In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing: limited effect on the morphology of the second meiotic spindle.
Cryopreservation of germinal vesicle (GV)-stage mouse oocytes results in a developmental block. As an approach to explain the failure in development, we have investigated the morphology of the second meiotic spindle after in-vitro maturation. Fully grown GV-stage mouse oocytes were collected from the ovaries of primed mice and kept in meiotic arrest with dibutyryl cyclic AMP. These oocytes were submitted to different variables of cryopreservation: (i) cooling to 22 degrees C or 0 degrees C; (ii) exposure to 1.5 M 1,2-propanediol at 22 degrees C or 0 degrees C; (iii) exposure to 1.5 M dimethylsulphoxide (DMSO) at 22 degrees C or 0 degrees C; (iv) ultrarapid freezing with 3.5 M DMSO/0.5 M sucrose; (v) exposure to a sucrose dehydration series according to the ultrarapid freezing protocol. The morphology of the second meiotic spindle was evaluated 16 h after release from meiotic arrest. We were able to demonstrate that following cooling, exposure to cryoprotectants or ultrarapid freezing of GV-stage mouse oocytes, a normal barrel-shaped spindle with the chromosomes in midplane position is found in 79-94% of oocytes except for two conditions with great exposure to dehydration stress. Exposure to DMSO at 0 degrees C or exposure to a sucrose dehydration series resulted in significantly lower percentages of barrel-shaped spindles, respectively 64% and 62%. The effect on spindle morphology has to be put into perspective, however, since the observed abnormalities were changes of spindle shape, such as elongation or reduction, which are assumed to be restorable, and since no polar organizational defects were found.(ABSTRACT TRUNCATED AT 250 WORDS)
Van der Elst
J
J
Centre for Reproductive Medicine, University Hospital, Vrije Universiteit Brussel, Belgium.
Nerinckx
S
S
Van Steirteghem
A C
AC
D000818:Animals; D003080:Cold Temperature / Q000009:adverse effects; D015925:Cryopreservation / Q000379:methods; D003451:Cryoprotective Agents / Q000009:adverse effects*; D004121:Dimethyl Sulfoxide / Q000009:adverse effects; D005260:Female; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008853:Microscopy; D009865:Oocytes / Q000166:cytology / Q000254:growth & development*; D019946:Propylene Glycol; D011409:Propylene Glycols / Q000009:adverse effects; D008941:Spindle Apparatus / Q000187:drug effects* / Q000648:ultrastructure
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D019946:Propylene Glycol; D004121:Dimethyl Sulfoxide