Brewis I A, Clayton R, Browes C E, Martin M, Barratt C L, Hornby D P, Moore H D
Department of Molecular Biology and Biotechnology, The University of Sheffield, UK.
Mol Hum Reprod. 1998 Dec;4(12):1136-44. doi: 10.1093/molehr/4.12.1136.
Protein tyrosine phosphorylation and induction of the acrosome reaction (AR) in non-capacitated and capacitated human spermatozoa was investigated in response to recombinant human zona pellucida glycoprotein (rhZP3) produced by Chinese hamster ovary cells transfected with a plasmid containing human ZP3 cDNA. rhZP3-containing medium promoted the AR in a high proportion of capacitated spermatozoa (48.6 +/- 3.2%; P < 0.01) compared with control (no rhZP3) samples (14.8 +/- 2.1%). However, rhZP3-containing medium did not cause increased acrosomal exocytosis in non-capacitated spermatozoa (16.8 +/- 3.0%). Induction of the AR was associated with increased tyrosine phosphorylation of a 95 +/- 5 kDa epitope only in capacitated spermatozoa. A dose-dependent increase in the protein phosphorylation of a 95 kDa epitope in response to rhZP3 was detected by [gamma-32P]-ATP labelling of detergent-solubilized sperm proteins. When spermatozoa were co-incubated with monoclonal antibody 97.25 (mAb 97.25) recognizing a 95 kDa tyrosine kinase epitope, there was no rhZP3 induction of tyrosine phosphorylation of the 95 kDa protein. Such co-incubation also markedly inhibited the AR (23.9 +/- 3.1%). These results support the model that initial interaction of the fertilizing spermatozoon with ZP3 involves the tyrosine phosphorylation of a 95 kDa tyrosine kinase protein and that this requires capacitation.
研究了重组人透明带糖蛋白（rhZP3）对未获能和获能的人类精子中蛋白质酪氨酸磷酸化及顶体反应（AR）诱导的影响。rhZP3由转染含人ZP3 cDNA质粒的中国仓鼠卵巢细胞产生。与对照（无rhZP3）样本（14.8±2.1%）相比，含rhZP3的培养基能促进高比例获能精子发生顶体反应（48.6±3.2%；P<0.01）。然而，含rhZP3的培养基并未使未获能精子的顶体外排增加（16.8±3.0%）。仅在获能精子中，顶体反应的诱导与95±5 kDa表位的酪氨酸磷酸化增加有关。通过[γ-32P] -ATP标记去污剂溶解的精子蛋白，检测到rhZP3诱导下95 kDa表位的蛋白磷酸化呈剂量依赖性增加。当精子与识别95 kDa酪氨酸激酶表位的单克隆抗体97.25（mAb 97.25）共同孵育时，rhZP3未诱导95 kDa蛋白的酪氨酸磷酸化。这种共同孵育也显著抑制了顶体反应（23.9±3.1%）。这些结果支持以下模型：受精精子与ZP3的初始相互作用涉及95 kDa酪氨酸激酶蛋白的酪氨酸磷酸化，且这一过程需要获能。
