Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil.
Department of Veterinary Clinical Sciences, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil.
Biol Reprod. 2017 Jan 1;97(6):835-849. doi: 10.1093/biolre/iox148.
Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy nonlactating Holstein cows to determine the protein expression profile of FF at key stages of follicular development. Follicles were individually aspirated in vivo at predeviation (F1 ∼ 7.0 mm), deviation (F1 ∼ 8.5 mm), postdeviation (F1 ∼ 12.0 mm), and preovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FFs from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labeled with TMTsixplex and quantified using liquid chromatography-mass spectrometry/mass spectrometry. A total of 143 proteins were identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P < 0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Among the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function.
牛卵泡液 (FF) 构成卵泡的微环境，包含各种具有生物活性的蛋白质。我们进行了一项研究，涉及 18 头健康的非泌乳荷斯坦奶牛，以确定卵泡发育关键阶段 FF 的蛋白质表达谱。在卵泡发育的前偏离期 (F1 ∼ 7.0mm)、偏离期 (F1 ∼ 8.5mm)、后偏离期 (F1 ∼ 12.0mm) 和排卵前阶段，通过测量卵泡雌二醇和孕酮浓度，分别在体内对单个卵泡进行抽吸。从 9 头奶牛中选择 FF 进行蛋白质组学分析。在耗尽白蛋白后，将三批 Pooled FF 进行还原、烷基化，并用胰蛋白酶消化。所得肽段用 TMTsixplex 标记，并通过液相色谱-质谱/质谱进行定量。共鉴定出 143 种蛋白质，并将其分配到各种生物学过程中，包括对刺激的反应和代谢过程。在不同阶段 (P < 0.05) 发现 22 种差异表达的蛋白质，表明卵泡内的变化随着发育而发生，预期的偏离时间对调节蛋白质表达至关重要。例如，在卵泡发育的早期阶段，发现卵泡抑素、抑制素、糖胺聚糖、spondin-1、纤维蛋白原和抗雄激素抗体的浓度较高。相比之下，在偏离后，载脂蛋白 H、α2-巨球蛋白、纤溶酶原、抗凝血酶-III 和免疫球蛋白增加。在差异丰度蛋白中，发现 19 种与类固醇生成有关。途径分析确定了主要与急性期反应信号、凝血系统、补体系统、肝/视黄醇 X 受体激活以及一氧化氮和活性氧的生物合成相关的蛋白质。差异表达的蛋白质提供了对卵巢卵泡微环境中与大小相关的蛋白质变化的深入了解，这些变化可能影响卵泡功能。
