Gastal G D A, Aguiar F L N, Alves B G, Alves K A, de Tarso S G S, Ishak G M, Cavinder C A, Feugang J M, Gastal E L
Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale, IL, USA.
Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS, USA.
Theriogenology. 2017 Jul 15;97:139-147. doi: 10.1016/j.theriogenology.2017.04.029. Epub 2017 Apr 23.
Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.
卵巢组织冷冻保存可在不确定的时间段内保留女性的生育潜力。本研究的目的是比较使用慢速冷冻和玻璃化方法时冷冻保护剂（CPA；二甲基亚砜，DMSO；乙二醇，EG；和丙二醇，PROH）的效率，并评估培养7天后冷冻保存的马卵巢组织的活力。对新鲜和冷冻保存的卵巢片段进行评估，观察腔前卵泡形态、基质细胞密度、表皮生长因子受体（EGFR）、Ki-67、Bax和Bcl-2蛋白表达以及DNA片段化情况。使用EG进行玻璃化时，形态正常的腔前卵泡比例最高，而使用DMSO时比例最低（分别为76.1±6.1%和40.9±14.8%；P<0.05）。在慢速冷冻中，尽管DMSO形态正常卵泡的比例最高（77.7±5.8%），但未观察到各CPA之间存在差异。使用EG进行玻璃化时，EGFR和Ki-67的荧光强度更高。无论冷冻保存处理如何，DMSO的Bax/Bcl-2比值最高（P<0.05）；然而，解冻后各处理之间的DNA片段化情况相似（P>0.05）。体外培养后，慢速冷冻和玻璃化方法之间正常卵泡的比例相似（P>0.05）；然而，玻璃化的基质细胞密度高于慢速冷冻（P<0.05）。总之，马卵巢组织成功实现了冷冻保存，分别使用DMSO和EG进行慢速冷冻和玻璃化方法时，解冻后卵巢组织中细胞的活力增加。因此，这些结果与生育力保存计划相关。
