Acetylcholine induces Ca²⁺ oscillations via m3/m4 muscarinic receptors in the mouse oocyte

Changes in intracellular Ca²⁺ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca²⁺ release from the endoplasmic reticulum (ER) via InsP₃ receptors (InsP₃R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (ml to m4) are involved in the generation of Ca²⁺ oscillations in native mouse oocytes. ACh (10 μM) elicited regular Ca ²⁺ oscillations similar to those induced by sperm in their temporal characteristics. The Ca²⁺ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794±119 to 2023±755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca²⁺ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca²⁺ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca ²⁺ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an InsP₃-receptor antagonist (xestospongin C), confirming that ACh generates Ca²⁺ oscillations via the PLC-InsP ₃ (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca²⁺ oscillations mainly via the PI pathway in mouse oocytes.