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| 抗缪勒管激素对绵羊卵泡颗粒细胞增殖和凋亡的影响及其作用机制研究 |
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张宇1,2,3, 张宁1,2,3, 宋宇琨1,2,3, 王俊兰1,2,3, 张家新1,2,3
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1.内蒙古农业大学 动物科学学院，内蒙古 呼和浩特 010018;2.内蒙古自治区羊遗传育种与繁殖重点实验室，内蒙古 呼和浩特 010018;3.农业农村部肉羊遗传育种重点实验室，内蒙古 呼和浩特 010018
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| 摘要: |
| 【目的】研究抗缪勒管激素（anti-Müllerian hormone，AMH）对绵羊有腔卵泡颗粒细胞生长的影响，并探究其作用机制。【方法】收集绵羊不同直径（＜3 mm、3～5 mm、＞5 mm）有腔卵泡卵泡液与颗粒细胞，利用实时荧光定量PCR（RT-qPCR）检测颗粒细胞AMH及其Ⅱ型受体（AMHRⅡ）mRNA表达水平，用ELISA法检测颗粒细胞和卵泡液中AMH蛋白质量浓度；将绵羊有腔卵泡颗粒细胞用含有不同质量浓度AMH（0（对照组），0.5，5和50 ng/mL）的培养基培养48 h，利用CCK-8法检测细胞增殖水平，用流式细胞仪检测培养细胞凋亡率，用RT-qPCR检测凋亡相关基因（Caspase 3、BCL-2、BAX）和增殖相关基因（PCNA）mRNA表达水平。用流式细胞仪检测AMH对颗粒细胞内SMAD1/5/8、磷酸化SMAD1/5/8（P-SMAD1/5/8）、CREB、磷酸化CREB（P-CREB）蛋白表达水平的影响。通过EdU细胞增殖试验，检测SMAD1/5/8抑制剂LDN193189和CREB抑制剂666-15对颗粒细胞增殖水平的影响。【结果】随着卵泡直径的增大，颗粒细胞内AMH转录水平及蛋白质表达量均显著下降（P＜0.05），AMHRⅡ mRNA在直径3～5 mm的中腔卵泡颗粒细胞中表达量显著高于小卵泡（直径＜3 mm）及大卵泡（直径＞5 mm）颗粒细胞（P＜0.05）；随着卵泡直径的增大，卵泡液中AMH蛋白质量浓度显著降低（P＜0.05）。与对照组相比，5和50 ng/mL AMH显著抑制了颗粒细胞增殖（P＜0.05），PCNA mRNA相对表达量显著降低。与对照组相比，5和50 ng/mL AMH组细胞凋亡率显著升高（P＜0.05），BCL-2 mRNA相对表达水平显著下降（P＜0.05），而Caspase 3、BAX mRNA相对表达水平显著上升（P＜0.05）。经5 ng/mL AMH培养的颗粒细胞，SMAD1/5/8与CREB总蛋白水平与对照组无显著差异（P＞0.05），但P-SMAD1/5/8蛋白水平显著高于对照组（P＜0.05），P-CREB蛋白水平显著低于对照组（P＜0.05）。LDN193189处理显著促进了颗粒细胞内PCNA表达（P＜0.05），而666 15处理显著抑制了颗粒细胞内PCNA表达（P＜0.05）；EdU检测结果显示，与AMH组相比，LDN193189处理颗粒细胞增殖水平显著提高（P＜0.05），666-15处理颗粒细胞增殖水平显著降低（P＜0.05）。【结论】绵羊有腔卵泡颗粒细胞中AMH分别通过SMAD1/5/8、CREB通路调节颗粒细胞增殖和凋亡，进而在卵泡发育过程中发挥重要作用。 |
| 关键词: 绵羊 抗缪勒管激素 颗粒细胞 有腔卵泡 |
| DOI：10.13207/j.jnwafu.2026.01.013 |
| 分类号: |
| 基金项目:内蒙古自治区自然科学基金项目（2023MS03001）；国家重点研发计划项目（2021YFD1300901） |
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| Effect of anti-Müllerian hormone on proliferation and apoptosis of ovine follicular granular cells and its mechanism |
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ZHANG Yu1,2,3, ZHANG Ning1,2,3, SONG Yukun1,2,3, WANG Junlan1,2,3, ZHANG Jiaxin1,2,3
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1.College of Animal Science,Inner Mongolia Agricultural University,Hohhot，Inner Mongolia 010018,China;2.Inner Mongolia Key Laboratory of Sheep & Goat Genetics Breeding and Reproduction, Hohhot，Inner Mongolia 010018,China;3.Key Laboratory of Genetics and Breeding of Meat Sheep,Ministry of Agriculture and Rural Affairs,Hohhot，Inner Mongolia 010018,China
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| Abstract: |
| 【Objective】This research aimed to investigate the effects of anti-Müllerian hormone (AMH) on the growth of granular cells in ovine follicles and its mechanism.【Method】The follicular fluid and granular cells of different diameter (small:＜3 mm,medium:3-5 mm,large:＞5 mm) antral follicles of sheep were collected,and the expression of AMH and its type Ⅱ receptor (AMHR Ⅱ) in granular cells were detected by real-time fluorescence quantification PCR(RT-qPCR).The concentration of AMH protein in granular cells and follicular fluid was detected by ELISA.The ovine follicular granular cells were cultured in medium with different mass concentrations of AMH (0 (control group),0.5,5 and 50 ng/mL) for 48 h,and the cell proliferation level was detected by CCK-8 assay.The apoptosis rate of cultured cells was detected by flow cytometry,and the mRNA levels of apoptosis-related genes (Caspase 3,BCL-2,BAX) and proliferation-related genes (PCNA) were detected by RT-qPCR.The expression levels of SMAD1/5/8,phosphorylated SMAD1/5/8,CREB and phosphorylated CREB proteins in granular cells were detected by flow cytometry.The effects of SMAD1/5/8 inhibitor LDN193189 and CREB inhibitor 666-15 on granular cell proliferation were examined by EdU cell proliferation assay.【Result】AMH transcription level and protein expression in granular cells were significantly decreased (P＜0.05),with the increase of follicle diameter,and AMHR Ⅱ mRNA expression in medium antral follicle granular cells of 3-5 mm was significantly higher than that in small follicle (diameter＜3 mm) and large follicle (diameter＞5 mm) granular cells (P＜0.05).With the increase of follicle diameter,the concentration of AMH protein in follicular fluid significantly decreased (P＜0.05).Compared with control group,5 and 50 ng/mL AMH significantly inhibited the proliferation (P＜0.05) of granular cell,the relative expression level of PCNA mRNA was significantly decreased.Compared with the control group,the apoptosis rates in 5 and 50 ng/mL AMH groups were significantly increased (P＜0.05),the relative expression level of BCL-2 mRNA was significantly decreased (P＜0.05),and the relative expression levels of Caspase 3 and BAX mRNA were significantly increased (P＜0.05).For granular cells cultured with 5 ng/mL AMH,the total protein levels of SMAD1/5/8 and CREB had no significant difference compared to the control group (P＞0.05),but the levels of P-SMAD1/5/8 proteins were significantly higher than that of the control group (P＜0.05).And the levels of P CREB were significantly lower than that in the control group (P＜0.05).LDN193189 treatment significantly promoted the expression of PCNA in granular cells (P＜0.05),while 666-15 treatment significantly inhibited the expression of PCNA in granular cells (P＜0.05).EdU test results showed that compared with AMH group,LDN193189 treatment significantly increased granular cell proliferation level (P＜0.05),and 666 15 treatment significantly decreased granular cell proliferation level (P＜0.05).【Conclusion】AMH regulates granular cell proliferation and apoptosis of ovine antral follicles through SMAD1/5/8 and CREB pathways,which plays an important role in ovine antral follice development. |
| Key words: sheep anti-Müllerian hormone(AMH) granular gells antral follicle |
