Sato Akane, Sarentonglaga Borjigin, Ogata Kazuko, Yamaguchi Mio, Hara Asuka, Atchalalt Khurchabiling, Sugane Naoko, Fukumori Rika, Nagao Yoshikazu
University Farm, Faculty of Agriculture, Utsunomiya University, Tochigi 321-4415, Japan.
Department of Animal Production Science, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo 183-0054, Japan.
J Reprod Dev. 2018 Feb 27;64(1):83-88. doi: 10.1262/jrd.2017-145. Epub 2017 Dec 7.
The maturation rate of canine oocytes during in vitro maturation (IVM) needs to be improved. The present study was designed to evaluate the effects of insulin-like growth factor-1 (IGF-1) on the IVM of canine oocytes. Ovaries were obtained by ovariohysterectomy and were sliced to release cumulus-oocyte complexes (COCs). In Experiment 1, the effects of different concentrations of IGF-1 on the nuclear maturation of oocytes was investigated. The COCs were cultured in a modified medium (mTCM199) with IGF-1 (0, 0.5, 5, 10, and 50 µg/ml). At the end of the 48 h culture, oocytes were fixed and stained to evaluate their nuclear stage. Supplementation with 50 µg/ml IGF-1 induced a significantly higher metaphase II (MII) rate (P < 0.05) compared to the 0 and 0.5 μg/ml IGF-1 groups. In Experiment 2, the expression levels of insulin receptor (INSR), IGF-1 receptor (IGF-1R), and IGF-2 receptor (IGF-2R) genes, localized to canine oocytes and cumulus cells, were investigated before and after IVM. The expression level of IGF-1R in cumulus cells after IVM was higher than that before IVM (P < 0.05). In Experiment 3, it was investigated whether an inhibitor of PTEN (phosphatase and tensin homolog), bpV, affects the nuclear maturation of oocytes. Regardless of bpV supplementation at a concentration of 0.2 to 200 µmol/l, there was no significant difference in the proportion of oocytes that reached the MII stage. These results indicated that IGF-1 has a favorable effect on the IVM of canine oocytes, possibly through the stimulation of the Ras/MAPK pathway via IGF-1R expressed in cumulus cells.
犬类卵母细胞在体外成熟（IVM）过程中的成熟率有待提高。本研究旨在评估胰岛素样生长因子-1（IGF-1）对犬类卵母细胞IVM的影响。通过卵巢子宫切除术获取卵巢，并将其切片以释放卵丘-卵母细胞复合体（COCs）。在实验1中，研究了不同浓度的IGF-1对卵母细胞核成熟的影响。将COCs培养于添加了IGF-1（0、0.5、5、10和50μg/ml）的改良培养基（mTCM199）中。在48小时培养结束时，固定并染色卵母细胞以评估其核阶段。与0和0.5μg/ml IGF-1组相比，添加50μg/ml IGF-1诱导的中期II（MII）率显著更高（P<0.05）。在实验2中，研究了定位于犬类卵母细胞和卵丘细胞的胰岛素受体（INSR）、IGF-1受体（IGF-1R）和IGF-2受体（IGF-2R）基因在IVM前后的表达水平。IVM后卵丘细胞中IGF-1R的表达水平高于IVM前（P<0.05）。在实验3中，研究了PTEN（磷酸酶和张力蛋白同源物）抑制剂bpV是否影响卵母细胞核成熟。无论bpV的添加浓度为0.2至200μmol/l，达到MII期的卵母细胞比例均无显著差异。这些结果表明，IGF-1对犬类卵母细胞的IVM具有有利影响，可能是通过刺激卵丘细胞中表达的IGF-1R的Ras/MAPK途径实现的。
