Fertility Center of CHA Gangnam Medical Center, CHA University, Gangnam-gu, Seoul, Republic of Korea.
Hum Reprod. 2012 Jun;27(6):1768-80. doi: 10.1093/humrep/des092. Epub 2012 Mar 28.
Oocyte activation is a crucial step that comprises the release of the oocyte from meiotic arrest, pronuclear formation and subsequent embryo development. Oocytes are activated by repetitive increases in the intracellular concentration of free Ca(2+), Ca(2+) oscillations, which are triggered during fertilization by the introduction of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce Ca(2+) oscillations or oocyte activation. We first purified recombinant human PLCZ1 (hPLCZ1) protein and evaluated its Ca(2+) oscillation activity in mouse and human oocytes with the view to investigate its application in the clinic for assisted oocytes activation in lieu of chemical agents.
Recombinant hPLCZ1 was synthesized using the Escherichia coli system, and subjected to immunoblot analysis with anti-PLCZ1 and anti-His tag antibodies. Ca(2+) oscillations by microinjection of recombinant hPLCZ1 into mouse or human oocytes were examined by Ca(2+) monitoring with Fluo 4. Ploidy of the oocytes with recombinant hPLCZ1 injection was confirmed with fluorescence in situ hybridization.
A band of 68 kDa on recombinant protein was detected with both antibodies. Injection of recombinant hPLCZ1 induced Ca(2+) oscillations in a dose-dependent manner in both mouse and human oocytes. These oscillations, which closely resembled those initiated by the sperm upon fertilization, triggered activation and cleavage in oocytes of both species, although further development of the mice embryos was low. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. Microinjection of recombinant hPLCZ1 into ICSI-failed human oocytes rescued fertilization failure in five of eight attempts.
Repeated Ca(2+) oscillations and oocyte activation were induced in mouse and human oocytes by microinjection of recombinant hPLCZ1 synthesized in E. Coli. Injection of recombinant protein could thus provide a biological solution for inducing artificial activation of oocytes.
卵母细胞激活是一个关键步骤，包括卵母细胞从减数分裂阻滞中释放、原核形成和随后的胚胎发育。卵母细胞通过细胞内游离 Ca(2+)浓度的反复增加而被激活，Ca(2+) 震荡，这是在受精过程中由精子特异性磷脂酶 C ζ 1 (PLCZ1)的引入触发的。最近的研究表明，缺乏 PLCZ1 表达或表达 PLCZ1 突变体的患者的精子无法诱导Ca(2+) 震荡或卵母细胞激活。我们首先纯化了重组人 PLCZ1(hPLCZ1)蛋白，并在小鼠和人卵母细胞中评估了其Ca(2+) 震荡活性，以期研究其在临床上替代化学药物用于辅助卵母细胞激活的应用。
使用大肠杆菌系统合成重组 hPLCZ1，并使用抗 PLCZ1 和抗 His 标签抗体进行免疫印迹分析。通过 Fluo 4 监测Ca(2+)，检查重组 hPLCZ1 微注射入小鼠或人卵母细胞后Ca(2+) 震荡。注射重组 hPLCZ1 后的卵母细胞的倍性通过荧光原位杂交确认。
两种抗体均在重组蛋白上检测到 68 kDa 的条带。重组 hPLCZ1 的注射以剂量依赖性方式诱导小鼠和人卵母细胞中Ca(2+) 震荡。这些震荡与精子受精时引发的震荡非常相似，引发了两种物种卵母细胞的激活和分裂，尽管小鼠胚胎的进一步发育较低。PLC 抑制剂 U73122 阻断了 hPLCZ1 引发震荡的能力。在 8 次尝试中的 5 次中，将重组 hPLCZ1 微注射入 ICSI 失败的人卵母细胞中挽救了受精失败。
在大肠杆菌中合成的重组 hPLCZ1 微注射入小鼠和人卵母细胞中诱导了重复的Ca(2+) 震荡和卵母细胞激活。因此，注射重组蛋白可以为诱导卵母细胞人工激活提供一种生物学解决方案。
