| 摘要: |
| 为研究双调蛋白(AREG)对绵羊卵丘细胞(Cumulus cells，CCs)葡萄糖代谢的影响，采集来源于中腔卵泡和小腔卵泡的卵丘-卵母细胞复合体(COCs)，利用荧光定量PCR检测中、小腔卵泡卵丘细胞葡萄糖代谢相关基因表达；添加AREG进行卵丘细胞的体外培养和卵母细胞的体外成熟(IVM)，检测卵丘细胞的增殖，测定培养液或细胞中的葡萄糖、乳酸、NADPH和ATP含量。结果表明:1)来源于小腔卵泡的卵丘细胞G6PDH、PFKL和PFKM的mRNA表达量以及细胞增殖能力显著低于中腔卵泡卵丘细胞(P<0.05)；2)较低浓度(10 ng/mL)的AREG显著提高了中腔卵泡卵丘细胞的增殖能力(P<0.05)而对小腔卵泡卵丘细胞无影响(P>0.05)，在生长分化因子9(GDF9)和骨形态发生蛋白15(BMP15)的协同作用下，10 ng/mL AREG可以显著提高两者的增殖能力(P<0.05)且两者间差异不显著(P>0.05)；3)在GDF9和BMP15的协同作用下，AREG可显著提高不同直径腔卵泡卵丘细胞培养液中的葡萄糖消耗、乳酸生成、NADPH生成以及细胞中的ATP含量(P<0.05)；4)小腔卵泡卵母细胞的IVM培养液中添加AREG+GDF9+BMP15，显著提高培养液中的葡萄糖消耗、乳酸生成及卵丘细胞中的ATP含量(P<0.05)且显著高于中腔卵泡(P<0.05)，显著提高了卵母细胞中的NADPH生成及ATP含量(P<0.05)且与中腔卵泡卵母细胞组差异不显著(P>0.05)。综上，在GDF9和BMP15的协同作用下，AREG可以增强绵羊卵丘细胞的葡萄糖代谢，并且对进行IVM的小腔卵泡COCs添加AREG+GDF9+BMP15可使其葡萄糖代谢达到中腔卵泡的水平。 |
| 关键词: 卵丘细胞 AREG 葡萄糖代谢 绵羊 小腔卵泡 |
| DOI：10.11841/j.issn.1007-4333.2022.11.11 |
| 投稿时间：2021-07-28 |
| 基金项目:国家重点研发计划(2021YFD1300901) |
|
| Effect of AREG on the glucose metabolism of sheep cumulus cells |
|
WANG Zhaochen1,ZHAO Yongchao1,DU Wei1,ZHANG Yu1,ZHANG Jiaxin1*,AN Lei2*
|
| (1.College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China;2.College of Animal Science and Technology, China Agricultural University, Beijing 100193, China) |
| Abstract: |
| In order to study the effect of amphiregulin(AREG)on glucose metabolism of sheep cumulus cells(CCs), cumulus-oocyte complexes(COCs)from medium and small antral follicles were collected. The fluorescent quantitative PCR was used to detect the expression of genes related to glucose metabolism in cumulus cells. AREG was added for in vitro culture of cumulus cells and in vitro maturation(IVM)of oocytes. The proliferation of cumulus cells was detected. The contents of glucose, lactic acid, NADPH and ATP in culture medium or cells were determined. The results showed that 1)The mRNA expression of G6PDH, PFKL and PFKM and the cell proliferation ability of cumulus cells derived from small antral follicles were significantly lower than those of cumulus cells from medium antral follicles(P<0. 05). 2)Lower concentration(10 ng/mL)AREG significantly increased the proliferation of cumulus cells from medium antral follicles(P<0. 05)but had no effect on cumulus cells from small antral follicles(P>0. 05). Under the synergistic effect of growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15), 10 ng/mL AREG significantly increased the proliferation of both(P<0. 05), and there was no significant difference between them(P>0. 05). 3)AREG+GDF9+BMP15 significantly increased glucose consumption, lactate production, NADPH production in cumulus cell culture medium and ATP content of cumulus cells from different diameter antral follicles(P<0. 05). 4)The addition of AREG+GDF9+BMP15 added to the IVM medium of small antral follicular oocytes significantly increased the glucose consumption and lactic acid production in the culture medium and ATP content in cumulus cells(P<0. 05), and it was significantly higher than that of medium antral follicles(P<0. 05). The NADPH production and ATP content of oocytes were significantly increased(P<0. 05)and there was no significant differences compared with the medium antral follicular oocytes(P>0. 05). In summary, under the synergistic effect of GDF9 and BMP15, AREG can enhance the glucose metabolism of sheep cumulus cells. Also for IVM, adding the combination of AREG, GDF9 and BMP15 to the COCs culture medium of small antral follicles can make their glucose metabolism reach the same level of medium antral follicles. |
| Key words: cumulus cells AREG glucose metabolism sheep small antral follicles |
