孕酮在诱导精子顶体反应IP3及PKC途径的机制研究及导入激动剂／抑制剂对顶体反应影响的研究

KEY ISSUES.During fertilization, spermatozoon binds to and penetrates the zona pellucdia (ZP). The penetration of ZP requires the release of acrosomal enzymes from spermatozoa by the process of acrosome reaction. Disordered acrosome reaction causes failure of sperm–ZP penetration resulting in infertility. In standard in-vitro fertilization (IVF), defective sperm-ZP penetration due to sperm abnormalities is a major cause of fertilization problem. Consistently, ZP-induced acrosome reaction has been reported to be highly correlated with fertilization rates in vitro. ..The identification of disordered acrosome reaction is a challenge to reproductive medicine specialists. It can occur in patients with normal semen analysis and ZP binding. A practical diagnostic method for these patients is not yet available. Most previous studies used ZP as an acrosome reaction inducer to diagnose disordered acrosome reaction. Although the method has proven to be clinically useful, the limited availability of human ZP makes the assay impractical for routine clinical use. ..HYPOTHESIS.Progesterone is present at high level at the site of fertilization and induces acrosome reaction in sperm of human and other species. However, little is known on its relationship with fertilization rates in vitro. The diagnostic potential of progesterone-induced acrosome reaction for disordered acrosome reaction is unclear. In this proposal, we hypothesize that progesterone-induced acrosome reaction is a useful indicator in addition to routine semen analysis for diagnosis of patients with disordered acrosome reaction. Three objectives will be provided to test the hypothesis. They are: 1) To compare the signal pathways mediated by ZP and progesterone on inducing acrosome reaction; 2) To establish the relationship between progesterone-induced acrosome reaction with the ability of sperm to penetrate the ZP and fertilization rate in vitro; and 3) To determine the role of signal transduction on defective ZP- and progesterone-induced acrosome reaction...LONG TERM IMPACT.Treatment of human infertility using assisted reproductive technology with standard IVF and intracytoplasmic sperm injection (ICSI) have been very successful. The choice between standard IVF and ICSI is generally based on the results of semen analysis. However, standard semen analysis provides only limited information about sperm fertilizing ability and disordered acrosome reaction can occur in patients with normal semen analysis. To improve the clinical management of these patients, it is important to diagnose disordered acrosome reaction by reliable test before commencing assisted reproduction treatment. The proposed project will study the use of progesterone-induced acrosome reaction as a sperm function test for predicting the fertilization potential of semen samples. It will also give indication to whether modulation of sperm acrosome reaction-associated signaling pathways can be applied clinically to enhance fertilization.

精子顶体反应是受精的必要条件，精子顶体反应缺陷会导致精子－透明带穿透失败，从而导致不育。人类透明带是诱导顶体反应的天然因子，我们的前期研究发现，孕酮也可以引起顶体反应，对于预测体外受精率有一定价值。我们打算从三方面对孕酮诱发的顶体反应机制及相关调控进行深入研究：（1）比较透明带和孕酮在诱导顶体反应信号途径方面的异同，对PKC等因子活性进行比较，并通过基于BioPORTER技术所引起的的IP3敏感的钙离子通道能力的变化对透明带和孕酮诱发的顶体反应的作用进行比较；（2）建立孕酮诱发的顶体反应与精子穿透透明带的能力和体外受精率之间的关系；（3）确定信号传导对透明带和孕酮诱发的缺陷性顶体反应的作用，观察对于DZPIAR患者的精液给予顶体反应相关的信号级联的激动剂后的精子-透明带穿透能力的变化。希望通过本研究为临床诊断DZPIAR提供方便、价廉、可靠的方法，并为受精缺陷患者的治疗提供新的思路。

肌醇1,4,5 -三磷酸 (Inositol 1, 4, 5-trisphosphate，IP3)敏感的钙离子通道为精子顶体反应信号通路中的关键分子，本项目应用新型膜渗透性工具BioPORTER® 为IP3敏感的钙离子通道直接参与人类精子顶体反应信号通路最终步骤提供了直接可靠的证据。同时，立足于临床迫切需解决的生育力保存问题，结合蛋白质组学分析拓展探索了冷冻复苏后精子功能下降的分子机制，及马尾松树皮提取物(Pinus Massoniana Bark Extract, PMBE)添加于精子冻存保护液对复苏后精子功能的改善作用及可能的机制。在研究中发现，在精子冻存液中添加PMBE可有效改善冷冻复苏后精子活力和顶体反应发生率的下降,其可通过清除冷冻复苏过程中大量产生的ROS来降低氧化应激水平。参与胞内肌醇合成的肌醇-3-磷酸盐合成酶1(Inositol-3-phosphate synthase 1, ISYNA1)在冷冻复苏后显著下降，而加入PMBE可以有效改善其下降的趋势，达到与冷冻前相近的水平。故推测PMBE可能是通过清除冷冻复苏过程中大量产生的ROS，降低氧化应激水平，从而使冷冻复苏过程中降解的ISYNA1减少，保护复苏后精子细胞内的肌醇生物合成，改善冷冻复苏后精子的顶体反应。本研究明确了BioPORTER®在人类精子体系中应用的安全性和有效性，为后续人类精子信号通路的研究提供了有效的分子工具，并提供了IP3敏感的钙离子通道参与人类精子顶体反应信号通路的直接证据，为后续的研究提供了一定的理论基础。此外，拓展探索的结果为PMBE添加于精子冻存液的后续临床应用提供了重要的理论基础，并为更好地保存生育力提供了可能的手段。项目资助发表SCI论文4篇，培养博士研究生3名，硕士研究生1名，其中1名已经取得博士学位，3名在读。项目投入经费57万元，支出33.3676万元，各项支出基本与预算相符。剩余经费23.6324万元，剩余经费计划用于本项目研究后续支出。