249 BOVINE GRANULOSA CELLS MRNA EXPRESSION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-α AND THE PROTO-ONCOGENE C-FOS

Abstract

PPARα and c-Fos are involved in regulation of gene expression and are known to be dependent on retinoic acid (RA), which in turn influences oocyte growth and developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003. Reproduction 125, 409–416), probably acting in part through granulosa cells. Peroxisome proliferator-activated receptor-α (PPARα) heterodimerizes with the retinoid receptor X (RXR), while c-Jun/c-Fos heterodimerizes with liganded retinoic acid receptors (RARs), then preventing formation of transcription factor activator protein 1 (AP-1) complexes capable of DNA binding. Cellular retinoic acid binding protein (CRABP) limits RA excess and regulates the transcriptional potential of RA;; CRABPII has been detected in rat granulosa cells from mature follicles and luteal cells. The aim of this study was to investigate PPARα, c-Fos and CRABPII mRNA expression in bovine granulosa cells. In parallel, other genes whose expression can be influenced by RA were analyzed: luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), aromatase and growth hormone (GH). Ovaries were collected at a local abattoir and kept in saline at 30–35°C. Granulosa cells were obtained by aspirating 2- to 7-mm antral follicle contents, pelleted at 700 g for 4 min and resuspended in RNA-later (Ambion^®). Total RNA was isolated with a NucleoSpin^® RNAII kit (Macherey-Nagel), and mRNA was reverse transcribed into single-stranded cDNA using a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). A PCR standard method was made using 1 μL of the cDNA as a template. All PCR primer couples were designed on the basis of the bovine sequence, but c-Fos and CRABPII primers were designed based on the human-murine sequences. Primers within the couple were located in different exons to distinguish DNA from RNA amplification. CRABPII was further investigated in bovine whole ovary, corpus luteum (CL) and liver, in a search for positive controls. Bovine β-actin, 18S and 28S were examined in each sample as positive controls for RNA isolation and cDNA synthesis efficiency. Ten μL of product were loaded into an agarose 2% gel in TBE buffer containing ethidium bromide, and were separated by horizontal electrophoresis. Gels were visualized with ultraviolet light and photographed using a digital camera. Gene expression in granulosa was demonstrated for PPARα, c-Fos, LHr, FSHr, aromatase, GH and controls (β-actin, 18S and 28S) but CRABPII gene did not express in granulosa cells, whole ovary, CL or liver under our experimental conditions. While lacking CRABPII expression remains intriguing, the expressed genes support a role of retinoid pathway within granulosa cells under both in vivo and in vitro conditions, because granulosa cells used in the present experiments were derived from follicles providing oocytes for IVM-IVF. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).