Temporal and Spatial Characterization of Hyaluronic Acid and Associated Transcriptional Activity During Early Porcine Embryo Development.

Abstract

Hyaluronic acid (HA), a member of the glycoaminoglycan family, has been shown to have positive effects on in vitro embryo development in mice, cows, and pigs. Furthermore, its addition to culture media has been shown to increase embryo development following transfer in mice and improve pregnancy rates resulting from embryo transfer in human populations with multiple previous failures. However, the mechanisms by which HA affects early embryo development is yet unknown, a situation further complicated by changing effects in response to both the concentration and molecular weight of the HA used. The objective of this experiment was to determine the normal status of HA within the porcine reproductive tract and resolve any temporal and spatial variations within the system which may affect embryo viability up to the blastocyst stage. Primiparous sows (n=21), on their second estrus following weaning, were randomly designated for sample collection at seven periods corresponding to the germinal vesicle (GV), metaphase II (MII), 2cell, 4cell, early morula, morula, and blastocyst stages of embryonic development. Timing of collection for each the post-ovulatory stages was based on detection of ovulation by real time transcutaneous ovarian ultrasound at 4 hour intervals; while the staging of the GV group was based on detection of standing estrus. A terminal hysterectomy was performed, and flushing of the oviduct and lower, middle, and upper thirds of the uterine horns relative to cervix carried out. Tissue samples from each of the uterine sections along with the ampula, isthmus and ampulary-ismic junction were collected and flash frozen in liquid nitrogen, while embryos were isolated from the flushings, imaged and frozen in pools of five embryos at like stages. HA content in each segment was determined through quantitative ELISA and concentrations adjusted to account for flush volume. Total RNA was extracted from tissue samples and embryos, DNase treated, and analyzed using real time PCR for determination of relative gene expression of HAS1, 2 and 3 and CD44 using 18S as an endogenous control. Expression was analysed using the ΔCT method for endometrial samples, and the residuals of 18S as a covariate for embryos. ELISA data was log transformed for normality and all data was analysed for temporal and spatial variation using the general linear model of SAS. Minimal spatial variation was found in the post-ovulatory stages, however temporal differences in HA concentration and gene expression were identified. ELISA results show elevated concentrations of HA at the GV stage that decrease rapidly at the 2cell stage and then increase toward the blastocyst stage. HAS1 and HAS3 were identified as the predominantly transcribed synthase forms in both the reproductive tract and embryos, while low level expression of HAS2 was detected in the reproductive tract irrespective of embryonic stage. Synthase gene expression appears to be reciprocal between the uterus and embryos, with highest expression peaking at the GV and 4 cell stages respectively. These results suggest HA does not play a significant role in the development of spatial asynchrony in the reproductive tract, but may play a significant role in temporal asynchrony. Furthermore, the complex interplay between embryonic and uterine expression profiles suggests an extracellular signaling role for HA during early development. (poster)