The present study aimed to assess the transcripts for the proteins that embryos require, histone 2a (H2a-FZ), heat shock 70 kDa protein 1A (HSP 70.1), zygote arrest 1 (ZAR-1), and maternal antigen (MATER), in bovine oocytes submitted to prematuration (PM) culture and/or in vitro maturation (IVM). Follicles (2–6 mm diameter) were aspirated from slaughterhouse-derived ovaries. Oocytes were selected and randomly distributed among treatments. For PM, oocytes were cultured 24 h in TCM-199 medium supplemented with 10 µm butyrolactone I, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin (BGV group). Part of the prematured oocytes were washed and transferred to IVM culture (BMII group). For IVM, oocytes were cultured in TCM-199 supplemented with 10% FCS, 5.0 µg mL–1 LH, 0.5 µg mL–1 FSH, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin for 22 h. As controls one group of oocytes was collected immediately after aspiration (GV group) and another group was matured in vitro (MII group) without undergoing prematuration. All cultures were carried out in 100-µL droplets of culture medium under mineral oil at 38.5�C and 5% CO2 in air. Oocytes from each treatment were denuded and frozen at 80�C (3 pools of 200 oocytes) in PBS with 0.1% polyvinyl alcohol (PVA) and 1 UI µL–1 RNase inhibitor. RNA was extracted using the RNeasy Protect Kit (Qiagen, S�o Paulo, Brazil) according to the manufacturer's instructions. The extracted RNA was used for reverse transcription by the enzyme Improm-II Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. cDNA was produced in a thermocycler for 60 min at 42�C, followed by warming to 70�C for 15 min and cooling to 4�C for freezing at –20�C. Relative quantification of the transcripts for the genes H2a-FZ, HSP 70.1, ZAR-1, and MATER was performed by real-time PCR using the SYBR GREEN Kit (Applied Biosystems do Brasil, Sao Paulo, Brazil) according to manufacturer's instructions. Data were analyzed by the REST� software (Relative Expression Software Tool; Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) 2005 BETA V1.9.9; a level of significance of 5% was considered to show differences among transcripts using H2a-FZ to normalize data. No differences were observed (P < 0.05) for the transcripts HSP 70.1, ZAR-1, and MATER, respectively, when comparing GV (1.0, 1.0, and 1.0), MII (0.37 � 0.1, 0.37 � 0.05, and 0.43 � 0.1), and GV with BGV (0.43 � 0.15, 0.54 � 0.2, and 0.33 � 0.25). However, a difference was detected (P < 0.05) between BGV (1.0, 1.0, and 1.0) and BMII (0.17 � 0.1, 0.13 � 0.05, and 0.3 � 0.2), but not between (P > 0.05) MII (1.0, 1.0, and 1.0) and BMII (0.52 � 0.25, 0.5 � 0.1, and 0.6 � 0.25) for transcripts HSP 70.1, ZAR-1, and MATER, respectively. A reduction in transcripts in group BMII may influence oocyte competence, reducing embryo development and/or quality.
Effect of Granulosa Cells Added to Culture Medium for in Vitro Maturation and Fertilization of Bovine Oocytes.
