250 EXPRESSION PATTERN OF p53 mRNA DURING THE ESTROUS CYCLE IN SWINE

Abstract

During each estrous cycle, more than 99% of the ovarian follicles undergo a degenerative process known as atresia. The p53 is an antiproliferative transcription factor that enhances the transcription rate of important genes in the apoptotic pathway. The aim of the current study was to investigate the pattern of p53 mRNA expression during estrous cycle in the pig ovary by real-time PCR technique. Sixteen prepubertal gilts (Landrace × Large White × Pietrain) were obtained from the pig farm at the Universidade Federal de Viçosa (Viçosa, MG, Brazil). The estrous cycle was synchronized with P.G. 600^® (Intervet/Schering-Plough Animal Health, Millsboro, DE, USA; 400 IU eCG and 200 IU hCG). The onset of estrus was checked twice a day using a mature boar. The gilts, n = 4 per group, were slaughtered on Days 0, 6, 12, and 18 of estrous cycle. Granulosa cells from follicles were collected by vacuum aspiration and washed in PBS by centrifugation at 5.000 × g for 6 min, and the RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The ovarian cortex was stored in RNAlater (Ambion, Austin, TX, USA) and frozen at -80°C. Its RNA was extracted from 30 mg using the same Kit. For each animal in each stage, a pool of equivalent amounts of RNA from granulosa cells and ovary cortex was reverse transcribed with SuperScript III/RNaseOUT Enzyme Mix (Invitrogen Life Technologies, Carlsbad, CA, USA) to evaluate gene expression for the ovary as a whole. Quantitative real-time PCR was performed using SYBR green fluorescent detection system on ABI Prism 7300 Sequence Detection Systems (Applied Biosystems, Foster City, CA, USA). The primers were designed from swine sequences available at GenBank. The linearity of amplification for p53 mRNA was similar to the endogenous control gene, glyceraldehyde-3-phosphate dehydrogenase. Reactions were performed using 200 nM primer and 100 ng of the cDNA per reaction for both genes. The thermal cycling conditions consisted of 40 cycles of 30 s of melting at 95°C followed by 30 s of annealing and extension at 60°C. After amplification, a melting curve analysis was performed to validate the absence of non-specific products. Gene expression data were presented using the 2ACt method (Livak and Schimittgen 2001). The results of gene expression were analyzed using linear regression after transformation ln(2ACt + 1) as the dependent variable and days of estrous cycle as independent variables using the general linear model procedure (SAS Institute, Inc., Cary, NC, USA). The mRNA expression was not affected by days of estrous cycle (P = 0.86). In rats, the expression of p53 mRNA in granulosa cells has been already described. Despite the fact that no difference has been found during the estrous cycle in this study, the expression of this messenger in the pig ovary seems to be undescribed until now. In the future, for a better understanding of p53 regulation in the pig, gene expression analysis in different follicle sizes and physiological status will be presented by our group to examine the expression patterns of this gene, as well as other related ones, included in this apoptotic pathway.