The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation.
BACKGROUND
Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells.


METHODS
Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure.


RESULTS
Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively.


CONCLUSIONS
Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.
Mullen
S F
SF
Comparative Medicine Center and Department of Veterinary Pathobiology, University of Missouri at Columbia, Columbia MO 65211, USA.
Agca
Y
Y
Broermann
D C
DC
Jenkins
C L
CL
Johnson
C A
CA
Critser
J K
JK
D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D005260:Female; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D016015:Logistic Models; D008677:Metaphase; D009865:Oocytes / Q000166:cytology* / Q000502:physiology; D009997:Osmotic Pressure; D008941:Spindle Apparatus / Q000187:drug effects / Q000502:physiology*
D006982:Hypertonic Solutions; D007038:Hypotonic Solutions