Induced Synaptotagmin (SYT) Protein Family Binds to Membrane SNAP25 to Facilitate Vesicle Secretion in Murine Granulosa/Cumulus Cells during Ovulation

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During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors via the exocytosis system. Exocytosis is controlled by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex consisting of proteins residing in the vesicle membrane and the plasma membrane. SNAP25 localizes on the plasma membrane whereas synaptotagmins (SYT) reside in secretion vesicle membranes. After Ca²⁺-induced binding of SNAP25 and SYT, the release of vesicle-contained factors is triggered. Therefore, we sought to determine if induction of Syt mRNA and binding of SYT to SNAP25 in granulosa and cumulus cells were involved in exocytosis during ovulation. After hCG stimulation, the expression levels of Syt1, Syt2, Syt4 and Syt6 were induced markedly in granulosa cells, whereas the levels of Syt3 and Syt7 mRNAs did not change dramatically. The levels of SYT1 protein were also increased 4 hr after hCG injection and remained increased until 16 hr post-hCG. Immunofluorescence using an SYT1 specific antibody showed increased staining for SYT1 in the granulosa cells and in the cumulus cells of hCG-primed mice. Additionally, the SYT1 antibody coimmunoprecipitated SNAP25 in granulosa cell lysates collected at 8-16 hr post-hCG. From these results, we conclude that the interactions of SYT1 and SNAP25 facilitate the exocytosis system in granulosa cells and cumulus cells during ovulation.