Slow cooling of human oocytes: ultrastructural injuries and apoptotic status.
OBJECTIVE
To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (DeltaPsim), and apoptotic status of fresh and cryopreserved oocytes.


METHODS
Experimental study.


METHODS
University biology research unit and private IVF unit.


METHODS
Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles.


METHODS
Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent DeltaPsim reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.


RESULTS
Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 +/- 1.94/10 microm vs. fresh 10.30 +/- 3.9/10 microm), swelling of smooth endoplasmic reticulum (F/T 0.084 +/- 0.03 microm(2) vs. fresh 0.040 +/- 0.02 microm(2)), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low DeltaPsim of pericortical mitochondria, but no signs of apoptosis.


CONCLUSIONS
Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.
Gualtieri
Roberto
R
Department of Structural and Functional Biology, University of Naples Federico II, Monte S Angelo, Naples, Italy. roberto.gualtieri@unina.it
Iaccarino
Mirella
M
Mollo
Valentina
V
Prisco
Marina
M
Iaccarino
Stefania
S
Talevi
Riccardo
R
D000328:Adult; D017209:Apoptosis / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D018871:Endoplasmic Reticulum, Smooth / Q000648:ultrastructure*; D005260:Female; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure*; D009865:Oocytes / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D013696:Temperature; D013997:Time Factors
D003451:Cryoprotective Agents; D020169:Caspases