From the Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003.
J Biol Chem. 2013 Dec 6;288(49):35307-20. doi: 10.1074/jbc.M113.489476. Epub 2013 Oct 15.
Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3(-)-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca(2+) and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.
受精能力是在女性生殖道中通过一种称为获能的过程获得的。获能对于运动的激活（例如超激活）和使精子为已知的顶体反应的胞吐过程做好准备是必需的。虽然 HCO3(-) 依赖性可溶性腺苷酸环化酶 Adcy10 在运动中发挥作用，但对于精子头部中 cAMP 的来源知之甚少。跨膜腺苷酸环化酶（tmAC）是 cAMP 的另一个可能来源。这些酶受刺激性异三聚体 Gs 蛋白调节；然而，哺乳动物精子中 Gs 或 tmAC 的存在一直存在争议。在这项研究中，我们使用 Western blot 和霍乱毒素依赖性 ADP-ribosylation 显示了精子头部 Gs 的存在。此外，我们表明，tmAC 特异性激活剂 forskolin 诱导 WT 和 Adcy10 缺失小鼠的精子中 cAMP 积累。这种增加被 tmAC 抑制剂 SQ22536 阻断，但不受 Adcy10 抑制剂 KH7 的阻断。尽管在精子头部检测到 Gs 免疫反应性和 tmAC 活性，但 PKA 仅存在于尾部，先前已经证明 Adcy10 位于尾部。与顶体定位一致，顶体反应后的精子中 Gs 反应性丧失，并且 forskolin能够增加细胞内 Ca(2+)并诱导顶体反应。总的来说，这些数据表明 cAMP 途径在精子中是分隔的，Gs 和 tmAC 在头部，而 Adcy10 和 PKA 在鞭毛中。
