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Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: implications for
Amy Jones1, Jonathan Van Blerkom, Patrick Davis
1Reproductive Biology Associates, 1150 Lake Hearn Dr., Suite 600, Atlanta, GA 30342, USA. hesikaij@aol.com
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.
Conclusion:
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.
Insights
Cryopreservation of human oocytes may impair mitochondrial function, leading to reduced embryo development. Thawed oocytes show altered mitochondrial polarity (DeltaPsim) and calcium (Ca2+) regulation, impacting fertilization success.
Area of Science:
- Reproductive Biology
- Cell Biology
- Developmental Biology
Background:
- Embryo development outcomes are lower for oocytes cryopreserved at the MII stage compared to the pronuclear stage.
- Investigating cryopreservation effects on oocyte mitochondrial polarity (DeltaPsim) and its impact on ATP levels and Ca2+ regulation is crucial.
Purpose of the Study:
- To determine if cryopreservation alters human oocyte mitochondrial polarity (DeltaPsim).
- To assess the impact of cryopreservation on intracellular ATP levels and Ca2+ regulation in human oocytes.
- To correlate these changes with oocyte competence for embryogenesis.
Main Methods:
- Fresh and thawed MII oocytes were analyzed using JC-1 for mitochondrial polarity and Fluo-4 for intracellular Ca2+ levels.
- ATP content was directly measured, and cortical granule status was assessed.
- Oocytes were exposed to ionophores to evaluate cytoplasmic Ca2+ response.
Main Results:
- Thawed MII oocytes exhibited significantly reduced pericortical J-aggregate fluorescence, indicating lower mitochondrial polarity.
- The ability of the cytoplasm to increase free Ca2+ upon ionophore exposure was significantly diminished in thawed oocytes.
- No significant differences in ATP content or cortical granule release were observed between fresh and thawed oocytes.
Conclusions:
- Cryopreservation is associated with an irreversible loss of high mitochondrial polarity (DeltaPsim) in MII oocytes.
- This loss may lead to defects in Ca2+ signaling post-insemination.
- Impaired Ca2+ signaling could negatively affect subsequent normal embryogenesis.
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