Roles of intracellular cyclic AMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa

Abstract

It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca²⁺ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.

Fig. 1.

Possible segment-specific cAMP signal transductions regulating transition of the flagellar movement pattern to hyperactivation in boar spermatozoa. ADCY10, adenylyl cyclase 10; cAMP, cyclic adenosine 3´,5´-monophosphate; PKA, protein kinase A (cAMP-dependent protein kinase); pS/pT, serine/threonine phosphorylation; PP, protein phosphatase; TK, tyrosine kinase; SYK, spleen tyrosine kinase; PTP, protein tyrosine phosphatase; pY, tyrosine phosphorylation; PLC, phospholipase C; PIP₂, phosphatidylinositol 4,5-bisphosphate; DAG, 1,2-diacylglycerol; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; PKC, protein kinase C; PI3K, phosphatidylinositol-3 kinase; PDK1, phosphoinositide-dependent protein kinase-1; CaM, calmodulin.

Fig. 2.

Detection of AMP-activated protein kinase (AMPK) in boar spermatozoa. For the immunodetection of the AMPK2α catalytic subunit protein, washed spermatozoa were incubated with cBiMPS and an inhibitor for PKA H-89 at 38.5 C. In panel A (Western blotting: a representative of three replicates), aliquots of each sperm suspension (1 × 10⁶ spermatozoa/lane) were recovered immediately before and after incubation, used for SDS-PAGE and transblotting to the membranes, treated with a diluted rabbit anti-phospho-AMPK α polyclonal antibody [Thr172, an active form, anti-phospho-AMPKα (pT172), 1:1,000-2,000] or with a diluted rabbit anti-phospho-AGC kinase substrate polyclonal antibody (anti-phospho-AGC kinase substrate, 1:5,000) and then treated with HRP-conjugated donkey anti-rabbit immunoglobulin polyclonal antibody (1:1,000–1:2,000). In addition, the tubulin was detected solely with HRP-conjugated mouse anti-α-tubulin monoclonal antibody (1:10,000, anti-α-tubulin) as loading controls. In panel B (indirect immunofluorescence: a representative of three replicates), aliquots of each sperm suspension (5 × 10⁵ spermatozoa/preparation) were recovered immediately after incubation for 180 min and treated with paraformaldehyde and Triton X-100. The fixed and membrane-permeated spermatozoa were blocked with bovine serum albumin (BSA) in PBS (blocking buffer), treated overnight at 4 C with the anti-phospho-AMPKα antibody (1:50) or a blocking buffer without the primary antibody (no primary antibody) and then treated with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulins (1:50). In each set of photographs, the upper photograph is from differential interference contrast microscopy, and the bottom photograph is from immunofluorescence microscopy.