title: Slow cooling of human oocytes: ultrastructural injuries and apoptotic status.
abstract: 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.
authors: Department of Structural and Functional Biology, University of Naples Federico II, Monte S Angelo, Naples, Italy. roberto.gualtieri@unina.it
authors: Roberto
authors: R
authors: Gualtieri
authors: Mirella
authors: M
authors: Iaccarino
authors: Valentina
authors: V
authors: Mollo
authors: Marina
authors: M
authors: Prisco
authors: Stefania
authors: S
authors: Iaccarino
authors: Riccardo
authors: R
authors: Talevi
mesh_terms: 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
chemical_list: D003451:Cryoprotective Agents; D020169:Caspases