Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre, John Radcliffe Hospital, Headington,Oxford OX3 9DU, UK.
Hum Reprod. 2011 Dec;26(12):3372-87. doi: 10.1093/humrep/der336. Epub 2011 Oct 18.
Mammalian oocyte activation occurs via a series of intracellular calcium (Ca(2+)) oscillations thought to be induced by a sperm-specific phospholipase C zeta (PLCζ). There is now strong evidence to indicate that certain types of human male infertility are caused by failure of the sperm to activate the oocyte in an appropriate manner. Molecular analysis of the PLCζ gene of a male patient with oocyte activation deficiency has previously identified a point mutation causing a histidine to proline substitution at PLCζ residue 398 (PLCζ(H398P)), leading to abnormal Ca(2+) release profiles and reduced oocyte activation efficiency.
In the present study, we used HEK293T cells to produce recombinant human wild-type PLCζ (PLCζ(WT)) protein which, upon microinjection into mouse oocytes, induced Ca(2+) oscillations characteristic of oocyte activation. Injection of recombinant PLCζ(H398P) was unable to elicit Ca(2+) oscillations in mouse oocytes. Loss of activity mutations, such as PLCζ(H398P) and an artificially induced frameshift mutation (PLCζ(ΔYC2)) did not affect Ca(2+) release when over-expressed in HEK293T cells, whereas PLCζ(WT) inhibited adenosine triphosphate-activated Ca(2+) release. Confocal imaging of fluorescently tagged PLCζ isoforms in HEK293T cells suggested a cytoplasmic pattern of localization, while quantitative analysis of fluorescence levels showed that PLCζ(WT) > PLCζ(H398P) > PLCζ(ΔYC2), indicating that loss of activity mutations may lead to protein instability. This was further indicated by the low proportion of sperm and the lower levels of total PLCζ immunofluorescence from the patient exhibiting PLCζ(H398P) compared with fertile controls.
We demonstrate, for the first time, the production of active recombinant human PLCζ protein which retained the ability to elicit characteristic Ca(2+) oscillations in mouse oocytes, an ability which was eliminated by an infertility-linked mutation. These findings advance our understanding of PLCζ, and provide a critical step forward in obtaining purified PLCζ protein as a potential therapeutic agent for oocyte activation deficiency.
哺乳动物卵母细胞的激活是通过一系列细胞内钙离子（Ca(2+)）振荡来实现的，这些振荡被认为是由精子特异性的磷脂酶 C ζ（PLCζ）诱导的。现在有强有力的证据表明，某些类型的男性不育是由于精子不能以适当的方式激活卵母细胞引起的。对卵母细胞激活缺陷男性患者的 PLCζ 基因进行分子分析，先前已确定一个点突变导致 PLCζ 残基 398 处的组氨酸突变为脯氨酸（PLCζ（H398P）），导致异常的 Ca(2+)释放谱和降低卵母细胞激活效率。
在本研究中，我们使用 HEK293T 细胞产生重组人野生型 PLCζ（PLCζ（WT））蛋白，将其微注射到小鼠卵母细胞中，可诱导卵母细胞激活的 Ca(2+)振荡。重组 PLCζ（H398P）的注射不能在小鼠卵母细胞中引起 Ca(2+)振荡。活性丧失突变，如 PLCζ（H398P）和人工诱导的移码突变（PLCζ（ΔYC2）），在 HEK293T 细胞中过度表达时不会影响 Ca(2+)释放，而 PLCζ（WT）抑制三磷酸腺苷激活的 Ca(2+)释放。在 HEK293T 细胞中用荧光标记的 PLCζ 同工型进行共焦成像显示细胞质定位模式，而荧光水平的定量分析表明 PLCζ（WT）> PLCζ（H398P）> PLCζ（ΔYC2），表明活性丧失突变可能导致蛋白质不稳定。这进一步表明，与生育力正常的对照组相比，表现出 PLCζ（H398P）的患者的精子比例较低，以及总 PLCζ 免疫荧光水平较低。
我们首次证明了活性重组人 PLCζ 蛋白的产生，该蛋白保留了在小鼠卵母细胞中引发特征性 Ca(2+)振荡的能力，而不育相关突变消除了这种能力。这些发现增进了我们对 PLCζ 的理解，并为获得纯化的 PLCζ 蛋白作为卵母细胞激活缺陷的潜在治疗剂提供了关键的一步。
