Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm
Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm
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DOI：
10.1095/biolreprod61.1.76
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发表时间：
1999-07-01
影响因子：
3.6
通讯作者：
Tezón, JG
中科院分区：
文献类型：
--
作者：
Visconti, PE;Stewart-Savage, J;Tezón, JG
获能是哺乳动物精子成功受精的先决条件。该过程通常在体外在限定的 NaHCO3 缓冲培养基中观察到，并且已被证明与 cAMP 代谢和蛋白质酪氨酸磷酸化的变化相关。在这项研究中，我们观察到，当 NaHCO3 被 4-(2-羟乙基)1-哌嗪乙磺酸 (HEPES) 取代时，仓鼠精子获能（衡量精子进行自发顶体反应的能力）并未发生。在精子已预孵育 3.5 小时的无 NaHCO3 培养基中添加 25 mM NaHCO3，在接下来的 4 小时内将自发顶体反应的百分比从 0% 增加到 80%。在含 NaHCO3 的培养基中添加阴离子转运阻滞剂，例如 4,4'-二异硫氰基-2,2'-二苯乙烯二磺酸盐 (DIDS) 或 4-乙酰氨基-4'-异硫氰酸二苯乙烯-2,2'-二磺酸 (SITS)，可抑制顶体反应，在 600 μM 时具有最大抑制作用，EC50 为 100 μM。增加细胞外或细胞内pH值不会在无NaHCO3培养基中诱导顶体反应。相反，添加500μM二丁酰cAMP（dbcAMP），单独或与100μM 1-甲基-3-异丁基黄嘌呤（IBMX）一起，诱导在无NaHCO3培养基中孵育的精子中的顶体反应。这些化合物还部分逆转了完全培养基中 DIDS 或 SITS 引起的顶体反应的抑制。与这些结果相反，IBMX 或 dbcAMP 不会在无 Ca2+ 培养基中孵育的细胞中诱导顶体反应。当仓鼠精子在不存在 NaHCO3 或存在 NaHCO3 和 DIDS 的情况下孵育时，cAMP 浓度显着低于在完全培养基中孵育的精子获得的值。蛋白质酪氨酸磷酸化也已被证明与许多物种获能的发生高度相关。在获能的第一个小时内，在完全培养基中观察到蛋白质酪氨酸磷酸化增加。在不存在 NaHCO3 的情况下，蛋白质酪氨酸磷酸化的增加延迟了 45 分钟，并且通过添加 dbcAMP 和 IBMX 克服了这种延迟。与对照培养基相比，在无 NaHCO3 的培养基中钙离子载体 A23187 诱导的顶体反应延迟了 2 小时。在 dbcAMP 和 IBMX 存在的情况下没有观察到这种延迟。综上所述，这些结果表明 cAMP 途径可能介导 NaHCO3 在仓鼠精子获能中的作用，并且蛋白质酪氨酸磷酸化是必要的，但对于完全获能还不够。
Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2,2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 mu M, and with an EC50 of 100 mu M. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium, In contrast, addition of 500 mu M dibutyryl cAMP (dbcAMP), atone or together with 100 mu M 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX, The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.