Fig 1.
Gene Expression of SNAP isoforms and NSF in mouse oocytes.
A. Detection of SNAPs mRNA. Lanes 1–3 and 10–11 were amplified using α-SNAP primers, lanes 4–6 using β-SNAP primers, and lanes 7–9 using γ-SNAP primers under the same experimental procedure. Agarose gel was stained with ethidium bromide. Lanes: M, molecular weight marker; 1,4, and 7,mouse brain; 2, 5, and 8, GV oocytes; 3, 6, and 9, PCR negative controls without cDNA; 10 and 11, RT-PCR negative controls without reverse transcriptase (RT) for brain and oocytes samples, respectively. B. Detection of NSF mRNA. All lanes were amplified using NSF primers and agarose gel was stained with SYBR safe. Lanes: M, molecular weight marker; 1,mouse brain; 2, mouse oocytes; 3, PCR negative control without cDNA; 4 and 5, RT-PCR negative control without RT for brain and oocytes samples, respectively. Shown are images representative of 3 independent experiments.
Fig 2.
Detection of α-SNAP, γ-SNAP and NSF by Western blot.
A. Left, Upper panels: Inmunoblot of α-SNAP: Protein extracts from equal numbers (150) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl2) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 1.25 μg) and recombinant His6-α-SNAP (α SNAP rec, 5 ng). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading. Lower panels: Immunoblot using anti-α-SNAP antibody preabsorbed with full lenght α-SNAP recombinant protein (α SNAP pb). Right, densitometry analysis of Western blots for α-SNAP (mean ± SEM, n = 4) showing α-SNAP protein expression level (α SNAP/β Tub ratio) relative to GV expression, set as 1. B. Left, Upper panels: Inmunoblot of γ-SNAP: Protein extracts from equal numbers (300) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl2) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 6 μg) and recombinant thrombine cleaved γ-SNAP-GST (γ SNAP rec, 0.2 μg). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading. Lower panels: Immunoblot using anti-γ-SNAP antibody preabsorbed with γ-SNAP control peptide (γ SNAP pb). Right, densitometry analysis of Western blots for γ-SNAP (mean ± SEM, n = 3) showing γ-SNAP protein expression level (γ SNAP/β Tub ratio) relative to GV expression, set as 1. C. Left, Upper panels: Inmunoblot of NSF: Protein extracts from equal numbers (200) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10 mM strontium chloride (SrCl2) were separated on a 15% SDS-PAGE gel. Positive controls: mouse brain (Brain, 3,5 μg) and recombinant His6-NSF (NSF rec, 75 ng). Immunoblot of β- actin (β Act) was performed as a control of protein loading. Lower panels: Immunoblot using anti-NSF antibody preabsorbed with NSF control peptide (NSF pb). Right, densitometry analysis of Western blots for NSF (mean ± SEM, n = 3) showing NSF protein expression level (NSF/ β Act ratio) relative to GV expression, set as 1. In all panels MW protein standards (x103) are indicated on the right and primary antibodies, on the left.
Fig 3.
α-SNAP, γ-SNAP, and NSF localization during meiotic maturation and MII oocytes activation.
α-SNAP (left panel), γ-SNAP (middle panel) and NSF (right panel) were immunodetected at different stages: GV-intact oocytes (GV), MII oocytes(MII), parthenogenetic activated MII oocytes with 10mM strontium chloride during 1 or 7 h post activation (SrCl2 (1h) and (7h), respectively), and two pronucleus (2PN) embryos after in vitro fertilization. Green, positive staining for primary α-/β-SNAP, γ-SNAP or NSF antibody detected by secondary antibodies conjugated to DyLight 488; red, cortical granules stained with LCA-Rhodamine; blue, DNA labeled with Hoechst 3342.A-C, panels show the fluorescence intensity profiles for α-SNAP, γ-SNAP and NSF (green) and cortical granules stained with LCA-Rhodamine (red). Fluorescence intensities were measured along dashed lines traced in each panel. The intensities of α-SNAP, γ-SNAP and NSF are indicated by green lines, and the intensity of cortical granules is indicated by red lines. Scale bar: 20 μm. D-F, cortical and cytoplasmic patterns of protein distribution. Cortical region was defined as the region of 10 μm thickness from the oocyte plasma membrane towards the oocyte centre. Those cells in which fluorescence decay at 10 μm were considered to present a cortical staining, and those cells which present high fluorescence at 10um towards the center and beyond were considered to present cytoplasmic staining. Percentage analysis was assessed. Number of analyzed cells for α-SNAP, γ-SNAP and NSF were, respectively: GV = 85,51,32; MII = 138,37,35; MII SrCl2 1h = 101, 36, 30; MII SrCl2 7h = 35, 27, 33; IVF = 13, 6, 9.
Fig 4.
Functional assay of cortical granule exocytosis.
A. Cortical granule exocytosis (CGE) was triggered bySrCl2 (10-40mM), A23187 (10–30 μM) or in vitro fertilization (IVF, cells fixed after 6 h co-incubation), and cortical granules (CG) were quantified. a, representative confocal images of oocytes subjected to different treatments, fixed and stained with FITC-LCA to label cortical granules. Scale bar: 20 μm. b, histogram shows CG density (% CG density/100 μm2) relative to untreated group (control) set as 100%. Data are shown as mean ± SEM from at least 3 independent experiments; numbers in parentheses below bars represents total number of activated MII oocytes (SrCl2, A23187) or embryos (IVF). ***, p ≤ 0.001 (Tukey’s test). B. a, schematic diagram depicting mounting of an MII oocyte partially compressed under a coverslip in order to provide a large flat field of cortical granules (purple dashed lines). b, confocal images on flat optical field, showing the biggest area containing cortical granules. Once image threshold was adjusted, this remained constant for all images within the same experiment. CG were computer-assisted counted from at least 4 non overlapping equal areas of 100 μm2 (red squares), and CG density/100 μm2 was determined as the mean of the counts. C. Cortical granules density is similar regardless the area size containing cortical granules and total fluorescence intensity. To show this, 58 serial pictures were taken with a spacingof 0.39μmin thezaxis (Z axis). CG density and fluorescence intensity measured as arbitrary intensity units (A.I.U) were plotted for each slice (Avg. Int./slice). D. Representative images for each slice (a-l) showed in C. F. Threshold of the images (a-l) shown in D, used for quantification of cortical granules. E. Tridimensional reconstruction of the MII oocyte from D panel showing cortical granules distribution. Top image, front view; bottom image, side view. Yellow and green colors represent images from slide and coverslip, respectively.
Fig 5.
Effect of recombinant wild type α-SNAP, mutant α-SNAP L294A, NEM, wild type NSFand mutant NSF D1EQ and on CGE.
A. MII oocytes were microinjected with wild type α-SNAP (αS WT) or mutant α-SNAP L294A (αS L294A) prior to CGE activation with 30 mM strontium chloride (SrCl2). Left, representative images of MII oocytes stained with FITC-LCA to label cortical granules. Right, histogram showing % CG density/100 μm2 relative to untreated group (Control) set as 100%. Data are shown as mean ± SEM from at least 5 independent experiments; numbers in parentheses represent total number of MII oocytes. ***, values compared to control, p ≤ 0.001;‡ ‡ ‡, values compared to SrCl2, p ≤ 0.001. Scale bar: 20 μm. B. MII oocytes were incubated in presence of different NEM concentrations prior to CGE activated by strontium chloride (SrCl2). Histogram shows % CG density/100 μm2 relative to untreated group (Control) set as 100%. Data are shown as mean ± SEM from at least 5 independent experiments.C. MII oocytes were microinjected with wild type NSF (NSF WT) or mutant NSF D1EQ (NSF D1EQ) prior to CGE activation with 30 mM strontium chloride (SrCl2). Left, representative images of MII oocytes stained with FITC-LCA to label cortical granules. Right, histogram showing % CG density/100 μm2 relative to untreated group (Control) set as 100%. Data are shown as mean ± SEM from at least 5 independent experiments; numbers in parentheses below bars represent total number of oocytes. ***, values compared to control, p ≤ 0.001;‡ ‡ ‡, values compared to SrCl2, p ≤ 0.001. Scale bar: 20 μm.
Fig 6.
Effect of microinjection of α-SNAP, γ-SNAP and NSF antibodies on CGE.
MII oocytes were microinjected with α-SNAP (A), γ-SNAP (B), and NSF (C) antibody and CGE was triggered with 30mM strontium chloride (SrCl2). Mouse and rabbit IgGs were microinjected as isotype controls at the same concentrations. Left: a-d, representative images of cells stained with FITC-LCA to label CG. e-h, representative images of oocytes subjected to inmunofluorescence protocol, were primary antibody was omitted, and secondary antibodies: Cy3 donkey anti-mouse in A, and Cy3 donkey anti-rabbit in B and C, were used to confirm proper microinjection of antibody or control isotype (red). Right,Histogram showing % CG density/100 μm2 relative to untreated group (Control) set as 100%. Data are shown as mean ± SEM from at least 4 independent experiments; numbers in parentheses below bars represent total number of oocytes. ***, values compared to control, p ≤ 0.001;‡ ‡ ‡, values compared to SrCl2, p ≤ 0.001. Scale bar: 20 μm.
Fig 7.
Working model for α-SNAP and NSF in CGE.
When MII oocyte is activated by fertilization or by parthenogenesis, α-SNAP (α SNAP) and N-ethilmaleimide sensitive factor (NSF) disassemble cis-SNARE complexes (red, in cortical granules; green and purple, in plasma membrane) making their components available to allow the formation of trans-SNARE complex (red-purple and red-green complexes) for membrane fusion and secretion. p. membrane: plasma membrane.