Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes

Abstract

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

Keywords: E-64; bovine oocytes; cathepsin B; developmental competence; in vitro maturation; ovum pick-up.

Figure 1

Effect of E-64 supplementation during in vitro maturation on the developmental rate and the quality of preimplantation embryos. Bovine COCs were collected separately from slaughterhouse ovaries of the same animal followed by in vitro maturation in the presence or absence of 1 μM of E-64. After in vitro fertilization, presumptive zygotes were cultured for 7 days. Cleavage (A) and blastocyst (B) rates were observed on days 2 and 7, respectively. Total number of oocytes used is 166. Total cell number of day 7 blastocysts was counted with DAPI (C). The apoptotic status of day 7 blastocysts was detected by TUNEL staining (D). Total number of embryos used is 44. The experiments were carried out at least three times. The data are expressed as mean ± SEM. Values with asterisks vary significantly, * p < 0.05, *** p < 0.001.

Figure 2

Effect of E-64 supplementation during in vitro maturation on the developmental competence of ovum pick-up-derived immature oocytes. Bovine COCs were recovered from live animals using ultrasound-guided transvaginal ovum pick-up technique. Recovered COCs were in vitro matured in the presence or absence of 1 μM of E-64. After in vitro fertilization, putative zygotes were cultured for 7 days. (A) Cleavage rate was evaluated on day 2 while the blastocyst rate was evaluated on day 7. Shown are representative images. (B) Quantifications of the cleavage rate. (C) Quantifications of the blastocyst rate. Total number of oocytes used is 98. The experiments were carried out five times. The data are expressed as mean ± SEM; Student’s t-test was used to analyze the data. Values with asterisks vary significantly, * p < 0.05.

Figure 3

Effect of E-64 supplementation during in vitro maturation on the quality of preimplantation embryos. Bovine COCs were recovered from live animals using ultrasound-guided transvaginal ovum pick-up technique. Recovered COCs were in vitro matured in the presence or absence of 1 μM of E-64 followed by IVF and culture. (A) Day 7 blastocysts were examined using TUNEL staining (green) to assess the apoptotic status and DAPI (blue) to assess the total cell number. Shown are representative images. (B) Quantification of the average total cell number. (C) Quantification of the percentage of TUNEL positive cells. Total number of oocytes used is 46. The experiments were carried out three times. The data are expressed as mean ± SEM; Student’s t-test was used to analyze the data. Values with asterisks vary significantly, ** p < 0.01, *** p < 0.001.

Figure 4

Schematic diagram of the role of cathepsin B and its inhibition by E-64 on the developmental competence and quality of bovine oocytes recovered by ultrasound-guided ovum pick-up technique.