Effect of Supplementation of Cryoprotectant Solution with Hydroxypropyl Cellulose for Vitrification of Bovine Oocytes

Abstract

The technology of successful cryopreservation is a very important factor in research and commercial applications. However, the survival and development of the vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. This study investigated the effect of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. Oocytes exposed to 0, 10, 50, and 100 µg/mL HPC were named the 0, 10, 50, and 100 HPC groups, respectively. Samples were thawed via sequential incubation in Dulbecco's phosphate-buffered saline (D-BPS) supplemented with 10% fetal bovine serum and decreasing concentrations of sucrose (1, 0.5, 0.25, and 0.125 M) for 1 min each time. After thawing, VT oocytes were treated at 0.05% hyaluronidase, and cumulus cells were removed by mechanical pipetting. The oocytes were washed with HEPES-buffered Tyrode's medium and incubated in a droplet of previously cultured in vitro maturation medium for 1 h to recover. The survival rate of the oocytes was significantly higher in the 50 HPC group (84.2%) than in the 0 (75.4%), 10 (80.4%), and 100 (75.5%) HPC groups. The reactive oxygen species (ROS) levels of the non-VT and 50 HPC groups were lower than the 0, 10, and 100 HPC groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA expression levels of antiapoptotic genes (BCl2) was higher in the non-VT than in the other groups. The mRNA level of a stress-related gene (Hsp70) was lower in the 50 HPC than in the other groups. At day 8, the developmental capacity of embryos obtained via parthenogenetic activation (PA) was determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate of the non-VT group was significantly higher, but the blastocyst development rate and total cell number per blastocyst did not significantly differ between the non-VT and 50 HPC groups. The mRNA levels of proapoptotic genes (Bax and Caspase-3) and a stress-related gene (Hsp70) were higher in the 0 HPC group than in the non-VT and 50 HPC groups. In conclusion, supplementation of vitrification solution with HPC improves the survival rate of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA.

Keywords: bovine oocytes; cryoprotectant; hydroxypropyl cellulose; solution vitrification.

Figure 1

Morphologies of oocytes in the 0 (A,a), 10 (B,b), 50 (C,c), and 100 (D,d) HPC groups. (A–D): before vitrification, (a–d): after thawing. Bar, 100× in (A–D) and (a–d).

Figure 2

Comparison of levels of ROS in HPC (0, 10, 50, and 100) groups and the non-VT group. Fluorescence images of ROS (A,a–e). ROS activity was detected with DCHFDA (green). Fluorescence intensity of DCHFDA (B). * p < 0.05 compared with the non-VT group.

Figure 3

Relative mRNA expression levels of genes related to apoptosis (Bax and Bcl2), demethylation (Dnmt3a), and stress (Hsp70) in non-VT and VT oocytes. * p < 0.05 compared with the non-VT group.

Figure 4

Morphologies of embryos obtained via PA in the non-VT, 0 HPC, and 50 HPC groups. Images of cleaved embryos at day 2 ((A), non-VT; (B), 0 HPC; and (C), 50 HPC) and blastocysts at day 8 ((a), non-VT; (b), 0 HPC; and (c), 50 HPC). Bar, 100 µm.

Figure 5

mRNA expression levels of genes (Dnmt3a, Hsp70, Caspase-3, Glut-5, Interferon-tau, HSF, and Bax) related to development potential in blastocysts produced via PA in the non-VT, 0 HPC, and 50 HPC groups. The internal standard used β-actin gene. * p < 0.05 compared with the non-VT group. ** p < 0.05 compared with the non-VT and 0 HPC groups.