Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for developmental competence.
BACKGROUND
Current outcome results with embryos derived from thawed MII human oocyes are significantly lower than with embryos cryopreserved at the pronuclear stage. Here, we investigated whether freezing-thawing was associated with changes in oocyte mitochondrial polarity (DeltaPsim) that could influence competence by altering ATP levels or the ability of the cytoplasm to regulate intracellular Ca2+.


METHODS
Fresh and thawed uninseminated and unfertilized MII oocytes were stained with the DeltaPsim-specific probe JC-1 to detect clusters of high-polarized mitochondria (J-aggregate positive) and with the Ca2+- specific probe Fluo-4 to measure changes in intracellular levels of this cation. ATP content per oocyte was measured directly and cortical granules were visualized with a cortical granule-specific probe.


RESULTS
A significant difference between fresh and thawed MII oocytes existed for pericortical J-aggregate fluorescence and for the ability of the cytoplasm to increase free Ca2+ in response to ionophore exposure. No significant difference in ATP contents was measured and cryopreservation was not associated with an apparent release of cortical granules.


CONCLUSIONS
Irreversible loss of high DeltaPsim in thawed oocytes may be associated with defects in Ca2+ signalling after insemination and could have downstream consequences for normal embryogenesis.
Jones
Amy
A
Reproductive Biology Associates, 1150 Lake Hearn Dr., Suite 600, Atlanta, GA 30342, USA. hesikaij@aol.com
Van Blerkom
Jonathan
J
Davis
Patrick
P
Toledo
Andrew A
AA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001562:Benzimidazoles; D002118:Calcium / Q000378:metabolism; D002232:Carbocyanines; D002467:Cell Nucleus / Q000502:physiology; D015925:Cryopreservation; D003594:Cytoplasmic Granules / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008677:Metaphase; D008928:Mitochondria / Q000502:physiology*; D009865:Oocytes / Q000166:cytology* / Q000502:physiology*
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D000255:Adenosine Triphosphate; D002118:Calcium