Location: Reproduction Research
Title: Molecular cloning and characterization of heparanase mRNA in porcine placenta throughout gestation Authors
Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: February 20, 2008
Publication Date: September 1, 2008
Citation: Miles, J., Vallet, J., Freking, B. 2008. Molecular cloning and characterization of heparanase mRNA in porcine placenta throughout gestation [abstract]. Biology of Reproduction. Special Issue:125-126 (Abstract #305). Technical Abstract: The porcine placenta is classified as epitheliochorial and is composed of a folded-bilayer consisting of intact epithelium from the endometrium and trophectoderm embedded in loose stroma. As pregnancy progresses, the fold width increases and becomes more complex providing greater surface area for feto-maternal exchange. The placental stroma is a complex extracellular matrix (ECM) composed of several glycosaminoglycans including heparan sulfate proteoglycan (HSPG). Heparanase (HPSE) is an endoglycosidase that degrades HSPG resulting in modification of the ECM. The objective of this study was to clone cDNA encoding porcine HPSE and characterize the expression pattern of HPSE mRNA in porcine placentas throughout gestation. White crossbred gilts were unilaterally hysterectomized-ovariectomized at 160 days of age and mated at estrus. At slaughter, placental tissues were collected and snap-frozen from the smallest and largest fetuses in each litter at Day 25, 45, 65, 85, and 105 of gestation (n = 3-5 litters per day). Whole-cell RNA from a Day 105 placenta of a large fetus was reverse transcribed (rt) and subjected to PCR with primers designed to amplify the coding region of HPSE based on EST sequences for the pig. Two cDNAs were identified relating to HPSE, an abundant cDNA of 1528 bp (6 of 7 positive clones) and a rarer cDNA of 1661 bp (1 of 7 positive clones). The abundant cDNA (HPSEa) included most of the protein coding region and corresponded to a predicted protein of 508 amino acids that shares 79.6% and 79.1% identity with human and bovine HPSE, respectively. The rarer cDNA (HPSEb) contained an additional exonic region in the N-terminal region that introduced several stop codons resulting in a predicted truncated protein of 81 amino acids. Real-time rt-PCR was used to measure expression of HPSEa and HPSEb in placentas from the smallest and largest fetuses from Day 25 to 105 of gestation. All data were adjusted based on the PCR efficiencies for each gene obtained from a relative standard curve using pooled cDNA and normalized to GAPDH expression levels. Analysis for the effects of gestational day and fetal size on gene expression was performed using MIXED model procedures using gilt within day as a random effect. The relationship of expression of HPSEa and HPSEb was also assessed by regression analysis. Expression of both HPSEa and HPSEb significantly (P < 0.001) increased as pregnancy advanced with the greatest expression in placentas at Day 85 and 105 of gestation. There was no significant effect of fetal size for either HPSEa or HPSEb expression. Expression of HPSEa was 32 times greater (P < 0.001) than HPSEb expression, and the relationship was linear (P < 0.001). These findings suggest that HPSE may be involved in modification of the ECM by degrading HSPG, especially during late gestation in the pig placenta. Whether the truncated form of HPSE corresponding to HPSEb is translated, and its possible role during pregnancy, remains to be determined.