Sperm attachment and penetration competence in the human oocyte: a possible aetiology of fertilization failure involving the organization of oolemmal lipid raft microdomains influenced by the ΔΨm of subplasmalemmal mitochondria  Jonathan Van Blerkom, Kyle Caltrider  Reproductive BioMedicine Online  Volume 27, Issue 6, Pages 690-701 (December 2013) DOI: 10.1016/j.rbmo.2013.09.011 Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Sperm on oolemma in unpenetrated oocytes and GM1 phenotypes. (A) An example of the typical unfertilized MII human oocyte examined in this study. (B–G) Detection of spermatozoa (arrows) on oolemma by bright field (B), phase contrast (C), differential interference contrast optics (A, E, and F) and DAPI fluorescence (D and G); SP=sperm head; Arrow, ZP=zona pellucida. (H–J) Projection of fully compiled SLCM section series showing punctate and uniformly distributed Alexa-488-CTB fluorescence reporting the ganglioside GM1 in an intact (H) and zona-free (I) MII human oocyte and the pattern of GM1 lipid raft microdomain distribution at four locations (J 1–4) in an MII oocyte fixed after staining with CTB and imaged by SLCM; the GM1 phenotypes are suggested to be consistent with fertilization of the MII human oocyte. (K–Q) Representative SLCM images of GM1 microdomain distributions reported by CTB in oocytes with spermatozoa (arrows) on oolemma at GM1-positive sites but which failed to penetrate; arrow head indicates a portion of the MII chromosomes; asterisks denote regions where GM1 microdomains reported by fluorophore-conjugated CTB were sparse or below levels of detection and where no sperm docking or attachment was observed. Bars=10μm (A, E, H–Q), 15μm (B), 5μm (C, F, and G). Reproductive BioMedicine Online 2013 27, 690-701DOI: (10.1016/j.rbmo.2013.09.011) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Interactions between male and female human gametes with respect to GM1 and CD9 lipid raft microdomains. (A–D) SLCM images of sections (1μm) showing interactions between the sperm membrane (N, DAPI-stained nucleus) and GM1 microdomains on the oolemma (arrows) of a zona-free human oocyte within seconds of contact: 10s (A), 20s (B), 30s (C) and 40s (D). (E–K) GM1 microdomain organization and densities that appear to be consistent (E, F, and I) or inconsistent (G, H, J, and K) with sperm docking or post-attachment penetration of the oolemma. F and G are high-magnification views of regions (highlighted in E and H, respectively) of comparatively high and low GM1 lipid raft microdomain density, respectively that in surface area that may be related to the ability of the spermatozoa to remain docked to the on the oolemma. I, J and K are higher magnification views of the density of GM1 microdomains (arrows) that a spermatozoon might encounter on the oolemma that are consistent (I) or inconsistent (J and K), respectively, with fertilization; K shows a GM1 macrodomain or island (arrow in inset). (L–O) Epifluorescence (L) and SLCM (M–O) of CD9 immunofluorescence alone (L, M, N) or after co-staining for GM1 microdomains (O) detected by fluorophore-conjugated CTB. The asterisks in N denote areas of the oolemma where CD9 immunofluorescence was undetectable. Bars=4μm (A, C, and L), 3μm (B), 5μm (D) 10μm (E and M), 1μm (G and J), 8μm (H) 0.5μm (I), 1μm (H), 0.4μm (K) and 6μm (H). Reproductive BioMedicine Online 2013 27, 690-701DOI: (10.1016/j.rbmo.2013.09.011) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Relationship between GM1 and CD9 phenotypes and ΔΨm in the subplasmalemmal cytoplasm and fertilization competence in meiotically mature human oocytes. (A) Projection of a fully compiled section series of a human MII oocyte dual stained for CD9 (red, arrows) and GM1 (green; arrows indicate small GM1 islands. (B–F) GM1 macrodomains against a background of uniform CD9 immunofluorescence at different positions during the full rotation of a dual-stained unfertilized MII human oocyte; These GM1 phenotypes are inconsistent with sperm attachment or penetration. (G–S) Typical phenotypes of unfertilized MII human oocytes strain with the potentiometric fluorescent probe JC1 and fluorophore-conjugated CTB and examined for ΔΨm and GM1 distributions by light microscopy (G), conventional epifluorescence (H, J, M, P, R) and SLCM (I, K, L, N, O, Q, S); G–I, J–L, M and N, P and Q, and R and S show the same oocytes. Patterns of ΔΨm distribution (orange, J-aggregate fluorescence, JA) could be normal (H) or abnormal (J, M, P, R, arrows) in mitochondria in the subplasmalemmal cytoplasm in MII human oocytes. The asterisk in J denotes a focal zone of high-potential (J-aggregate-positive) mitochondria where the corresponding oolemma also showed focal staining for GM1 microdomains (asterisk, L); a similar situation occurred for the zone of JA-positive mitochondria between the arrows in J, where the corresponding cytoplasm was found to be GM1 positive. The arrow(s) in K, M–P and S denote GM1 microdomains detected by positive CTB fluorescence. The differential intensity of CTB fluorescence parallels differential ΔΨm in the corresponding subplasmalemmal cytoplasm. For some oocytes, there was no detectable subplasmalemmal J-aggregate fluorescence (arrow in R) and the corresponding oolemma was largely devoid of GM1, as indicated by CTB fluorescence (S). Bars=8μm (A and L), 10μm (B–K, M, P, R, and S), 6μm (N) 3μm (O) and 9 μm (Q). Reproductive BioMedicine Online 2013 27, 690-701DOI: (10.1016/j.rbmo.2013.09.011) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions