Onions V J, Mitchell M R P, Campbell B K, Webb R
Division of Agricultural and Environmental Sciences, School of Biosciences, University of Nottingham, Sutton Bonington, Loughborough, Leicestershire, LE12 5RD, UK.
Hum Reprod. 2008 Mar;23(3):606-18. doi: 10.1093/humrep/dem414. Epub 2008 Jan 23.
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.
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.
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.
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.
据推测，冷冻保存会导致细胞凋亡，进而造成卵巢组织的基质损伤和卵泡丢失。本研究调查了整个绵羊卵巢冷冻保存后的组织活力，并研究了抗凋亡剂鞘氨醇-1-磷酸（S-1-P）对卵巢冷冻保存效率的影响。
对整个绵羊卵巢进行冷冻灌注，并进行慢速冷冻、快速解冻冷冻保存，然后进行一系列功能活力测试。然后，将冷冻保存培养基中20微摩尔（-1）S-1-P的效果与对照冷冻保存培养基和未冷冻组织进行评估。
颗粒细胞活力（通过台盼蓝评估）未受到显著影响，然而，冷冻保存后，指示细胞增殖的Ki67表达降低（P<0.05）。补充S-1-P后，颗粒细胞活力不受冷冻保存或S-1-P添加的影响。在冷冻保存的和新鲜的皮质组织中均观察到显示DNA合成的溴脱氧尿苷摄取，并且活力染色5（6）羧基荧光素二乙酸琥珀酰亚胺酯显示许多存活的小卵泡。冷冻保存增加了动脉内皮破坏（P<0.01），但未增加内弹性膜破裂或静脉损伤。然而，补充S-1-P并未改善卵巢或血管组织的存活。
这些结果对于整个卵巢冷冻保存是令人鼓舞的，表明细胞活力得以维持，然而，它们不支持在此浓度下添加S-1-P来改善冷冻保存后的组织活力。
