br Funding This work was supported by intramural

Funding This work was supported by intramural funding of the Department of Internal Medicine I-Cardiology, University Hospital Aachen, RWTH Aachen.
Acknowledgments
Introduction Cyclic adenosine monophosphate (cAMP) is a central second messenger that controls a plethora of vital functions. The strategies used to attribute cellular responses to cAMP typically rely on increasing cAMP levels with membrane-permeant analogs, phosphodiesterase inhibitors, and/or protein G/adenylyl cyclase stimulators. While these strategies have been fruitful, the irrefutable proof that a mechanism is mediated by cAMP can only be achieved if the said mechanism/cellular response is abolished when endogenous cAMP is unavailable. To the best of our knowledge, only two strategies have been designed to deplete Sobetirome of cAMP. One of them consists of introducing a phosphodiesterase in permeabilized cells to digest cAMP [9]. The other applies a genetically encoded buffer (named cAMP sponge) based on the high-affinity cAMP-binding sites of the regulatory subunit of the cAMP-dependent protein kinase (PKA) to deplete cAMP from the cytosol of transfected cells [33]. Exocytosis of the acrosome, or acrosome reaction (AR), is a regulated secretion event that sperm must undergo in order to fertilize the egg. Each sperm contains a single, very large, and electron dense acrosome that, in response to exocytosis inducers, first swells to contact the cell membrane, second becomes attached to and fuses with it and third sheds entirely, together with the portion of plasma membrane that surrounds it. These events are coupled to complex calcium signaling. Typically, AR triggers evoke a transient influx of calcium into the cytosol through plasma membrane channels; this event initiates complex signaling cascades that lead to intracellular calcium mobilization. Emptying of intracellular reservoires causes the opening of store operated calcium channels in the plasma membrane that elicits a sustained calcium signal. An influx of external calcium into sperm driven by calcium ionophores resembles the latter and induces the AR. So does the addition of CaCl2 to cells with their plasma membrane permeabilized with toxins such as streptolysin O (SLO [1], [26], [68]) or perfringolysin O [42]. In the first part of this paper, we describe a permeable, recombinant version of the cAMP sponge that we introduced into live human sperm through a mechanism known as protein transduction. Thanks to this tool, we demonstrate that AR triggers require cAMP to induce swelling of the acrosome and to activate pathways that mobilize calcium from the intra-acrosomal pool. In particular, relevant for sperm biologists are the observations that progesterone requires cAMP to achieve the AR and the elucidation of the signal transduction pathways involved in the exocytotic response to this hormone. In certain neurons, neuroendocrine, and exocrine acinar cells, cAMP potentiates calcium-dependent exocytosis, whereas in various non-neuronal cells, cAMP is the principal trigger of regulated secretion [10], [54], [58]. Until not too long ago, it was thought that the effects of cAMP in regulated exocytosis were mediated by PKA through phosphorylation of relevant substrates. More recently, we learned that cAMP modulates exocytosis via PKA-dependent and/or PKA-independent mechanisms. The latter are mediated by guanine nucleotide exchange factors (GEFs) activated by cAMP (Epacs, reviewed by [6], [12], [22], [32], [53]). Many processes of stimulus-secretion coupling are regulated by both Epac and PKA; how do they cross talk is still an open question. In sperm, the Epac-selective cAMP analog 8-(p-chlorophenylthio)-2 prime-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP) induces exocytosis per se, while the PKA-selective analog N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP) does not [8]. Furthermore, calcium-induced AR in SLO-permeabilized human sperm is mediated by cAMP/Epac and independent of PKA [9].