182 OSTEOPONTIN GENE EXPRESSION IN IMMATURE AND MATURE SWINE CUMULUS CELLS AND OOCYTES

Abstract

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found in both the female and the male reproductive tract that is believed to facilitate reproduction. It was recently reported in swine that OPN improves in vitro fertilization (Hao et al. 2006 Biol. Reprod. 75, 726–733). In bovine, OPN improves the efficiency of in vitro embryo production, and it has been detected on the zona pellucida (ZP) of immature and mature oocytes (Monaco et al. 2007 J. Anim. Sci. 85, 529 abst). This study was designed to evaluate the gene expression of OPN (SPP1) in immature and mature swine cumulus cells and oocytes. Ovaries from a local slaughterhouse were collected on three different days. Half of the immature cumulus–oocyte complexes (COCs) from each day were vortexed in 1 mL TCM-199 HEPES containing 10 mg mL^–1 hyaluronidase to separate immature cumulus cells, while the other half of the COCs were maturated for 40-42 h and then the cumulus cells were separated. RNA from cumulus cells and oocytes was extracted and genomic DNA was removed by DNase treatment. cDNA was synthesized using 100 ng of RNA and diluted 50% with DNase–RNase-free water. The RT-PCR product from cumulus cells and oocytes was run in a 2% agarose gel stained with ethidium bromide to verify the presence of SPP1. Relative mRNA abundance between immature and mature cumulus cells was assessed by SYBR green real-time RT–PCR run in triplicate using a 6-point twofold dilution standard curve. GAPDH was used as internal control. Primers for SPP1 and GAPDH were designed spanning an exon/exon junction. The presence of a single and specific PCR product was assessed by gel electrophoresis (a single band was expected at 100 and 90 bp, respectively, for SPP1 and GAPDH), dissociation curve, and sequencing. A t-test was used to assess differences between immature (n = 3) and mature (n = 3) cumulus cells. SPP1 was detected in agarose gel in both immature and mature oocytes and cumulus cells. Cumulus cells presented a Ct for SPP1 ranging from 20 (immature) to 29 (mature). Immature cumulus cells showed a 32-fold larger mRNA abundance compared to the mature cumulus cells (P < 0.05) when data were transformed using a standard curve. Results showed expression of SPP1 in porcine oocytes and cumulus cells. However, maturation significantly decreased the expression of SPP1 in cumulus cells. The presence of SPP1 mRNA in oocytes and cumulus cells and the larger mRNA abundance before maturation may suggest a role of this protein prior to maturation of oocytes. Additional studies will be required to determine the specific role of SPP1 in oocyte maturation in the pig.