134 Trichostatin A as an aging agent during in vitro fertilization in pig oocytes

Abstract

An aged oocyte is one that was not fertilized during the optimal time window after ovulation and potentially has diminished fertilization and embryonic development success. Oocytes (n = 2562) were matured with or without trichostatin A (TSA; 100 ng mg⁻¹), a known meiotic inhibitor, for 24 or 48 h (OMI), then for an additional 16 h (OMII) without TSA or hormones. Oocytes were measured for meiosis before maturation (n = 95), after OMI (n = 365), and after OMII (n = 230) as well as cumulus cell expansion (CCE). Oocytes (n = 800) were fertilized with frozen–thawed boar sperm and potential embryos were evaluated for IVF characteristics and cleavage and blastocyst formation 48 and 144 h after IVF, respectively. Apoptosis was determined in the maturing oocytes by levels of reactive oxygen species (ROS; n = 476), mitochondrial electrochemical potential gradient dissipation (n = 405), caspase 3 (n = 405), phosphatidylserine (n = 453), and DNA breakdown (n = 192). Data were analysed by ANOVA and Tukey’s test. Oocytes matured without TSA for 48 h had a significantly higher (P < 0.05) percentage of oocytes at the MI stage of meiosis compared to all other treatment groups (16.00 ± 6.80) and oocytes matured without TSA for 24 h had a significantly higher (P < 0.05) percentage of oocytes at the MII stage of meiosis compared to all other treatment groups (14.7 ± 11.30). Oocytes matured with or without TSA for 48 h had significantly less (P < 0.05) CCE compared to oocytes matured without TSA for 24 h. Supplementation of TSA to OMI significantly decreased (P < 0.05) fertilization penetration rates compared with not supplementing TSA for 24 h. Percent of embryos cleaved by 48 h and those reaching blastocyst by 144 h after IVF were significantly higher (P < 0.05) in oocytes matured for 24 h compared with those matured for 48 h. Oocytes matured without TSA for 24 h generated significantly different (P < 0.05) levels of ROS compared with oocytes matured with TSA for 48 h. Oocytes matured without TSA for 48 h had significantly higher (P < 0.05) mitochondrial membrane potential compared with all other treatments. Levels of caspase 3 activation and phosphatidylserine (which translocates in response to apoptosis) differed (P < 0.05) between treatments. Results from the TUNEL assay indicate oocytes matured through OMII with a short OMI and TSA supplemented (23.8 ± 2.99%) and through OMI with a long OMI and TSA supplemented (24.3 ± 1.11%) had significantly greater (P < 0.05) percent of oocytes with fragmented DNA than the other treatments, except for oocytes matured through OMII with a long OMI and TSA supplemented (35.0 ± 3.55%), which was significantly greater (P < 0.05) than all other treatments. Results indicate that use of TSA to stimulate aging in pig oocytes is a valid and a reliable option.