heat stress during IVM did not alter relative abundance of transcripts examined in oocytes or resulting embryos. However, abundance of 18S, 28S, and GDF9 decreased in oocytes during IVM (P < 0.05). A general trend also existed for abundance to decrease as development after IVF progressed (oocyte >4- to 8-cell > blastocyst). In surrounding cumulus, HSP70 was increased by 41�C at 12 h but reduced by 24 h of IVM compared to controls (P < 0.003). Regardless of IVM temperature, PAP was higher at 12 and 24 h compared to GV stage (P < 0.0001). A stepwise decrease occurred in cyclin B1 from GV stage to 24 h (P < 0.0001). During IVM, no differences were observed in cumulus for 18S or 28S; GDF9 and BMP15 were not detectable. In the context of our study, transcripts examined were not necessarily informative of developmental potential of heat-stressed oocytes. However, increased HSP70 expression in cumulus following exposure to 41�C suggests that consequences of heat stress on oocytes may be mediated, in part, by surrounding cumulus. The results of this study are a first step toward identifying maternal transcripts in oocytes and surrounding cumulus that may be targets for development of therapeutic strategies to improve oocyte quality.