Acrosome formation-associated factor is involved in fertilization.
OBJECTIVE
To investigate the effects of a novel acrosome formation-associated factor (Afaf) on fertilization by its regulation of acrosomal exocytosis and endosomal trafficking.


METHODS
Controlled laboratory study.


METHODS
Institution-affiliated state key laboratory.


METHODS
ICR mice.


METHODS
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.


METHODS
Number of penetrated sperms, in vitro fertilization rate. Acrosomal exocytosis index, relative Tf fluorescence.


RESULTS
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.


CONCLUSIONS
Acrosome formation-associated factor might play an important role in membrane trafficking during acrosome formation and participate in fertilization.
Hu
Xiao-Qian
XQ
The State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China.
Ji
Shao-Yang
SY
Li
Yin-Chuan
YC
Fan
Cui-Hong
CH
Cai
Huan
H
Yang
Jun-Ling
JL
Zhang
Chun-Ping
CP
Chen
Min
M
Pan
Zhi-Fang
ZF
Hu
Zhao-Yuan
ZY
Gao
Fei
F
Liu
Yi-Xun
YX
D000177:Acrosome / Q000378:metabolism*; D020101:Acrosome Reaction; D000818:Animals; D001692:Biological Transport; D020013:Calcium Signaling; D011992:Endosomes / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D034622:RNA Interference; D013084:Sperm-Ovum Interactions; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
C512717:Eqtn protein, mouse; D008565:Membrane Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D014168:Transferrin