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Submitted to: Biology of Reproduction Abstracts
Publication Type: Abstract Only Publication Acceptance Date: 2/20/2008 Publication Date: 9/1/2008 Citation: Vallet, J., Miles, J., Freking, B. 2008. Placental hyaluronidase expression during gestation in swine [abstract]. Biology of Reproduction. Special Issue:217-218 (Abstract #694). Interpretive Summary: Technical Abstract: Previous results have shown that microscopic folds of the epithelial bilayer of the porcine placenta deepen as gestation advances and that epithelial folds of placenta associated with small fetuses deepen more than placenta of large fetuses. These modifications likely provide more surface area for maternal/fetal exchange of nutrients. The folded bilayer is embedded in stromal tissue that previous results indicate is partially composed of hyaluronan. Thus, we hypothesized a role for hyaluronidases in the deepening of the folded epithelial bilayer of the porcine placenta. To explore this role, oligonucleotide primers were designed to amplify the coding regions of porcine hyaluronidase (HYAL) 1 and 2, based on previously reported porcine cDNA sequences. Placental RNA isolated from tissue from day 85 of gestation was subjected to reverse transcription, polymerase chain reaction (rtPCR). The products were cloned using TA cloning and sequenced. Two cDNAs corresponding to alternatively spliced mRNA forms of HYAL 1 were obtained (1379 bp and 1552 bp). The 1379-bp form was similar to the previously described porcine HYAL 1 cDNA. The 1552-bp form was novel and differed from the shorter cDNA by the failure of removal of putative intron 5 (based on the human gene structure, 173 bp) during splicing. Inclusion of the intron introduced a stop codon, truncating the HYAL 1 protein (338 predicted amino acids versus 420 predicted amino acids for the 1372-bp cDNA). A single cDNA for HYAL 2 was obtained, similar to previously described porcine HYAL2 cDNA. Relative expression of each HYAL form was measured by real time rtPCR using total RNA isolated from placental tissues of the smallest and largest fetuses in each litter recovered at slaughter on day 25, 45, 65, 85, and 105 (n = 3 to 5 litters per day) of gestation from unilaterally hysterectomized-ovariectomized (UHO) gilts. Results (after using GAPDH mRNA measurement as a covariate) indicated that expression of HYAL 2 and the 1379-bp form of HYAL 1 did not differ in placenta throughout pregnancy or between the largest and smallest fetuses. Expression of the 1552-bp form of HYAL 1 was greater in the placentas of the smallest fetuses during late gestation (day 65 to 105) compared to the largest fetuses (0.83, 0.76, and 0.86 relative units for small fetuses on day 65, 85, and 105 versus 0.24, 0.39, and 0.43 relative units on day 65, 85, and 105 for large fetuses). Relative hyaluronidase activity was also measured in placental tissue homogenates from the largest and smallest fetuses from UHO gilts on day 25, 45, 65, 85, and 105 of gestation using hyaluronan zymography. Two molecular weight forms were observed (70 kd and 55 kd). The 70-kd form was similar in molecular weight to previously reported HYAL 1. The 55-kd form was novel and was the dominant form. Both forms increased during gestation and were greater in placenta of the smallest fetuses compared to the largest fetuses. These data are consistent with a role for HYAL 1 in placental microscopic fold development during gestation and in response to intrauterine crowding. |
