Proceedings of The Physiological Society
University College Dublin (2009) Proc Physiol Soc 15, C21
Possible Link Between Estrogen Levels, Estrogen Receptors and the Tumor Suppressor Gene p53 During Gestation in Rat Placenta
A. Elfarra1, M. Al-Bader1, S. Mohan1, L. Jacob1
1. physiology, faculty of medicine, kuwait university, Kuwait, Kuwait.
Introduction: Estrogen is essential for initiation and maintenance of pregnancy in the rat. However, high levels of estrogens during pregnancy may have a specific growth-retarding effect on the placenta (1). Consequently, there has to be a control mechanism that enables the placenta to proliferate regardless of the otherwise high inhibitory levels of circulating estradiol. This may partially be mediated through a decrease in estrogen receptor (ER), which has been reported before (2), and a parallel decrease in p53 expression as a link between the expression of these two genes has been reported (3). Therefore, we hypothesize that a decrease in placental ER protein expression correlates with a decrease in placental p53 expression during pregnancy. This study was designed to investigate whether the placental expression of p53 changes during pregnancy in rat. Methodology: Pregnant Sprague-Dawley dams were stunned and killed by cervical dislocation at 16, 19 and 21 days gestation (dg). Placental tissues from each litter were collected (four pregnancies at each gestational age [n = 4] and three to four placental tissues per pregnancy were pooled). Gene and protein expression of p53 were studied using real-time PCR (ReT-PCR) and Western blotting and immunodetection methodologies. Taqman probes specific for p53 and for two housekeeping genes, 18S and beta-glucuronidase (BGLUC) were used. For p53 protein expression, tissues were homogenized to obtain nuclear and cytosolic fractions (verification of nuclear and cytoplasmic fractions was done using Histone H4 antibody and glucose-6-phosphate dehydrogenase (G6PD) assay, respectively. P53 and actin were analyzed in these fractions in addition to the total homogenate fraction. Results: Placentae weight increased significantly between 16 dg and 19 dg (p=0.001), and 16 dg and 21 dg (p=0.01), while there was no significant increase between 19 and 21 dg. Both 18S and BGLU were found to be suitable housekeeping genes as their expression was not changed with gestation. The expression of p53 decreased significantly by 19 dg (p<0.05) and increased by 21 dg (p<0.05). As for protein expression, p53 was detected in both homogenate and nuclear fractions with very faint bands in the cytosolic fraction at 16 dg only. There was a trend for p53 protein to decrease by 19 dg in both homogenate and nuclear fractions, however, this was not statistically significant. Conclusion: According to our results, we found that the expression of p53, at least at the mRNA level, decreased at 19 dg allowing the placenta to increase in weight while at 21 dg the p53 expression increased suppressing placental growth. This was reflected in the weights that we obtained.
Where applicable, experiments conform with Society ethical requirements