INSERM U846, Stem-cell and Brain Research Institute, Bron, France.
Hum Reprod. 2012 Jun;27(6):1811-21. doi: 10.1093/humrep/des100. Epub 2012 Mar 27.
Whole ovary cryopreservation has been suggested as a means to preserve fertility. In animal models, autologous cryopreserved ovary transplants frequently undergo thrombosis and a method to assess the vascular viability of cryopreserved ovaries would be valuable. We developed a staining method using methylthiazolyl blue tetrazolium (MTT, a metabolic marker) to assess the pedicle metabolism of whole ovaries vitrified using cryoprotectant called 'VS4'.
Whole sheep ovaries were perfused with MTT (1 g/l). In one group, ovarian tissue lesions were induced by immersing the ovarian pedicle in medium at 53°C or 65°C or in liquid nitrogen prior to MTT perfusion. In the second group, several metabolic substrates (d-glucose, l-glucose and pyruvic acid) and inhibitors [2-deoxy-d-glucose for d-glucose metabolism, azide for mitochondrial respiration and diphenyleneiodonium (DPI) for NADPH oxidase (an effector of the pentose phosphate pathway)] were added to the MTT stain. The third group was subjected to VS4 ± vitrification/warming prior to MTT perfusion. Pedicle MTT staining was assessed qualitatively by histological examination of frozen sections or quantified at 564 nm after solubilization in alcohol.
MTT strongly and reproducibly stained the vascular smooth muscle. Heating at 53°C or 65°C or cooling in liquid nitrogen significantly diminished MTT staining by 48% (P = 0.001, n = 10), 94% (P = 0.0002, n = 10) and 94% (P = 0.0002, n = 10), respectively. MTT staining was affected by d-glucose metabolism: absence of d-glucose, substitution of unmetabolized l-glucose for d-glucose or addition of 2-deoxy-d-glucose significantly decreased MTT staining by 44% (P < 0.01, n = 10), 45% (P < 0.01, n = 10) and 29% (P < 0.01, n = 10), respectively. Pyruvic acid failed to correct the MTT staining decrease induced by d-glucose deprivation and azide did not decrease MTT staining, suggesting that MTT staining could be independent of mitochondrial metabolism. Adding DPI significantly inhibited MTT staining by 25% (P < 0.001, n = 10), suggesting involvement of the pentose phosphate pathway's effectors. Compared with controls, VS4-vitrified/warmed pedicles showed significantly less MTT staining (-30%, P < 0.005, n = 10), with unstained foci, whereas unvitrified VS4-exposed pedicles showed no difference.
MTT can serve as a qualitative and quantitative vascular viability marker.VS4 vitrification caused alterations in ovarian vascular metabolism. MTT staining should allow accurate comparisons of whole-organ cryoprotection protocols.
全卵巢冷冻保存被认为是一种保存生育能力的方法。在动物模型中，自体冷冻保存的卵巢移植经常发生血栓形成，因此评估冷冻保存卵巢的血管活力的方法将是有价值的。我们开发了一种使用噻唑蓝（MTT，一种代谢标志物）染色的方法来评估使用称为“VS4”的冷冻保护剂冷冻的整个卵巢的花梗代谢。
将 MTT（1 g/l）灌注到整个绵羊卵巢中。在一组中，通过将卵巢花梗浸入 53°C 或 65°C 的培养基中或浸入液氮中来诱导卵巢组织损伤，然后再进行 MTT 灌注。在第二组中，添加了几种代谢底物（d-葡萄糖、l-葡萄糖和丙酮酸）和抑制剂[用于 d-葡萄糖代谢的 2-脱氧-d-葡萄糖、用于线粒体呼吸的叠氮化物和用于 NADPH 氧化酶（戊糖磷酸途径的效应物）的二苯基碘（DPI）]到 MTT 染色中。第三组在进行 VS4±冷冻/解冻后进行 MTT 灌注。通过冷冻切片的组织学检查对花梗 MTT 染色进行定性评估，或在用酒精溶解后在 564nm 处进行定量评估。
MTT 强烈且可重复地染色血管平滑肌。53°C 或 65°C 加热或在液氮中冷却分别使 MTT 染色减少 48%（P = 0.001，n = 10）、94%（P = 0.0002，n = 10）和 94%（P = 0.0002，n = 10）。MTT 染色受 d-葡萄糖代谢影响：缺乏 d-葡萄糖、用未代谢的 l-葡萄糖替代 d-葡萄糖或添加 2-脱氧-d-葡萄糖分别使 MTT 染色减少 44%（P < 0.01，n = 10）、45%（P < 0.01，n = 10）和 29%（P < 0.01，n = 10）。丙酮酸未能纠正 d-葡萄糖剥夺引起的 MTT 染色减少，叠氮化物也没有减少 MTT 染色，这表明 MTT 染色可能独立于线粒体代谢。添加 DPI 使 MTT 染色显著减少 25%（P < 0.001，n = 10），这表明戊糖磷酸途径的效应物参与其中。与对照组相比，VS4 冷冻/解冻后的花梗 MTT 染色明显减少（-30%，P < 0.005，n = 10），出现未染色焦点，而未暴露于 VS4 的花梗则无差异。
MTT 可作为血管活力的定性和定量标志物。VS4 冷冻导致卵巢血管代谢改变。MTT 染色应允许对整个器官冷冻保护方案进行准确比较。
