1. Ganglioside-enriched microdomains define an oolemma that is functionally polarized with respect to fertilizability in the mouse 
Jonathan Van Blerkom, Sarah Zimmermann 
Reproductive BioMedicine Online 
Volume 33, Issue 4, Pages (October 2016)
DOI: /j.rbmo
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2. Figure 1
(A–E) Differential interference contrast images of the same second metaphase mouse oocyte during the first 10 min after insemination at low concentration (around 200 sperm/ml). A region of the oolemma (between arrows A–D) where the first polar (PB) has formed is non-permissive for sperm docking, the boundary of which is outlined by white dots in (E); (F) mouse sperm pre-loaded with 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) (arrowhead, lower right) shows no docking in the non-permissive region of the oolemma that lies between the white arrows at 10 min after insemination (oocytes in E and F are from the same cohort). Magnification bars (µm) in A and D also apply to B, C and E respectively; (G–J) in this 8-s post-insemination sequence (2000 sperm/ml), the sperm indicated by a white arrow in panel G after first contacting the oolemma (t = 0 s) progressively glides over (panels H, I) and exits the membrane (panel J) in the non-permissive domain. Scale bar in panel G applies to panels H, I and J. In contrast, numerous spermatoza (SP) have docked in the permissive domain on the left-hand side of the oocyte. The black arrows are positional markers showing that the oocyte itself has not moved during imaging; (K–M) a different pattern of interaction between gamete membranes is shown in panels K to M that is characterized by a darting motion of the head (asterisk) in the non-permissive zone until it reached the permissive oolemma, where it docks. The white arrow and bar in each panel are positional markers showing that changes in sperm position were not caused by rotation of the oocyte (insemination concentration, around 2000/ml). Black scale bar in M also applies to K, L and N; (N) Docking of a DAPI stained sperm (SP) at a nearly 90° angle (similar to the docked sperm above asterisk in panel M) on an oolemma stained with Alexa Fluor 488-conjugated beta-subunit of cholera toxin (CTB) showing an apparent focal loss of GM1 microdomains at the docking site (insemination concentration, around 200/ml). The occurrence of typical CTB-positive microdomains in the permissive oolemma is shown at higher magnification in the insert, with the specific domains indicated by the arrow extending from panel N (scale bar is in µm).
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3. Figure 2
(A) The absence of detectable GM1 Alexa Fluor 555-conjugated cholera toxin B (CTB) fluorescence identifies a perinuclear region of the mouse oolemma at second metaphase (MII) that is deficient in the ganglioside GM1 and is unable to dock sperm. This ‘non-permissive’ region occurs between the arrows in panels A1–A3, where panel A1 (8-µm midline section) is an oocyte stained while intact; A2 is the same oocyte as in A1 imaged after removal of the pellucida (ZP) with acidic Tyrode's solution; panel A3 (fully compiled section series) is similar to the oocyte A2 but imaged after fixation. The white asterisk in each panel denotes the perinuclear domain where the first polar body would reside. Scale bar in A1 applies to A2 and A3; (B) shows sperm docking on the oolemma of a MII oocyte stained with Alexa Fluor 594-conjugated cholera toxin B (red fluorescence), following incubation with around 1000 DAPI-stained sperm/mL (blue fluorescence) for 5 min. This image is a laser-scanning confocal microscopy (LSCM) projection of one hemisphere of the oocyte; (C) shows the submicron-to-micron-sized GM1 microdomains (punctate green fluorescence) observed throughout the docking permissive region of the oolemma stained with Alexa Fluor 488-CTB. The insert in C is a structured illumination microscopic image showing the typical density of GM1 microdomains reported by beta-subunit of cholera toxin (CTB) in an approximate 45-µm2 area of the docking permissive oolemma; (D and E) show the interaction between sperm head (blue fluorescence, N) and GM1 microdomains (GM1) for an intact sperm (D) or an acrosome-reacted sperm (E) where docking occurred at different angles. In D similar GM1 microdomains (right hand arrow and arrowhead at lower-left) without or with an adjacent sperm docked are indicated; (F) the absence of detectable fluorophore-conjugated CTB fluorescence after polar body formation (asterisk denotes position of first polar body which was lost during zona removal) marks the non-permissive oolemma; (G) breaks in an otherwise continuous pattern of oolemmal CTB fluorescence occur (arrows) above the meiotic chromosomes as the MII meiotic spindle develops (c, chromosomes, panel G); (H) when the non-permissive region is fully formed after polar body abstriction, the transition between the region of oolemma that can dock sperm (SP, left of asterisk in panel H) and is unable to dock sperm (non-permissive domain, right of asterisk) is typically an abrupt one. The insert in panel H is a representative example of a single CTB-positive GM1 microdomain that is characteristic of those populating the permissive oolemma, and resembles the insert in Figure 1N; (I) The magnification is of the same oocyte as in panel H, increased by around 20% to better demonstrate the transition; (J) in a low percentage of MII oocytes examined, the first polar body (PB1) was CTB positive and the non-permissive CTB-deficient zone (region between arrows in panel J) was larger than normal, suggesting an atypical process of polar body formation and abstriction; (K1–3) the evolution of a CTB-negative domain during the early stages of polar body extrusion observed by time-lapse LSCM of CTB stained oocytes before (t = 0) and during the first 5 min of first polar body formation (asterisk), which is outlined by the white dots in panels K2 and K3. The arrows are positional markers of GM1 microdomains at varying distances from the site of polar body emergence. Scale bar in K1 applies to K2 and K3; (L–N) The distribution of cholesterol detected by filipin III in a MII oocyte is shown in the compiled image of a 20-µm LSCM section series (L) and at higher magnification (M, N). A strong fluorescent fillipin III signal indicates normal cholesterol levels in the permissive oolemma (arrow extending from oolemma in panel L to oolemma in panel N) and a relatively weak signal in the non-permissive oolemma (arrow extending from region between asterisks to oolemma in panel N) indicating a reduced cholesterol content. The spherical FIII-positive structure beneath the permissive oolemma in panel L and at higher magnification in panel M is likely a cluster of cisternae of the smooth surfaced endoplasmic reticulum (SER) Scale bar in M applies to N. (O and P) show the typical distribution of annexin 2 (A2) immunofluorescence in the oolemma (fully compiled LSCM section series) before (O) and after (P) MII is completed Scale bar in O applies to P; (Q) when double stained for GM1 with Alexa Flour-555-conjugated CTB (red fluorescence) and anti-A2 (A2, green fluorescence), both identifiable in the permissive domain (black asterisk, panels O and P) and shown at higher magnification in Q (1 µm LSCM section); (R) At MII, only A2 was detected in the non-permissive oolemma (white asterisk in panel P) and at a much reduced density (arrow extending from panel P to R, A2 green fluorescence); (S) is a projection of a fully compiled LSCM section series showing the typical distribution of CD9 immunofluorescence between the GV and MII stages; (T and U) individual sections through the midline of a GV and MII stage oocyte, respectively (red fluorescence = CD9; blue fluorescence = DAPI stained DNA surrounding oocyte nucleolus, n; oocyte nucleus = GV); (V) a similar continuous distribution of oolemmal FOLR4 fluorescence occurred at MII as observed by LSCM, which included the non-permissive domain and first polar body membrane (arrow) (W and X): colloidal gold immunostaining for transmission electron microscopy showed FOLR4 localized specifically to oolemma in the non-permissive region (arrow W; ×9200) and on microvilli in the permissive region (arrow X; ×9200) in the same oocyte. Scale bar in W applies to X; (Y) shows a focal thickening of actin microfilaments (arrow) beneath the first polar body in an MII oocyte stained for F-actin with fluorophore-conjugated phalloidin in which the relative fluorescence intensity was increased by about 20% during LSCM image acquisition.
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4. Figure 3
With the exception of the brightfield images in panels A,H,N,O and Z, all other images were taken by laser-scanning confocal microscopy (LSCM). The insemination concentration used in studies shown in panels A to Q was about 700–1000 motile sperm/ml; (A–C) the response of the oolemma to sperm docking. The asterisks in panels A and H denote the approximate mid-point of the non-permissive domain. The arrows that extend from two sperm docked at the permissive region in panel A to panels B and C show a corresponding focal reduction in Alexa Fluor 488 conjugated cholera toxin B (CTB) fluorescence, indicating ganglioside GM1, 15 s after docking was first observed in these zona-free second metaphase (MII) oocytes; (D-G) shows a docked sperm (arrow) in a zona-free oocyte fixed at 20 s after docking (similar to the one in panel A) that has been labelled with filipin III (FIII), an autofluorescent cholesterol reporter (arrow, panel E). When compared with relative FIII fluorescent intensity in the adjacent oolemma (asterisk), an apparent reduction in cholesterol occurs where the sperm has docked. The subplasmalemmal network of actin microfilaments at the site of docking is markedly attenuated or disrupted within the first 60 s of sperm docking as indicated by reduced or absent anti-F actin immunofluorescence (arrow, 3F, 30 s after docking, sperm mechanically displaced); (G) is a 5-µm LSCM section at a docking site in an oocyte similar to the one in panel F showing an apparent reduction in the subplasmalemmal actin microfilament density at the site of sperm docking (4′, 6-Diamidino-2-phenylindole dihydrochloride stained, blue fluorescent sperm nucleus at 30 s) after staining with Alexa-Fluor 546-phalloidin; (H-M) a reduction in blue FIII fluorescence indicating a reduction in cholesterol content was clearly evident at the sites of sperm docking. The sperm indicated by a blue arrow in H was docked at nearly 90o angle to the oolemma and was easily displaced by a gentle stream of medium and fixed within 30 s. This arrow extends into panel I and shows reduced FIII blue fluorescence at this site after displacement. The sperm indicated by a green arrow in panel H was also displaced by 30 s in the same oocyte, and the region denoted by the green arrow in J shows that annexin II immunofluorescence was undetectable by LSCM at this docking site. No apparent reduction in the relative immunofluorescence intensity of CD9 (K, 1 min) or FOLR4 (L, 30 s) was evident after sperm docking (white arrow extending from sperm in panel H to panel L, SP), or for several minutes after stable attachment was observed, e.g. FOLR4, panel M, 5 min; (N–Q) are two hemispheres of the same oocyte stained with FIII at 45 s after docking showing the permissive domain indicated by docked sperm (asterisks in N) and their absence in the non-permissive domain (indicated by a white arrow in O). The arrows extending from panels N to P show the local diminution of membrane cholesterol reported by blue FIII fluorescent signal resulting from a single (right hand side) and multiple, closely spaced docked sperm (left hand side). The normal relative intensity of FIII fluorescence in the permissive domain where no docking occurred is indicated by the white arrow (FIII) in panels P and Q. Scale bar in P applies to Q. The black arrow in panel Q marks the apparent transition from the permissive to the non-permissive oolemma (right of black arrow); (R–V) show the effects of cholesterol chelation with methyl-beta-cyclodextrin (MβCD) on the organization of GM1 lipid raft microdomains reported by fluorophore-conjugated CTB (R–T) and sperm docking (U and V). R and S are projections of a fully compiled LSCM section series of an untreated oocyte stained with Alexa Fluor 488-CTB and one stained with Alexa Fluor 555-CTB after a 5-h exposure to MβCD, respectively. T is a cross-section of the oocyte shown in panel S showing scant CTB fluorescence (arrows). Scale bar in S applies to T. After 7 h in the presence of MβCD, CTB fluorescence is sparse (asterisk, red fluorescence in U) and after a 30-min insemination at around 1000 sperm/ml, the few sperm that docked did so at the residual GM1 microdomains reported by CTB, e.g., black arrow in panel U. The region between the white arrows above the MII spindle is the non-permissive domain where CTB fluorescence is normally undetectable. Figure V is a higher magnification image of a sperm (SP) docked at a GM1 positive microdomain (GM1) in the oocyte show in panel U. Exposure to MβCD under the conditions used had no apparent effects on the organization or relative intensity of anti-CD9 (W) or anti-FOLR4 (X) immuno-fluorescence. The effects of cholesterol chelation on CTB fluorescence were reversible after culture in normal medium, as shown by the return of normal oolemmal CTB staining fluorescence (red oolemmal fluorescence, panel Y), except in the non-permissive domain above the MII spindle (arrow panel Y); (Z) after insemination at around 1000 sperm/ml, numerous sperm (SP) were docked on the oolemma within the first few minutes.
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5. Figure 4
Staining with the mitochondrial-specific potentiometric dye JC1 shows the localization of hyperpolarized, orange fluorescent, J-aggregate positive organelles in the sub-plasmalemmal cytoplasm of an second metaphase (MII) mouse oocyte. Hypopolarized mitochondria fluoresce green. (A) is a typical untreated JC-1 stained MII oocyte showing hyperpolarized, J-aggregate positive mitochondria populate the subplasmalemmal cytoplasm in the docking permissive oolemma (small arrow), but are hypopolarized in the non-permissive oolemma associated with first polar body (PB1, region between large arrows); (B) shows hyperpolarized subplasmalemmal mitochondria in the permissive region (arrow) and hypopolarized mitochondria in the non-permissive region (between asterisks) after 7 h of culture in the presence of the cholesterol chelator methyl-beta-cyclodextrin; (C–D) staining with Mitotracker Green-FM (C, 8 µm laser-scanning confocal microscopy section taken at the midline of the interface between the polar body and oolemma) and transmission electron microscopy (D, 6000×) confirmed the presence of mitochondria (M) in subplasmalemmal cytoplasm associated with the non-permissive region of the oolemma; (E) shows a completely circumferential population of hyper-polarized mitochondria (arrows) in the subplasmalemmal cytoplasm of a typical human MII oocyte where the entire oolemma is permissive for sperm docking, including the region beneath the first polar body (PB1).
Reproductive BioMedicine Online  , DOI: ( /j.rbmo )
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