| In vitro Induction and Detection of Acrosomal Exocytosis in Human Spermatozoa
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Author:
Date:
2020-07-20
[Abstract] The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, spermatozoa are then capable of penetrating the outer vestments of the oocyte and initiating fertilization. Accordingly, the failure of spermatozoa to complete an acrosome reaction represents a relatively common etiology in male infertility patients, and the ability to induce acrosomal exocytosis has found clinical utility in the evaluation of sperm fertilizing capacity. Here, we firstly describe protocols for driving the capacitation of human ...
[摘要] [摘要 ] 顶体反应是高度调控的胞吐事件，引发精子成功受精。诱导后，顶体胞吐作用通过一束囊泡波进行，该囊泡波辐射穿过精子头部，使顶体囊泡不稳定并导致顶体内容物释放。脱去顶体后，精子便能够穿透卵母细胞的外衣并开始受精。因此，在男性不育患者中，精子不能完成顶体反应代表了相对普遍的病因，并且诱导顶体胞吐作用的能力已在评估精子受精能力中发现了临床用途。在这里，我们首先描述驱动人类精子获能的方案 在体外使用化学成分确定的培养基以引发细胞完成顶体胞吐作用。然后，我们描述了常规用于诱导结合了生理激动剂（即甾体激素，孕酮）或药理试剂（即二价阳离子离子载体，A23187）的顶体胞吐作用的方法。最后，我们描述了组织化学和免疫荧光技术的应用，这些技术可用于研究顶体反应的完成。这样的协议对于临床和男科学研究实验室中的精子功能测试具有重要的诊断实用性。
[背景 ] 顶配体是雄性配子所特有的，是一种溶酶体样的膜细胞器，装饰在精子头部的前部区域，并由顶体内膜和外膜描绘（Hermo 等人，2010a和2010b）。如此形成的顶体囊泡封装了无数的结构和酶促组分，将其分隔为可溶或不可溶级分（Guyonnet 等，2012； Guyonnet 等，2014）。这些中的后者，称为顶体基质，用作稳定的支架，其允许在顶体反应期间受控释放受精所必需的基质相关蛋白（Guyonnet ...
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| Sample Preparation for X-ray Micro-computed Tomography of Woody Plant Material and Associated Xylem Visualisation Techniques
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Author:
Date:
2016-03-20
[Abstract] Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow ...
[摘要] 短旋转枝（SRC）柳树的组织结构的变化是驱动木质纤维素糖产量的差异的主要因素，但是该组织的大部分生理学和发育是未知的。传统的切片在木质组织中既困难又具有破坏性;然而，诸如三维X射线微计算断层扫描（μCT）扫描的技术可以用于移动生物学研究者超越组织变化的传统二维评估，而不必破坏性地切割细胞。这种技术不能取代传统的微观技术，而是可以仔细地与传统方法整合，以改进植物生物学世界的三维探索。以下程序概述了用于3D X射线μCT和相关木质部染色和可视化技术的柳树的制备，特别是在凝胶状纤维（g-纤维）发育期间的次生木质部程序性细胞死亡（PCD）延迟。这里的许多染色技术可转移到其他木本物种，如杨树和桉树。
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| Immunostaining Protocol: P-Smad2 (Xenograft and Mice)
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Author:
Alexandre Calon, Elisa Espinet, Sergio Palomo-Ponce, Daniele V. F. Tauriello, Mar Iglesias, María Virtudes Céspedes, Marta Sevillano, Cristina Nadal, Peter Jung, Xiang H.-F. Zhang, Daniel Byrom, Antoni Riera, David Rossell, Ramón Mangues, Joan Massague, Elena Sancho and Eduard Batlle,
Date:
2014-05-05
[Abstract] Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF-beta responses when injected into nude mice as shown by lack of p-SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells.
[摘要] 转移依赖于在TGF-β刺激时由肿瘤微环境表达的基因程序。 当注射到裸鼠中时，CRC（结肠直肠癌）细胞系不诱导强烈的基质TGF-β应答，如肿瘤相关基质细胞中缺乏p-SMAD2积累所示。 为了在异种移植物中强化高TGF-β信号传导，我们设计CRC细胞系以分泌活性TGF-β。 从HT29-M6TGF-β，KM12L4aTGF-β细胞和SW48TGF-β细胞获得的皮下肿瘤含有丰富的p-SMAD2 + 基质细胞。
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