71 Removal of hypotaurine from porcine embryo culture medium decreases message for pro-apoptotic genes but does not affect development at low oxygen tension

Abstract

Hypotaurine (HT) is a routine additive to embryo culture medium, acting primarily as an oxygen radical scavenger. However, the practice of culturing embryos at ~5% O2 has been widely adopted because this is more physiologically relevant to the oxygen tensions detected in the oviduct and uterus. Thus, the utility of HT may be diminished as fewer oxygen radicals are produced during culture at 5% O2. The objective here was to determine the effects of removing HT from our porcine embryo culture medium (MU2) on the development of embryos incubated at a lower oxygen tension (5% O2) compared with atmospheric oxygen (~20% O2). Porcine cumulus-oocyte complexes were aspirated, matured, and fertilized with standard procedures from our laboratory. Presumptive zygotes were cultured in 1 of 4 conditions: MU2 with 5mM HT at 5% O2 (low O2 +HT; control), MU2 without HT at 5% O2 (low O2 −HT), MU2 with 5mM HT at 20% O2 (high O2 +HT), or MU2 without HT at 20% O2 (high O2 −HT). The percentage of presumptive zygotes that developed to the blastocyst stage on Day 6 and total number of nuclei in the blastocysts were determined. The RNA was extracted from pools of 30 blastocysts, and cDNA was synthesised for quantitative PCR for genes associated with HT synthesis, oxidative stress, and apoptosis. Damage to DNA was assessed by TUNEL staining of Day 6 blastocysts. Data were analysed for normality by using a Shapiro-Wilk test, and differences between means were detected by using 2-way ANOVA followed by Tukey’s honest significant difference test with P<0.05 considered significant. Embryos cultured with high O2 −HT had significantly decreased blastocyst development compared with all other groups (26.2±2.5% v. 36.9-41.7±3.3-4.3%). Embryos cultured with low O2 −HT had significantly more nuclei than those cultured with high O2 +HT (50.5±1.5v. 45.5±1.2). Notably, differences in blastocyst development (41.7±3.3% v. 36.9±3.3%) or total number of nuclei (50.0±1.8v. 50.5±1.5) were not detected between embryos cultured with low O2 +HT or low O2 −HT. The abundance of messages for genes involved in HT synthesis (cysteine sulfinic acid decarboxylase [CSAD]) and oxidative stress (superoxide dismutase 1 [SOD1] and glutathione peroxidase 6 [GPX6]) did not differ between groups. Contrarily, messages for 2 pro-apoptotic markers (BCL2 associated agonist of cell death [BAD] and caspase 3 [CASP3]) were significantly increased in embryos cultured with +HT regardless of oxygen tension; however, percentages of DNA-damaged nuclei in blastocysts after TUNEL staining were not different between groups (5.4-6.5±0.5-1.0%). These results indicate that HT is not necessary for porcine pre-implantation development at 5% O2 but is beneficial at atmospheric oxygen tension. Further investigation is required to confirm if HT promotes apoptosis in pre-implantation embryos. This research was supported by Food for the 21st Century at the University of Missouri and R01 HD080636.