Ovarian tissue viability following whole ovine ovary cryopreservation: assessing the effects of sphingosine-1-phosphate inclusion.
BACKGROUND
Cryopreservation is hypothesized to result in apoptosis, contributing to stromal damage and follicle loss in ovarian tissue. This study investigated tissue viability following whole ovine ovary cryopreservation and examined the effects of the anti-apoptotic agent sphingosine-1-phosphate (S-1-P) on ovarian cryopreservation efficiency.


METHODS
Whole ovine ovaries were cryoperfused and subjected to slow-freeze, rapid-thaw cryopreservation before a range of functional viability tests were performed. The effects of 20 micromol(-1) S-1-P, in the cryopreservation media, were then assessed against a control cryopreservation media and non-frozen tissue.


RESULTS
Granulosa cell viability (assessed by trypan blue) was not significantly affected, however, Ki67 expression, indicative of cellular proliferation, was reduced following cryopreservation (P< 0.05). Following S-1-P supplementation, granulosa cell viability was not affected by either cryopreservation or S-1-P inclusion. Bromodeoxyuridine uptake, demonstrating DNA synthesis, was seen in both cryopreserved and fresh cortical tissue and the viability stain, 5(6)carboxyfluorescein diacetate succinimidyl ester, showed many viable small follicles. Cryopreservation increased arterial endothelial disruption (P< 0.01), but not internal elastic lamina rupture or venous damage. However, S-1-P supplementation did not improve ovarian or vascular tissue survival.


CONCLUSIONS
These results are encouraging for whole ovary cryopreservation, demonstrating maintained cell viability, however, they do not support S-1-P inclusion at this concentration to improve tissue viability following cryopreservation.
Onions
V J
VJ
Division of Agricultural and Environmental Sciences, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, UK.
Mitchell
M R P
MR
Campbell
B K
BK
Webb
R
R
D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D005260:Female; D006107:Granulosa Cells / Q000502:physiology; D019394:Ki-67 Antigen / Q000096:biosynthesis; D008246:Lysophospholipids / Q000494:pharmacology*; D006080:Ovarian Follicle / Q000166:cytology; D010053:Ovary / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D012756:Sheep; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D046509:Tissue Culture Techniques; D020858:Tissue and Organ Harvesting / Q000379:methods
D019394:Ki-67 Antigen; D008246:Lysophospholipids; D018809:Proliferating Cell Nuclear Antigen; C060506:sphingosine 1-phosphate; D013110:Sphingosine