Cytoskeletal Changes of Vitrified Porcine Oocytes

This experiment was designed to examine the spindle organization, and chromosomal and microfilament distribution of vitrified porcine oocytes. The germinal vesicle -stage (GV stage) oocytes were aspirated from antral follicles (2-5 mm in diameter). All Metaphase II-stage (M II stage) oocytes used in the experiment were derived from GV oocytes matured in vitro. Either GV or M II stage oocytes were divided into three groups:the control group, the group treated with vitrification solution, and the vitrified group. Vitrified-warmed M II oocytes derived from maturation in vitro were directly used for Laser-Scanning Confocal Microscopy (LSCM); vitrified-warmed GV oocytes were firstly cultured for 44 h and then used for LSCM. Oocytes used for the experiment were fixed and stained by immunofluorescence and were then observed by LSCM. The percentage of GV stage oocytes treated with vitrification solution with normal spindle organization, chromosome alignment and actin filaments (F-actin) distribution was 42.9%, 89.6% and 28.6%, respectively. These were significantly higher than those from the vitrified group of GV stage oocytes (10.1%, 36.4% and 16.9%, respectively; P<0.05). The values for GV oocytes treated with vitrification solution were all significantly lower than those from the control, except for the percentage with normal chromosome alignment (79.5%, 93.1% and 72.3%, respectively, P<0.05). The percentage of M II stage oocytes from the group treated with vitrification solution and from the vitrified group with normal spindle organization, chromosome alignment and actin filaments (F-actin) distribution were 34.4% versus 12.9%, 61.3% versus 56.7%, and 47.9% versus 37.2%, respectively. These were significantly lower than those from the control (78.3%, 90.1% and 72.8%, respectively; P<0.05). Results from this experiment suggest that irreversible damage to the cytoskeleton of porcine GV and M II oocytes after vitrification could be an important factor affecting the maturation, fertility and subsequent development of the oocytes.

Keywords
Porcine; Oocytes; Vitrification; Cytoskeleton; Laser-scanning confocal microscopy