Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries.
The aim of this study was to develop a maturation protocol for immature oocytes and assess the protocol with cryopreserved oocytes. Nuclear maturation (mature spindle and aligned chromosomes) occurred irrespective of the treatment regime: 71-89% of oocytes matured in vitro had a normal spindle and chromosomes compared with 87% matured in vivo. Fertilization rates were not significantly different from those of in-vivo matured oocytes. Of the maturation treatment regimes investigated, the initial treatment producing best development to blastocyst (cytoplasmic maturation) involved a 2 h incubation in standard maturation medium (SMM) containing 7.5 IU follicle stimulating hormone (FSH) followed by 14 h in SMM plus 7.5 IU FSH:luteinizing hormone with follicular cells [62% (range 49-69)]. The addition of 1 ng/ml epidermal growth factor (EGF) in this protocol resulted in development [75% (range 71-81)] that was not significantly different from in-vivo matured oocytes [82% (range 73-90)]. Exposure of the oocytes to 1.5 M dimethylsulphoxide (DMSO) did not affect fertilization or development rates. Following a slow-cool/thaw freezing regime, 81% (range 74-89) of the oocytes were morphologically normal, i.e. had a spherical shape with an intact zona and oolemma; they had, however, lost their previously attached cumulus and corona cells. Maturation of frozen-thawed oocytes in the presence of EGF gave good fertilization rates but poor development rates [80% (range 77-86) and 37% (range 33-40) respectively]. In conclusion, the best maturation, both nuclear and cytoplasmic, occurred in the presence of a combination of gonadotrophins, EGF and follicular cells. Oocytes cryopreserved using a slow-cool/thaw regime can be matured to produce blastocysts after in-vitro fertilization.
Cooper
A
A
Department of Obstetrics and Gynaecology, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Paynter
S J
SJ
Fuller
B J
BJ
Shaw
R W
RW
D000818:Animals; D002467:Cell Nucleus / Q000502:physiology*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D002875:Chromosomes / Q000502:physiology; D015925:Cryopreservation; D003451:Cryoprotective Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000502:physiology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005314:Embryonic and Fetal Development / Q000502:physiology; D005260:Female; D005306:Fertilization / Q000502:physiology; D005307:Fertilization in Vitro; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000502:physiology*; D010053:Ovary / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D012016:Reference Values; D013268:Stimulation, Chemical
D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide