Galantino-Homer H L, Visconti P E, Kopf G S
Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, Philadelphia 19104-6080, USA.
Biol Reprod. 1997 Mar;56(3):707-19. doi: 10.1095/biolreprod56.3.707.
Mammalian sperm capacitation, defined as an obligatory maturational process leading to the development of the fertilization-competent state, results from a poorly understood series of morphological and molecular events. We report here that ejaculated bovine sperm, incubated under conditions that support capacitation in vitro, display a reproducible pattern of protein tyrosine phosphorylations that are regulated by a cAMP-dependent pathway. The appearance of these tyrosine phosphorylated proteins correlated temporally with the time course of capacitation induced by heparin, and these phosphorylations displayed a similar heparin concentration dependence. Glucose, which inhibits capacitation, inhibited these protein tyrosine phosphorylations in media containing heparin. The biologically active cAMP analogues (dibutyryl cAMP [db-cAMP], 8-bromo cAMP, sp-cAMPS) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced the same protein tyrosine phosphorylation patterns as seen with heparin. Moreover, these cAMP agonists could overcome the inhibition of the heparin-induced tyrosine phosphorylations by glucose. In contrast, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPS), a protein kinase A (PK-A) antagonist, blocked the capacitation-associated increases in protein tyrosine phosphorylation. This cAMP regulation of the protein tyrosine phosphorylation pattern is mediated by PK-A since N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide-dihydrochloride (H89), another inhibitor of PK-A, inhibited the heparin-induced protein tyrosine phosphorylation pattern in a concentration-dependent manner in either the absence or presence of db-cAMP, IBMX, and glucose. These data support a model for sperm capacitation that includes protein tyrosine phosphorylation as an important regulatory pathway, and a role for cAMP/PK-A in the regulation of this pathway leading to capacitation. These studies are the first to report a unique interrelationship between tyrosine kinase/phosphatase and cAMP signaling pathways at the level of PK-A in bovine sperm capacitation.
哺乳动物精子获能是一个导致具备受精能力状态的必要成熟过程，它源于一系列形态和分子事件，但人们对此了解甚少。我们在此报告，在支持体外获能的条件下孵育的射出牛精子，呈现出一种可重复的蛋白质酪氨酸磷酸化模式，该模式受cAMP依赖途径调控。这些酪氨酸磷酸化蛋白的出现与肝素诱导的获能时间进程在时间上相关，并且这些磷酸化表现出类似的肝素浓度依赖性。抑制获能的葡萄糖在含有肝素的培养基中抑制了这些蛋白质酪氨酸磷酸化。具有生物活性的cAMP类似物（二丁酰cAMP [db-cAMP]、8-溴cAMP、sp-cAMPS）和磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）诱导出与肝素相同的蛋白质酪氨酸磷酸化模式。此外，这些cAMP激动剂可以克服葡萄糖对肝素诱导的酪氨酸磷酸化的抑制作用。相反，蛋白激酶A（PK-A）拮抗剂Rp-腺苷-3',5'-环磷酸硫代酯（Rp-cAMPS）阻断了与获能相关的蛋白质酪氨酸磷酸化增加。这种cAMP对蛋白质酪氨酸磷酸化模式的调节是由PK-A介导的，因为另一种PK-A抑制剂N-[2-(对溴肉桂氨基)乙基]-5-异喹啉磺酰胺二盐酸盐（H89）在不存在或存在db-cAMP、IBMX和葡萄糖的情况下，均以浓度依赖的方式抑制肝素诱导的蛋白质酪氨酸磷酸化模式。这些数据支持了一个精子获能模型，该模型包括蛋白质酪氨酸磷酸化作为一个重要的调节途径，以及cAMP/PK-A在调节该途径导致获能中的作用。这些研究首次报道了在牛精子获能过程中，酪氨酸激酶/磷酸酶和cAMP信号通路在PK-A水平上存在独特的相互关系。
