【作者】 李洁；

【导师】 刘兴廷；

【作者基本信息】 广西大学 ， 畜牧学， 2025， 硕士

【摘要】 在性成熟母鸡卵巢中,每天从数个小黄卵泡(Small Yellow Follicles,SYFs)中选出一个进入最后快速的等级发育阶段,即卵泡选择。被选择进入等级发育的卵泡开始响应FSH的刺激,大量摄取富含脂质的卵黄前体物,体积迅速增大。卵黄前体物在肝脏细胞合成后,经血液运输到达并通过卵泡基膜,在通过颗粒细胞间隙后到达卵母细胞表面与受体LR8结合后入卵。研究表明颗粒细胞间紧密连接影响卵黄前体物入卵。NR5A2是核受体超家族的一员,广泛参与多种生物过程,研究发现NR5A2具有调节鸡颗粒细胞类固醇生成的作用,但对颗粒细胞间紧密连接的调节作用尚不清楚。本研究首先比较产蛋高峰期广西麻鸡卵泡选择前(SYF)、后(F5)颗粒细胞形态,卵黄前体物入卵,紧密连接,紧密连接相关蛋白和NR5A2表达水平的差异,随后分别对体外培养的SYF/F5颗粒细胞进行Nr5a2过表达/干扰,探究NR5A2对广西麻鸡卵泡选择中颗粒细胞间紧密连接相关蛋白的影响。研究结果如下:(1)卵泡选择前后紧密连接和NR5A2表达水平变化卵泡选择后颗粒细胞层逐渐由多层变为单层,颗粒细胞间隙增大,卵黄前体物入卵增多,紧密连接减少,紧密连接相关蛋白(ZO-1、OCLN、CLDN-1)表达水平和基因转录水平显著下调(P<0.05),NR5A2蛋白表达水平和基因转录水平显著上调,F1、F2、F3颗粒细胞NR5A2表达水平显著高于F4、F5、SYF颗粒细胞(P<0.05)。(2)干扰Nr5a2对F5颗粒细胞紧密连接相关蛋白的影响通过FOXL2免疫荧光染色验证成功分离培养SYF和F5颗粒细胞。干扰Nr5a2使F5颗粒细胞紧密连接相关蛋白(ZO-1、OCLN、CLDN-1)表达水平和基因转录水平显著上调,细胞增殖能力降低,凋亡水平升高(P<0.05)。(3)过表达Nr5a2对SYF颗粒细胞紧密连接相关蛋白的影响过表达Nr5a2使SYF颗粒细胞紧密连接相关蛋白(ZO-1、OCLN、CLDN-1)表达水平和基因转录水平显著下调,细胞增殖能力升高,凋亡水平降低(P<0.05)。综上所述,本研究发现NR5A2通过调节紧密连接相关蛋白的表达,调控卵泡选择中颗粒细胞间紧密连接。研究结果为进一步完善母鸡卵黄入卵调控网络提供数据支撑,并为从卵黄沉积角度探究提高产蛋量提供理论参考。更多还原

【Abstract】 In the ovaries of sexually mature hens,one small yellow follicle(SYF)is daily selected from a cohort of several follicles to enter the final rapid hierarchical developmental stage,known as follicle selection.The selected follicle begins to respond to the stimulation of FSH,entering hierarchical development initiates massive uptake of lipid-rich yolk precursors,resulting in rapid volumetric expansion.These yolk precursors,synthesized by hepatocytes,are transported via the bloodstream to the follicular basal lamina.After traversing the intercellular spaces between granulosa cells,they ultimately bind to the LR8 receptor on the oocyte surface for internalization.Studies have demonstrated that tight junctions between granulosa cells significantly influence yolk precursor uptake into the oocyte,a process regulated by multiple secretory factors.NR5A2,a member of the nuclear receptor superfamily,is widely involved in diverse biological processes.While studies have established its regulatory role in steroidogenesis within chicken granulosa cells,its potential function in modulating tight junction between granulosa cells remains unclear.In the present study,the morphological characteristics of granulosa cells,yolk precursor deposition,tight junction integrity,expression levels of tight junction associated proteins,and NR5A2 in pre-selection(SYF)and post-selection(F5)follicles from Guangxi Ma hens were explored during peak laying period.Subsequently,the Nr5a2 overexpression and knockdown experiments were performed in cultured SYF/F5 granulosa cells to investigate the regulatory effects of NR5A2 on tight junction associated proteins during follicular selection.The results are as follows:(1)Changes in tight junction integrity and NR5A2 expression levels during follicular selectionFollowing follicle selection,the granulosa cell layer undergoes a progressive transition from a multilayered to a monolayered organization,concomitant with expanded intercellular spaces,augmented yolk precursor deposition,and diminished tight junction integrity.Quantitative analyses revealed significant downregulation(P<0.05)in both protein abundance and m RNA expression levels of tight junction associated proteins(ZO-1,OCLN,and CLDN-1).In contrast,NR5A2 demonstrated marked upregulation at both translational and transcriptional levels(P<0.05),with granulosa cells from F1～F3 exhibiting substantially higher NR5A2 expression compared to those from F4～F5 and SYF(P<0.05).(2)Effects of Nr5a2 interference on the expression of tight junction associated proteins in F5 granulosa cellsFOXL2 immunofluorescence staining validated the successful isolation of SYF and F5 granulosa cells.The expression and transcription of tight junction associated proteins(ZO-1,OCLN,CLDN-1),the cell proliferation decreased and cell apoptosis increased significantly after Nr5a2 interference(P<0.05).(3)Effects of overexpression Nr5a2 on the expression of tight junction associated proteins in SYF granulosa cellsThe expression and transcription of tight junction associated proteins(ZO-1,OCLN,CLDN-1),the cell proliferation increased and cell apoptosis significantly decreased after Nr5a2 overexpression(P<0.05).In conclusion,the study demonstrated that NR5A2 modulates tight junction between granulosa cells during follicle selection by regulating the expression of tight junction associated proteins,thereby affecting yolk precursor deposition in hens.The results showed new insights into the regulatory network governing follicular selection,and establish a theoretical framework for improving egg production through manipulation of yolk deposition processes.更多还原