Grasa P, Coward K, Young C, Parrington J
Department of Pharmacology, University of Oxford, Oxford, UK.
Hum Reprod. 2008 Nov;23(11):2513-22. doi: 10.1093/humrep/den280. Epub 2008 Jul 24.
Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa.
A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro.
After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region.
PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.
近期研究表明，在哺乳动物中，受精时卵母细胞激活是由精子特异性磷脂酶C（PLCζ）触发的。我们研究了PLCζ在人类精子中的定位。
制备了针对人类PLCζ的多克隆抗体，并将其用于未获能和获能状态下射出的人类精子的免疫印迹和免疫荧光研究。还使用离子载体在体外诱导顶体反应。
通过免疫印迹验证抗PLCζ抗体的特异性后，免疫荧光研究表明，未获能精子中PLCζ的主要定位在赤道区，获能和离子载体处理后该模式保持不变。对混合样本的分析显示，约88%的未获能精子在赤道区表达PLCζ，而分别约35%和21%的精子在顶体或顶体后区表达额外的PLCζ群体。在约12%的精子的顶体后区观察到一群PLCζ。获能和离子载体处理后，顶体后PLCζ的细胞比例增加（P<0.05）。在单个样本中也发现了相同的趋势。完整顶体的存在与顶体区对PLCζ免疫反应性的精子比例之间存在强相关性（r = 0.716，P<0.0001）。
在精子头部的三个部位可不同程度地检测到PLCζ：赤道段和顶体/顶体后区。可育男性精子中PLCζ定位的变异性可能反映个体之间或射精内卵母细胞激活能力的差异。这种方法可能有助于研究PLCζ与某些类型男性不育之间的可能联系。
