Expression and regulation of the rat prostaglandin E2 receptor type 4 (EP4) in pregnant cervical tissue☆
Section snippets
Tissue and RNA extraction
For these studies, cervical tissue was surgically obtained from timed-pregnant Sprague-Dawley rats under pentobarbital sodium anesthesia (100 mg/kg) by use of a protocol approved by the Institutional Animal Care and Utilization Committee at the University of Chicago. Specifically, animals were killed in estrus and on gestational days 12, 16, 20, 21, and 22 (day of parturition), and day 0 post partum. All tissue was obtained before midday. Labor begins in the evening of day 22 with delivery
Results
Real-time RT-PCR measurements of cervical EP4 mRNA demonstrated increasing levels with advancing gestation (Fig 1). This increase was determined to be significant (ANOVA, P = .028). EP4 mRNA levels were relatively constant from estrus to day 21 of gestation. EP4 mRNA expression increased significantly on day 22 and postpartum day 0 over levels seen on day 12 and estrus (multiple comparisons, S-N-K method). EP4 mRNA expression doubles on day 22 compared with estrus levels.
EP4 Western blots of rat
Comment
Cervical ripening involves the remodeling of the extracellular matrix. Biochemical studies of the cervix have identified changes in the composition of the extracellular matrix with increases in collagenase activity.3., 20. Anatomic studies have demonstrated fragmentation of collagen fibrils and disruption of collagen bundles in the ripened cervix1., 20., 33. and in cervical tissue treated with PGE2.1., 3., 20. These changes were found to be similar in both the human and the rat in anatomic
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2008, American Journal of Obstetrics and GynecologyCitation Excerpt :MMP-2 protein expression was determined by Western blot as previously described.27 Ribonucleic acid (RNA) for real-time RT-PCR was prepared as previously described,28 using Trizol (Invitrogen, Carlsbad, CA) and SuperScript first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer's protocol. First-strand synthesis was performed using 4 μg of total RNA, random hexamer primers, and SuperScript II reverse transcriptase.
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Supported by University of Chicago Summer Research Stipend, NIH Grant for Basic Research, Society for Gynecological Investigation Summer Medical Student Grant, and the National Institute of Child Health and Human Development (grant No. HD01232-01).