Yoda Ayako, Oda Shoji, Shikano Tomohide, Kouchi Zen, Awaji Takeo, Shirakawa Hideki, Kinoshita Katsuyuki, Miyazaki Shunichi
Department of Physiology, Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo 162-8666, Japan.
Dev Biol. 2004 Apr 15;268(2):245-57. doi: 10.1016/j.ydbio.2003.12.028.
Sperm-specific phospholipase C zeta (PLC zeta) is known to induce intracellular Ca(2+) oscillations and egg activation when expressed in mouse eggs by injection of RNA encoding PLC zeta. We investigated the expression level and spatial distribution of PLC zeta in the egg in real time and in relation to the initiation and termination of Ca(2+) oscillations by monitoring fluorescence of a yellow fluorescent protein 'Venus' fused with PLC zeta. Ca(2+) oscillations similar to those at fertilization were induced at 40-50 min after RNA injection, when expressed PLC zeta reached 10-40 x 10(-15) g in the egg. PLC zeta-Venus increased up to 3 h and attained a steady level at 4-5 h. Interestingly, PLC zeta-Venus is accumulated to the pronucleus (PN) formed at 5-6 h and continuously increased there. Ca(2+) oscillations stopped in most eggs before initiation of the accumulation. A variant of PLC zeta that lacks three EF hand domains was much less effective in induction of Ca(2+) oscillations and little accumulated in the pronucleus, indicating a critical role of those domains. The ability of the accumulation to the pronucleus qualifies PLC zeta for a strong candidate of the Ca(2+) oscillation-inducing sperm factor, which is introduced into the ooplasm upon sperm-egg fusion and concentrated to the pronucleus after inducing egg activation.
已知精子特异性磷脂酶Cζ（PLCζ）通过注射编码PLCζ的RNA在小鼠卵母细胞中表达时，可诱导细胞内Ca(2+)振荡和卵母细胞激活。我们通过监测与PLCζ融合的黄色荧光蛋白“金星”的荧光，实时研究了PLCζ在卵母细胞中的表达水平和空间分布，以及与Ca(2+)振荡的起始和终止的关系。在RNA注射后40 - 50分钟，当卵母细胞中表达的PLCζ达到10 - 40×10(-15) g时，诱导出与受精时相似的Ca(2+)振荡。PLCζ-金星在3小时内持续增加，并在4 - 5小时达到稳定水平。有趣的是，PLCζ-金星在5 - 6小时形成的原核（PN）处积累，并在那里持续增加。在积累开始之前，大多数卵母细胞中的Ca(2+)振荡停止。缺少三个EF手结构域的PLCζ变体在诱导Ca(2+)振荡方面效果要差得多，并且在原核中几乎不积累，这表明这些结构域具有关键作用。向原核积累的能力使PLCζ成为Ca(2+)振荡诱导精子因子的有力候选者，该因子在精卵融合时被引入卵质，并在诱导卵母细胞激活后集中到原核。
