Dua Diksha, Nagoorvali D, Chauhan M S, Palta P, Mathur P, Singh M K
Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, 132001, India.
ICAR-Central Institute for Research on Goats, Makhdum, Mathura, 281122, India.
In Vitro Cell Dev Biol Anim. 2019 Mar;55(3):159-168. doi: 10.1007/s11626-019-00322-x. Epub 2019 Feb 8.
Parthenogenetically developed embryos are efficient sources of in vitro embryo production, having less ethical issue and being useful for investigating culture conditions/treatments, early developmental, genomic studies, and homonymous source of stem cells. Keeping its advantages in mind, we aimed to study the effects of different activating agents on embryo production and its quality and gene expression. In the present study, 1348 immature oocytes recovered were parthenogenetically developed to embryos. Usable-quality immature oocytes were collected by puncturing the surface follicles and matured in in vitro maturation (IVM) medium for 27 h in a humidified 5% CO incubator at 38.5°C. The matured oocytes were parthenogenetically activated by exposure to 5 μM calcium ionophore for 5 min or 7% ethanol for 7 min sequentially followed by 4 h incubation in 2 mM 6-DMAP and then in vitro cultured (IVC) in RVCL/G-2 medium for 8 days. Matured oocytes were activated by calcium ionophore, the cleavage rate observed was 76.67 ± 3.47%, and further they developed into 4-cell, 8-16-cell, morula, blastocyst, and hatched blastocyst with 85.30 ± 1.57%, 70.60 ± 2.00%, 45.05 ± 2.66%, 22.89 ± 2.40%, and 5.70 ± 1.97%, respectively. Whereas ethanol-activated oocytes showed cleavage rate of 87.60 ± 1.70% and further culture developed into 4-cell, 8-16 cell, morula, blastocyst, and hatched blastocyst with 86.14 ± 1.03%, 71.56 ± 2.21%, 40.90 ± 2.45%, 19.02 ± 1.26%, and 2.22 ± 0.38%, respectively. Blastocyst developed from calcium ionophore-activated oocytes showed significantly (P < 0.05) higher total cell number (282.25 ± 27.02 vs 206.00 ± 40.46) and a lower apoptotic index (2.42 ± 0.46 vs 4.07 ± 1.44) than blastocyst developed from ethanol-activated oocytes. The relative expression of anti-apoptotic genes (BCL2, BCL2A1, MCL) at different stages of embryos produced by either calcium ionophore or ethanol activation was found to be increased in earlier stages and decreased in later stages of embryonic development. Similarly, when these embryos were subjected to pro-apoptotic genes (BAX, BAD, BAK), expression was found to be slightly higher in blastocysts than other stages. This study shows that calcium ionophore-activated blastocysts were developmentally more competent than the ethanol-activated blastocysts.
孤雌生殖发育的胚胎是体外胚胎生产的有效来源，伦理问题较少，可用于研究培养条件/处理方法、早期发育、基因组研究以及干细胞的同源来源。考虑到其优势，我们旨在研究不同激活剂对胚胎生产及其质量和基因表达的影响。在本研究中，回收的1348个未成熟卵母细胞经孤雌生殖发育成胚胎。通过穿刺表面卵泡收集可用质量的未成熟卵母细胞，并在38.5°C、5%二氧化碳饱和湿度的培养箱中，于体外成熟（IVM）培养基中培养27小时。成熟卵母细胞依次暴露于5μM钙离子载体5分钟或7%乙醇7分钟进行孤雌激活，然后在2mM 6 - DMAP中孵育4小时，接着在RVCL/G - 2培养基中进行体外培养（IVC）8天。经钙离子载体激活的成熟卵母细胞，观察到的分裂率为76.67±3.47%，进一步发育为4细胞、8 - 16细胞、桑葚胚、囊胚和孵化囊胚的比例分别为85.30±1.57%、70.60±2.00%、45.05±2.66%、22.89±2.40%和5.70±1.97%。而经乙醇激活的卵母细胞分裂率为87.60±1.70%，进一步培养发育为4细胞、8 - 16细胞、桑葚胚、囊胚和孵化囊胚的比例分别为86.14±1.03%、71.56±2.21%、40.90±2.45%、19.02±1.26%和2.22±0.38%。由钙离子载体激活的卵母细胞发育而来的囊胚，其总细胞数显著高于（P<0.05）由乙醇激活的卵母细胞发育而来的囊胚（282.25±27.02对206.00±40.46），且凋亡指数更低（2.42±0.46对4.07±1.44）。发现由钙离子载体或乙醇激活产生的胚胎在不同阶段的抗凋亡基因（BCL2、BCL2A1、MCL）相对表达在胚胎发育早期增加，后期减少。同样，当这些胚胎接受促凋亡基因（BAX、BAD、BAK）检测时，发现其在囊胚期的表达略高于其他阶段。本研究表明，钙离子载体激活的囊胚在发育能力上比乙醇激活的囊胚更强。
