Cafe Shenae L, Anderson Amanda L, Nixon Brett
Priority Research Centre for Reproductive Science, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Hunter Medical Research Institute, Pregnancy and Reproduction Program, New Lambton Heights, NSW, Australia.
Bio Protoc. 2020 Jul 20;10(14):e3689. doi: 10.21769/BioProtoc.3689.
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 spermatozoa using chemically defined media in order to prime the cells for completion of acrosomal exocytosis. We then describe methodology routinely used for the induction of acrosomal exocytosis incorporating either a physiological agonist (, the steroidal hormone, progesterone) or pharmacological reagent (, the divalent cation ionophore, A23187). Finally, we describe the application of histochemical and immunofluorescence techniques that can be applied to study the completion of the acrosome reaction. Such protocols have important diagnostic utility for sperm function testing in both clinical and andrological research laboratories.
顶体反应是一种高度调控的胞吐事件，它使精子具备成功受精的能力。诱导后，顶体胞吐通过一波囊泡化进行，这波囊泡化从精子头部辐射开来，使顶体囊泡不稳定，导致顶体内容物释放。精子脱去顶体后，便能够穿透卵母细胞的外层膜并启动受精过程。因此，精子无法完成顶体反应是男性不育患者相对常见的病因，而诱导顶体胞吐的能力已在评估精子受精能力方面具有临床应用价值。在此，我们首先描述使用化学成分明确的培养基促使人类精子获能的方案，以便使细胞为完成顶体胞吐做好准备。然后我们描述常规用于诱导顶体胞吐的方法，该方法可采用生理激动剂（甾体激素孕酮）或药理试剂（二价阳离子载体A23187）。最后，我们描述可用于研究顶体反应完成情况的组织化学和免疫荧光技术的应用。此类方案在临床和男科学研究实验室的精子功能检测中具有重要的诊断用途。
