1. Acrosome formation-associated factor is involved in fertilization
Xiao-Qian Hu, M.D., Ph.D., Shao-Yang Ji, M.S., Yin-Chuan Li, Ph.D., Cui-Hong Fan, M.S., Huan Cai, M.S., Jun-Ling Yang, M.S., Chun- Ping Zhang, M.S., Min Chen, Ph.D., Zhi-Fang Pan, M.S., Zhao-Yuan Hu, B.S., Fei Gao, Ph.D., Yi-Xun Liu, Ph.D. 
Fertility and Sterility 
Volume 93, Issue 5, Pages (March 2010)
DOI: /j.fertnstert
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2. Figure 1
The inhibitory effect of anti-Afaf IgG on sperm penetration to ZP of prestained oocytes. The oocytes were preloaded with Hoechst before addition of the sperms and the indicated antibody inhibitors. The cells were then fixed with the glutaraldehyde, and the Hochest stained sperms (blue) in eggs were observed and countered under a fluorescence microscope. (A) Bright (left panel)- and dark (right panel)-field images of oocytes incubated with PBS (top), FITC-labeled control IgG (middle) and anti-Afaf IgG antibody (bottom). (B) The total number of bound and/or fused sperms was determined by the ZP binding assay. In each experiment, six to eight occytes were counted and three independent assays were performed. ∗Denotes significant difference (P<.05) by one-way analysis of variance followed by least significant difference (LSD) test.
Fertility and Sterility  , DOI: ( /j.fertnstert )
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3. Figure 2
The inhibitory effect of anti-Afaf IgG on in vitro fertilization (IVF) rate. IVF was performed according to the standard protocol as described in Materials and Methods. The mean and SD of three independent experiments were plotted against the IVF fertilization rate. In each experiment, at least 100 to 150 oocytes were used and the results were analyzed by two researchers independently (24). ∗Denotes significant difference (P<.01) from the PBS control at 6 and 42 hours.
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4. Figure 3
Acrosome formation-associated factor was required for Ca2+-triggered AE by acting upstream of acrosomal Ca2+ efflux. (A) Schematic diagram was used to demonstrate the treatment protocol of the sperms as described before (5). (B) A: In the control group without stimulation, Afaf was present on the acrosome. B: In the presence of 10 μM Ca2+ (Ca2+) stimulation, only a few sperms showed Afaf staining. C: In the presence of anti-Afaf and 10 μM Ca2+ (anti-Afaf–Ca2+), AE was dramatically inhibited. D(a): In the presence of NP followed by anti-Afaf and then Ca2+ and hν (NP–anti-Afaf–Ca2+–hν), AE was inhibited dramatically. D(b): In the presence of NP followed by Ca2+ and then anti-Afaf and hν (NP–Ca2+–anti–Afaf–hν), no inhibition of AE was observed. For each experiment, the data were normalized by subtracting the number of reacted spermatozoa in the negative control from all values, and the result was expressed as a percentage of the AE observed in the positive control. The acrosomal status of 200 spermatozoa was determined in randomly selected fields under a fluorescence microscope. (C) Representative staining of Afaf protein (green) and Hoechst staining (blue) is showed. ×400 magnification.
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5. Figure 4
Acrosome formation-associated factor had an interaction with SNAP25 in HeLa cells. The HeLa cells were simultaneously transfected with Afaf–myc and SNAP25–GFP plasmids. Afaf–myc expression is shown in purple, whereas SNAP25 is shown in green. Hochest was used for nuclei staining (blue). (A) Acrosome formation-associated factor and SNAP25 demonstrated significant overlap in expression throughout the cytoplasm in the HeLa cell (merge). They share a high degree of colocalization in the perinuclear region, but Afaf had a specific distribution in the cell membrane. (B) Acrosome formation-associated factor formed a complex with SNAP25. The protein complexes were analyzed using HeLa cell lysates for coimmunoprecipitation analysis. The GFP antibody was used to pull down the complex of SNAP25 and its potential interacting proteins. The anti-Myc antidody was used to confirm existence of Afaf–myc in the pull-down complex. Preprecipitated proteins from each treatment were severed as the input control. As Afaf has two transcripts, two bands in the input were identified by anti-myc antibody, and only the longer one was pulled down with SNAP25 by the anti-GFP antibody.
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6. Figure 5
Expression of BoNT/E LC interrupted Afaf distribution in PC12 cells. (A) PC12 cells were transfected with plasmids encoding GFP–SNAP25 wt or mutant D179K (green) and Afaf-myc (red) with or without BoNT/E LC as indicated. The distribution of GFP–SNAP25 (wt) in a perinuclear RE pool was altered by coexpression of BoNT/E (arrowheads). However, the perinuclear RE distribution of GFP-SNAP25 D179K was unaltered by coexpression of the toxin. The similar redistribution of Afaf–myc with SNAP25 wt or mutant was observed. The nuclei were stained with Hochest (blue). (B) Representative Western blot shows the effect of BoNT/E LC (light chain of botulinum neurotoxin E) expression on endogenous SNAP25 (marked end, top) and on GFP–SNAP25 (bottom) as described before (17). GFP–SNAP25 D179K, a mutated SNAP25, which was resistant to BoNT/E cleavage, was used here to show the size of uncleaved GFP–SNAP25 band. The asterisk denotes the bands of cleaved SNAP25 by BoNT/E.
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7. Figure 6
Acrosome formation-associated factor affected trafficking of Tf in endocytosis assay. (A) MiRNA-based interference of Afaf was prepared by inserting the Afaf-miR-1/3 into pcDNA6.2-GW/EmGFP-miR. The expression of Afaf miRNA and GFP mRNA was under the control of CMV promoter. (B) Western blotting showing knockdown effect of Afaf–miRNA-1 and Afaf–miRNA-3. HeLa cells were transfected with Afaf–Myc alone, Afaf–myc, and negative control miRNA (neg-miRNA) or Afaf–miRNA-1 or Afaf–miRNA-3 simultaneously. GFP signal confirmed the expression of miRNA. β-Actin served as the internal control. (C) Afaf decreased fluorescence of Tf in endocytosis assay. The representative pictures with uptake for 15 minutes were shown: (a) The endocytosis of Tf in the cells transfected with mock vector; (b) the endocytosis of Tf with Afaf–Myc; and (c) the merge picture with Hochest staining. The red denotes the vesicles of Tf, and the green represents Afaf–Myc. Arrowhead of (c) indicated the suppression of Afaf ovexpression on Tf trafficking. (D) Afaf–miRNA-3 rescued the inhibitory effect on endocytosis. HeLa cells were transfected with Afaf–Myc and neg-miRNA or Afaf–miRNA-3 simultaneously. The uptake of Tf for 5 minutes (upper panel) and 10 minutes (lower panel) was examined, respectively. The HeLa cells transfected with the neg-miRNA were shown (a, b, e, and f). Arrowheads of (a, e), indicated the inhibitory effect of Afaf–Myc on Tf's trafficking under this condition. The target miRNA were introduced as shown in (c, d, g, and h). Arrowheads of (c and g) showed the rescued effect and are similar to a normal endocytosis. Red color indicated the vesicles of Tf. The GFP proteins pointed the cells that expressed miRNA. Hochest stained the nucleus. (E) The fluorescence intensity of Tf staining was quantified and normalized to the cellular area in at least 40 randomly selected cells as described before (37). The bars of mock and Afaf represent the Tf uptake of the cells transfected with mock or Afaf plasmid, and the bars of Afaf plus negative miRNA or miRNA-3 mean the Tf uptake in the Afaf overexpressed cells with the irrelevant or Afaf-specific miRNA, respectively. Values are means ± SD, with asterisk corresponding to P<.01 for comparison with mock-transfected by t test. The mean fluorescence of mock-transfected cells was set as 1.
Fertility and Sterility  , DOI: ( /j.fertnstert )
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