Acrosome formation-associated factor is involved in fertilization

ObjectiveTo investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking.DesignControlled laboratory study.SettingInstitution-affiliated state key laboratory.SubjectsICR mice.Intervention(s)Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afaf's action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake.Main Outcome Measure(s)Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence.Result(s)The Afaf antibodies were capable of significantly inhibiting sperm penetration of the eggs, therefore reducing the rate of in vitro fertilization. Acrosome formation-associated factor was involved in calcium-triggered AE by acting upstream of the calcium efflux from the acrosome inside. Acrosome formation-associated factor might exert an interaction with SNAP25, which is a crucial component in both exocytosis and endosomal trafficking. Acrosome formation-associated factor was also involved in the endocytic pathway by down-regulating Tf endocytosis in the HeLa cells, and the miRNA-mediated RNAi could rescue this alternation induced by Afaf.Conclusion(s)Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization. To investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking. Controlled laboratory study. Institution-affiliated state key laboratory. ICR mice. Sperm penetration assay and in vitro fertilization experiment were performed to study the effects of the Afaf antibody on acrosome reaction and fertilization. Acrosome exocytosis (AE) with streptolysin O (SLO) permeabilization was conducted to test the Afaf's action in calcium events. Colocalization and coimmunoprecipitation was done to determine the interaction between Afaf and SNAP25 (synaptosome-associated protein of 25,000 daltons). Transferrin (Tf) uptake assay was performed to demonstrate the impact of Afaf on endosomal pathway. RNAi was used to rescue the inhibition of Afaf on Tf uptake. Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence. The Afaf antibodies were capable of significantly inhibiting sperm penetration of the eggs, therefore reducing the rate of in vitro fertilization. Acrosome formation-associated factor was involved in calcium-triggered AE by acting upstream of the calcium efflux from the acrosome inside. Acrosome formation-associated factor might exert an interaction with SNAP25, which is a crucial component in both exocytosis and endosomal trafficking. Acrosome formation-associated factor was also involved in the endocytic pathway by down-regulating Tf endocytosis in the HeLa cells, and the miRNA-mediated RNAi could rescue this alternation induced by Afaf. Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization.