Lee Hanna, Elahi Fazle, Lee Joohyeong, Lee Seung Tae, Hyun Sang-Hwan, Lee Eunsong
Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea.
Department of Animal Life Science and Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea.
Theriogenology. 2017 Mar 15;91:1-8. doi: 10.1016/j.theriogenology.2016.12.015. Epub 2016 Dec 18.
This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles < 3 mm in diameter (SAFCOCs). SAFCOCs were untreated (control) or treated with 20 μM CIL and/or 50 μM FSK for 2 days for IVG. Next, IVG oocytes were cultured for maturation and then induced for parthenogenesis (PA) or used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation of oocytes was significantly lower in the control (49.6 ± 9.3%) group than in other groups (67.2 ± 5.0-79.8 ± 7.9%). The cumulus expansion score after IVG-IVM was significantly higher in the control and CIL group than in the FSK and CIL + FSK groups. CIL significantly increased mean diameter SAF-derived oocytes (120.0 ± 0.5 μm) compared to the control, FSK, and CIL + FSK (114.8 ± 0.5-116.7 ± 0.6 μm) and showed a comparable level of intracellular glutathione (GSH) contents (0.84 ± 0.07 pixels/oocyte) to medium antral follicle (MAF)-derived oocytes (1.00 ± 0.08 pixels/oocyte), but was higher than those of oocytes treated with FSK and CIL + FSK (0.29 ± 0.05 and 0.37 ± 0.05 pixels/oocyte, respectively). CIL treatment significantly increased blastocyst formation (55.1 ± 4.7%) after PA relative to the control (29.4 ± 6.4%), FSK (34.8 ± 7.1%), and CIL + FSK (41.1 ± 5.2%). A higher proportion of oocytes treated with CIL, FSK, and CIL + FSK (73.3 ± 1.7-82.8 ± 9.1%) remained at the germinal vesicle stage after IVG culture than control oocytes (40.0 ± 5.0%). Following SCNT, blastocyst formation of SAFCOCs treated with CIL (22.4 ± 6.3%) was higher than that of oocytes (0-10.4 ± 5.3%) in control, FSK, and CIL + FSK, but similar to that (25.3 ± 3.5%) of MAF-derived COCs not cultured for IVG. The cAMP level of SAFCOCs before IVG was 0.1 ± 0.03 fmol/oocyte. After 2 days of IVG culture, cAMP level was increased significantly by treatment with FSK and CIL + FSK (3.0 ± 0.57 and 12.1 ± 0.62 fmol/oocyte, respectively) relative to the control and CIL treatment (0.1 ± 0.03 and 0.3 ± 0.04 fmol/oocyte, respectively). Our results demonstrate that CIL treatment during IVG improves the low developmental competence of SAFCOCs to levels comparable to MAFCOCs by allowing oocyte growth while inhibiting premature meiotic maturation, probably via maintenance of cAMP concentrations at appropriate levels.
本研究旨在评估环磷酸腺苷（cAMP）调节剂西洛酰胺（CIL）和福斯高林（FSK）在体外生长（IVG）期间对直径小于3mm的小窦卵泡来源的卵丘-卵母细胞复合体（COCs）的生长、成熟和胚胎发育的影响。对SAFCOCs不进行处理（对照），或用20μM CIL和/或50μM FSK处理2天以进行IVG。接下来，将IVG卵母细胞培养以使其成熟，然后诱导孤雌生殖（PA），或用作体细胞核移植（SCNT）的受体卵质体。对照组卵母细胞的核成熟率（49.6±9.3%）显著低于其他组（67.2±5.0 - 79.8±7.9%）。IVG-IVM后的卵丘扩展评分在对照组和CIL组中显著高于FSK组和CIL + FSK组。与对照、FSK和CIL + FSK组（114.8±0.5 - 116.7±0.6μm）相比，CIL显著增加了SAF来源的卵母细胞的平均直径（120.0±0.5μm），并且其细胞内谷胱甘肽（GSH）含量（0.84±0.07像素/卵母细胞）与中等大小窦卵泡（MAF）来源的卵母细胞（1.00±0.08像素/卵母细胞）相当，但高于用FSK和CIL + FSK处理的卵母细胞（分别为0.29±0.05和0.37±0.05像素/卵母细胞）。相对于对照（29.4±6.4%）、FSK（34.8±7.1%）和CIL + FSK（41.1±5.2%），CIL处理显著增加了PA后的囊胚形成率（55.1±4.7%）。IVG培养后，用CIL、FSK和CIL + FSK处理的卵母细胞（73.3±1.7 - 82.8±9.1%）处于生发泡期的比例高于对照卵母细胞（40.0±5.0%）。SCNT后，用CIL处理的SAFCOCs的囊胚形成率（22.4±6.3%）高于对照、FSK和CIL + FSK组中的卵母细胞（0 - 10.4±5.3%），但与未进行IVG培养的MAF来源的COCs的囊胚形成率（25.3±3.5%）相似。IVG前SAFCOCs的cAMP水平为0.1±0.03 fmol/卵母细胞。IVG培养2天后，与对照和CIL处理（分别为0.1±0.03和0.3±0.04 fmol/卵母细胞）相比，用FSK和CIL + FSK处理显著提高了cAMP水平（分别为3.0±0.57和12.1±0.62 fmol/卵母细胞）。我们的结果表明，IVG期间的CIL处理通过促进卵母细胞生长同时抑制过早减数分裂成熟，可能通过将cAMP浓度维持在适当水平，将SAFCOCs的低发育能力提高到与MAFCOCs相当的水平。
