ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, India.
ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, India.
Theriogenology. 2022 Jan 15;178:1-7. doi: 10.1016/j.theriogenology.2021.10.024. Epub 2021 Oct 25.
The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 μm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.
本研究旨在建立一种绵羊腔前卵泡的玻璃化方案，使解冻后的卵泡保持活力，并评估不同玻璃化处理对凋亡和发育相关基因表达的影响。腔前卵泡通过组织学方法从卵巢皮质切片中分离出来。分离的腔前卵泡（200-300μm）被随机分为四组。第 1 组-对照组新鲜腔前卵泡（256 个卵泡）；第 2 组-玻璃化处理 A（259 个卵泡）（玻璃化溶液 1（VS1）-胎牛血清（FBS）10%，乙二醇（EG）：1.8M，二甲基亚砜（DMSO）：1.4M，蔗糖-0.3M 4 分钟；VS2-FBS10%，EG：4.5M，DMSO：3.5M，蔗糖：0.3M 45 秒），第 3 组-玻璃化处理 B（235 个卵泡）（VS1-FBS20%，EG：1.3M，DMSO1.05M 15 分钟，VS2-FBS20%，EG：2.7M，DMSO：2.1M 5 分钟）和第 4 组-玻璃化处理 C（248 个卵泡）（VS1-甘油（Gly）：1.2M 3 分钟，VS2-Gly：1.2M，EG：3.6M 3 分钟，VS3-Gly3M，EG：4.5M 1 分钟）。腔前卵泡置于相应的玻璃化处理中，然后立即浸入液氮（-196°C）中。一周后，用台盼蓝排斥法分析卵泡的活力，并进行基因表达分析。结果表明，与对照组相比，低浓度的冷冻保护剂（玻璃化处理 B）对腔前卵泡的活力有负面影响。对照组（87%）、处理 A 组（79%）和处理 C 组（75%）的活力率无显著差异。与对照组相比，来源于玻璃化处理 B 的有活力的腔前卵泡（73%）百分比显著降低（P<0.05%）。玻璃化处理 B 组凋亡基因 BAK 的表达较高。其他凋亡相关基因（Bcl2L1、BAD、BAX、Caspase 3 和 Annexin）的表达在各组之间无显著差异。GDF-9 和 BMP-15 等发育能力基因的表达模式在玻璃化处理 A 和 C 中分别较高（P<0.05）。与对照组相比，玻璃化处理 B 组的 NOBOX 基因表达显著增加。我们得出结论，玻璃化处理 A 和处理 C 都是有效的绵羊腔前卵泡玻璃化方法。
