181 The effects of aging in the oocyte induced by trichostatin A

Abstract

Successful fertilization and embryonic development decreases for an aged oocyte that is not fertilized during the optimal window of time after ovulation. If fertilization of an aged oocyte does occur, the developing embryo is prone to have a delay through meiosis and a decrease in cleavage and blastocyst formation. Trichostatin A (TSA) prohibits the breakdown of the germinal vesicle during meiosis. The objective of this study was to create an invitro model to study the effects of aging in pig oocytes. Oocytes (n=1489) were matured with or without TSA (100ngmL−1) for 24 or 48h followed by an additional 16h of maturation without TSA. A portion of the oocytes were evaluated for stage of meiosis (MI and MII) before maturation (n=95), after the first part of maturation (OMI; 24 or 48h, n=363), or after completing maturation (OMII; 16h, n=230). The remaining oocytes (n=801) were fertilized for 6 to 8h and potential embryos developed. A portion of the embryos were evaluated for fertilization characteristics 12h after IVF (n=400). Remaining embryos (n=401) were evaluated for cleavage by 48h and blastocyst formation by 144h, after IVF. Data were analysed using ANOVA and Tukey's test. Oocytes matured without TSA for 64h had a significantly higher (P<0.05) percentage of oocytes at the MI stage of meiosis compared with all other treatment groups (16.00±6.80), and oocytes matured without TSA for 40h had a significantly higher (P<0.05) percentage of oocytes at the MII stage of meiosis compared with all other treatment groups (14.7±11.30). The results (Table 1) indicate that supplementation of TSA to OMI significantly decreased fertilization penetration rates compared with not supplementing TSA for 24h. However, the percent of embryos cleaved by 48h after IVF was significantly higher in oocytes matured for 40h compared with those matured for 64h. The percent of embryos reaching the blastocyst stage of development by 144h after IVF was significantly lower in oocytes matured for 64h compared with those matured for 40h without TSA. In conclusion, these results suggest that supplementation of TSA during early maturation delays meiosis and decreases fertilization, and increasing maturation time causes a decrease in early embryonic development. By supplementing TSA during early maturation and increasing the length of maturation by 24h, we have created an invitro model to study the effects of aging in pig oocytes. Table 1.Fertilization and developmental characteristics of oocytes1 Amount of TSA added during OMI (ngmL−1) Length of OMI (h) Oocytes penetrated (%) Polyspermicoocytes2 (%) Oocytes with MPN2 (%) Cleavage (%) Day 7 blastocyst (%) 0 24 83.00±3.80a 15.70±4.00 75.90±4.70 77.20±4.20a 27.70±4.20a 0 48 72.00±4.50ab 20.80±4.80 76.40±5.00 58.00±5.00bc 12.00±5.00b 100 24 65.00±4.80b 30.80±5.80 73.80±5.50 68.00±4.70ab 21.00±4.70ab 100 48 56.00±5.00b 30.40±6.20 62.50±6.50 46.00±5.00c 8.00±5.00b a,bMeans with different superscripts in the same column differ (P<0.05). 1TSA, trichostatin A; OMI, oocyte maturation 1. 2Percent of the number of oocytes penetrated.