Research Team of Intracellular Signaling and Technology of Reproduction (SINTREP), Department of Biochemistry and Molecular Biology and Genetics, University of Extremadura, Cáceres 10071, Spain.
J Cell Biochem. 2010 Jan 1;109(1):65-73. doi: 10.1002/jcb.22393.
The cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase-3alpha (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase-resistant cell permeable cAMP analogue 8Br-cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro-32-0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM- or 8Br-cAMP-stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility.
cAMP 依赖性蛋白激酶（PKA）、蛋白激酶 C（PKC）和磷脂酰肌醇 3-激酶（PI3K）途径控制着精子细胞的大多数相关功能，包括运动性。最近，我们证明糖原合酶激酶-3α（GSK3A）的磷酸化状态也是控制猪精子运动性的关键事件。然而，GSK3A 丝氨酸磷酸化（抑制）的上游调节剂在雄性生殖细胞中仍知之甚少。这项工作研究了 PKA、PKC 和 PI3K 途径在猪精子中 GSK3A 磷酸化中的作用。在获能培养基（TCM）或磷酸二酯酶抗性细胞通透的 cAMP 类似物 8Br-cAMP 中，GSK3A 的 Ser21 磷酸化显著增加，同时猪精子的运动性也显著增加。这些作用在精子预先孵育 PKA 抑制剂 H89 或 PKC 抑制剂 Ro-32-0432 后被阻断。PI3K 抑制剂 LY294002 增加了精子的运动参数和基础 GSK3A 磷酸化，但不影响 TCM 或 8Br-cAMP 刺激的 GSK3A 磷酸化。PI3K 抑制作用是通过增加猪精子中的细胞内 cAMP 水平介导的，并且被 PKA 抑制剂 H89 抑制。总之，我们证明了 PKA、PKC 和 PI3K 途径在猪雄性生殖细胞中相互作用，以关键调节 GSK3A 磷酸化，进而控制细胞运动性。此外，我们的结果表明，PI3K 位于 PKA 的上游，而 PKA 位于 PKC 的上游，在这个调节级联中。我们的发现有助于阐明调节精子细胞最重要的功能之一——运动性的分子机制。
