鹅催乳素糖基化对等级前卵泡发育的影响及其调控机制

Poor egg production performance is the bottleneck in goose industrial development, and the rate of egg production depends upon the growth and development of ovarian pre-hierarchical follicles. Prolactin (PRL) plays an important role in regulating follicle development, but both its structural properties and biological functions are influenced by glycosylation. Results from our previous research suggested that goose pituitary mainly secrets two major PRL isoforms, i.e. non-glycosylated (NG-) and glycosylated (G-) PRL, and the proportion of G-PRL to NG-PRL significantly varies during different reproductive states. Nevertheless, it still remains largely unknown how glycosylation modification of PRL influences its role during pre-hierarchical follicular development. Therefore, the main objectives of this research are: 1) to isolate and purify both NG-PRL and G-PRL from geese pituitaries using immunoprecipitation and lectin affinity chromatography and to compare the similarities and differences between their structures, immune and biological activities; 2) to investigate the effects of NG-PRL and G-PRL on the morphology and physiological functions of pre-hierarchical follicular somatic cells according to the concentration and the ratio of glycosylation and 3) to identify differently expressed lncRNAs, mRNAs and miRNAs in follicular cells treated without or with certain concentration and glycosylation ratio of NG-PRL and G-PRL and to build PRL glycosylation-based lncRNA-mRNA-miRNA molecular regulatory network using high throughput whole transcriptome sequencing. This research will contribute to a better understanding of the relationship between PRL glycosylation and ovarian follicular development as well as the potential regulatory mechanisms, and will provide some new insights or practical guidance into inhibiting broodiness and enhancing egg production performance in geese.

鹅产蛋性能差是制约其产业化发展的瓶颈，产蛋量高低取决于卵巢内等级前卵泡的生长发育能力。催乳素（PRL）对卵泡发育具有重要调控作用，但其结构和功能受到糖基化修饰的影响。本项目组前期研究发现鹅垂体主要分泌非糖基化（NG-）和糖基化（G-）PRL，且PRL糖基化水平（即G-PRL：NG-PRL比值）随繁殖周期不同呈显著波动变化。但是，PRL如何根据其糖基化水平波动影响等级前卵泡的发育尚不清楚。本项目拟通过免疫沉淀、凝集素亲和层析、LC-MS/MS等技术首先从鹅垂体中分离纯化及鉴定NG-PRL和G-PRL，进一步通过卵巢卵泡、膜细胞和颗粒细胞培养模型明确不同剂量和糖基化水平PRL对等级前卵泡细胞形态和功能的影响，并运用全转录组测序构建PRL糖基化调控等级前卵泡发育的分子网络。本研究对揭示PRL糖基化与卵泡发育关系及其调控机制具有重要理论意义，并对抑制鹅就巢性、提高产蛋性能具有重要理论和实践意义。

鹅产蛋性能差是制约其产业化发展的瓶颈，产蛋量高低取决于卵巢内等级前卵泡的生长发育能力。催乳素（PRL）对卵泡发育具有重要调控作用，但其结构和功能受到糖基化修饰的影响。然而，PRL及其糖基化修饰如何影响鹅等级前卵泡的发育尚不清楚。本项目旨在：1）分离纯化及鉴定鹅天然PRL蛋白（nPRL）和糖基化催乳素蛋白（G-PRL），明确糖基化修饰对PRL结构和活性的影响；2）揭示PRL及其糖基化修饰对鹅等级前卵泡细胞功能的影响及其调控机制。通过本项目的实施，已获得的研究结果主要包括：1）构建了鹅PRL原核表达载体，分离纯化出鹅PRL重组蛋白（rPRL），以鹅rPRL为抗原制备出兔抗鹅PRL特异性抗体，采用自制的免疫亲和层析柱高效分离纯化出足量nPRL，采用自制的凝集素亲和层析柱进一步从nPRL中成功分离纯化出天然G-PRL，并采用一系列技术手段对nPRL、G-PRL进行了纯度和结构分析，明确了糖基化修饰对PRL结构、免疫和生物学活性的影响；2）揭示了PRL 总蛋白、mRNA及其两种亚型（即NG-PRL、G-PRL）在胚胎至出壳早期鹅垂体中的发育性表达变化，揭示了PRL受体（PRLR）在不同发育阶段鹅垂体、卵巢及卵泡组织和细胞中的表达模式，证实PRL-PRLR信号通路在调控鹅垂体、卵巢及卵泡早期发育过程中发挥着重要作用；3）采用RNA-Seq、ATAC-Seq等高通量测序技术筛选并鉴定出调控鹅垂体、卵巢及卵泡发育的关键通路和基因，并揭示了部分关键基因及miRNAs对鹅等级前和等级卵泡发育的影响机制；4）基于已纯化出的鹅rPRL、nPRL和G-PRL，系统比较分析了不同浓度鹅rPRL、nPRL和G-PRL以及同剂量下不同糖基化水平的鹅nPRL对等级前和等级卵泡颗粒细胞活性、增殖凋亡、脂滴沉积和类固醇激素合成的影响。综上，本项目研究结果不仅揭示了PRL及其糖基化修饰对鹅等级前和等级卵泡发育的影响机制，为生产上抑制鹅就巢性、提高产蛋性能提供了新的思路；而且由此建立的鹅nPRL和G-PRL分离纯化技术与方法，对于分离提纯家禽组织中其他天然蛋白、糖基化蛋白具有较好的参考价值，有助于开展蛋白翻译后修饰的功能研究及其生产应用。