Shin Hyeji, Lee Yongjin, Lee Joohyeong, Lee Seung Tae, Lee Geun-Shik, Lee Eunsong
Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea.
Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon, 24341, South Korea.
Theriogenology. 2021 Apr 15;165:37-43. doi: 10.1016/j.theriogenology.2020.12.018. Epub 2021 Feb 24.
The objective of this study was to evaluate the effects of reducing the sodium chloride content in in vitro growth (IVG) medium to 61.6 mM on in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium with 108 mM NaCl (αMEM-108) or porcine zygote medium (PZM) containing 61.6 mM (PZM-61.6) or 108 mM (PZM-108) NaCl. These media were further supplemented with 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 10% (v/v) fetal bovine serum. After IVG culture, oocytes were matured for 44 h in our standard IVM medium. The IVG culture in PZM-61.6 significantly increased nuclear maturation (88.0 ± 2.2%) of SAF oocytes compared to that in PZM-108 (77.3 ± 3.9%) or αMEM-108 (75.9 ± 3.8%). After parthenogenesis (PA), the proportions of blastocysts, based on the number of metaphase II (MII) oocytes, induced for PA were not different among IVG oocytes cultured in PZM-61.6 (50.2 ± 3.0%), PZM-108 (46.8 ± 2.9%), or αMEM-108 (45.6 ± 2.9%). The IVM oocytes derived from IVG in PZM-61.6 showed increased perivitelline space (PVS) (12.1 ± 0.6 μm) and intra-oocyte glutathione (GSH) content (1.19 ± 0.04 pixels/oocyte) compared to PVS (8.0 ± 0.5 and 7.4 ± 0.4 μm) and GSH (1.03 ± 0.04 and 1.00 ± 0.04 pixels/oocyte) of oocytes derived from PZM-108 and αMEM-108, respectively. The IVG culture in PZM-61.6 stimulated meiotic resumption after IVG and faster nuclear progression after IVM than that in αMEM-108. After somatic cell nuclear transfer (SCNT), the blastocyst formation of SAF oocytes grown in PZM-61.6 (17.8 ± 3.3%) was higher than that of oocytes grown in PZM-108 (7.5 ± 2.7%) but not different from that of oocytes in αMEM-108 (11.4 ± 3.4%). Regardless of the different osmotic pressures, nuclear maturation was significantly increased by IVG culture in PZM with reduced NaCl (86.8 ± 2.3 and 84.9 ± 4.2% in PZM-61.6 and PZM-61.6 with sorbitol, respectively) than in PZM-108 (70.5 ± 3.4%). Blastocyst formation was not affected by the differences in NaCl content and osmotic pressure of the IVG medium, whereas the mean number of cells in blastocysts was significantly higher following IVG culture in PZM-61.6 than in the other groups. In conclusion, the results demonstrate that, following SCNT in pigs, IVG culture of SAF oocytes in a medium with a reduced NaCl concentration stimulates oocyte maturation and improves subsequent embryonic development.
本研究的目的是评估将体外生长（IVG）培养基中的氯化钠含量降低至61.6 mM对直径小于3 mm的小腔卵泡（SAF）来源的猪卵母细胞的体外成熟（IVM）和胚胎发育的影响。将SAF卵母细胞在含有108 mM NaCl的α-最小必需培养基（αMEM-108）或含有61.6 mM（PZM-61.6）或108 mM（PZM-108）NaCl的猪合子培养基（PZM）中培养2天以诱导IVG。这些培养基进一步补充有1 mM二丁酰环磷酸腺苷（dbcAMP）和10%（v/v）胎牛血清。IVG培养后，卵母细胞在我们的标准IVM培养基中成熟44小时。与PZM-108（77.3±3.9%）或αMEM-108（75.9±3.8%）相比，PZM-61.6中的IVG培养显著提高了SAF卵母细胞的核成熟率（88.0±2.2%）。孤雌生殖（PA）后，基于用于PA的中期II（MII）卵母细胞数量，PZM-61.6（50.2±3.0%）、PZM-108（46.8±2.9%）或αMEM-108（45.6±2.9%）中培养的IVG卵母细胞诱导形成的囊胚比例没有差异。与分别来自PZM-108和αMEM-108的卵母细胞的卵周隙（PVS）（8.0±0.5和7.4±0.4μm）和卵母细胞内谷胱甘肽（GSH）含量（1.03±0.04和1.00±0.04像素/卵母细胞）相比，PZM-61.6中IVG产生的IVM卵母细胞显示出卵周隙增加（12.1±0.6μm）和卵母细胞内谷胱甘肽含量增加（1.19±0.04像素/卵母细胞）。与αMEM-108相比，PZM-61.6中的IVG培养在IVG后刺激减数分裂恢复，在IVM后核进展更快。体细胞核移植（SCNT）后，在PZM-61.6中生长的SAF卵母细胞的囊胚形成率（17.8±3.3%）高于在PZM-108中生长的卵母细胞（7.5±2.7%），但与αMEM-108中的卵母细胞（11.4±3.4%）没有差异。无论渗透压如何不同，与PZM-108（70.5±3.4%）相比，在NaCl含量降低的PZM中进行IVG培养可显著提高核成熟率（PZM-61.6和添加山梨醇的PZM-61.6中分别为86.8±2.3%和84.9±4.2%）。囊胚形成不受IVG培养基中NaCl含量和渗透压差异的影响，而PZM-61.6中IVG培养后囊胚中的平均细胞数显著高于其他组。总之，结果表明，在猪进行SCNT后，在NaCl浓度降低的培养基中对SAF卵母细胞进行IVG培养可刺激卵母细胞成熟并改善随后的胚胎发育。
