MAIA, Fc receptor-like 3, supersedes JUNO as IZUMO1 receptor during human fertilization - PubMed (original) (raw)

. 2022 Sep 9;8(36):eabn0047.

doi: 10.1126/sciadv.abn0047. Epub 2022 Sep 7.

Michaela Frolikova 1, Lukas Ded 1, Jiri Cerny 2, Pavla Postlerova 1 3, Veronika Palenikova 1, Ondrej Simonik 1, Zuzana Nahacka 4, Krystof Basus 1, Eliska Valaskova 1, Radek Machan 5, Allan Pacey 6, Zuzana Holubcova 7 8, Pavel Koubek 9, Zuzana Ezrova 4, Soojin Park 10, Ruiwu Liu 11, Raghavendran Partha 12, Nathan Clark 13, Jiri Neuzil 4 14, Masahito Ikawa 10, Kent Erickson 15, Kit S Lam 11, Harry Moore 16, Katerina Komrskova 1 17

Affiliations

MAIA, Fc receptor-like 3, supersedes JUNO as IZUMO1 receptor during human fertilization

Jana Vondrakova et al. Sci Adv. 2022.

Abstract

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor-like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on human oolemma and played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

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Figures

Fig. 1.

Fig. 1.. OBOC assay for human sperm binding and FcRL3 ERC with gamete recognition proteins.

(A) OBOC library structure (c,

d

-cysteine; X, 19

l

-eukaryotic amino acids except

l

-cysteine). (B) Sperm-OBOC assay. (C) Sperm-bead binding (inset: human egg). (D) Sperm-bead binding (higher magnification). (E) Peptide hits and proportions assigned to functions. (F) Sperm (five donors) consistently bound to four specific bead aliquots incubated under specific conditions (fig. S1). Bead 16 hit for FcRL3. (G) Sperm bound to resynthesized cAMWNEDc peptide–beads during incubation. (H) Sperm-bound bead (higher magnification, bottom left corner; representative sperm highlighted by green cross) and “naked” bead (*). (I) IZUMO1 on human sperm. (J) Acrosomal antigen 18.6 on human sperm (control). (K) Antibody inhibition of sperm binding to resynthesized beads (n = repetitions) (16). (L) Antibody inhibition of sperm fusion to zona-free hamster eggs (n = animals, each of 10 to 15 oocytes). (M) Gamete interaction proteins on sperm (IZUMO1, SPACA6, DCST1, TMEM95, DCST2, and FIMP) and egg (JUNO, FcRL1, and FcRL3) experienced similar gene evolutionary histories, as shown by ERC sequence analysis. Color intensity reflects the strength of ERC value for that pair of genes. NA, not available.

Fig. 2.

Fig. 2.. MAIA immunolocalization and interaction in human oocytes.

(A to C) MAIA on human mature metaphase II (MII) stage oocyte (red), β-tubulin (green), nuclear counterstain (Hoechst 33342), confocal microscopy; (A) single plane, (B) a maximum intensity projection visualization of MAIA localization and (C) differential interference contrast (DIC). (D to F) Colocalization between protein pairs (D) MAIA-JUNO, (E) MAIA-CD9, and (F) JUNO-CD9 on human MII oocyte explained by preferential localization of the proteins in, or close to, the cell membrane (fig. S2). (G to I) PLA on human MII oocyte for protein pairs (G) MAIA-JUNO, (H) MAIA-CD9, and (I) JUNO-CD9 with appropriate negative control (NC) α-tubulin–β1 integrin and positive control (PC) α-tubulin–β-tubulin. (J) Pearson correlation coefficient for the protein pairs corresponding to colocalization analysis (fig. S2). (K) Representative transmission electron microscopy image of MAIA (big dots and green arrows) and JUNO (small dots and magenta arrows) localized on the microvilli/oolemma of MII oocyte.

Fig. 3.

Fig. 3.. Sperm binding assessment in relation to cell fusion.

(A) Immunostaining of MAIA (green) and JUNO (red) cotransfected CHO cells; (B) with fused human sperm head (white arrow); nuclear counterstain (Hoechst); (C) merged with DIC; (D) Imaris Surface Render analysis (fig. S4 and movie S4). (E) Representative image of sperm captured by lifetime confocal microscopy (green) displaying a range of tail beating reflecting the status of sperm-cell fusion (movie S5); white arrow, fusing sperm (slow tail beating); white asterisks, nonfusing sperm (fast tail beating); white tips (fused sperm, static tail without beating). (F to I) AI object identification. (F) Representative image of captured tail beating pattern used for AI training. (G) Example of novel neuronal network training masks for human sperm tail beating pattern quantification. (H) Quantification of sperm tail beating amplitude calculated from the degree angle as low, medium, and high. (I) Representative charts of degree angle change. (J and K) Representative snapshot of a video recording of sperm kinematic parameter analysis assessed by CASA with phase contrast (yellow cross, static sperm–bound sperm with low tail beating; blue track, medium-progressive swimming sperm; green track, rapid-swimming sperm; red track, rapid-progressive swimming sperm); (J) MAIA-JUNO cotransfected HEK293T (HEK) cells (movie S6); (K) nontransfected HEK cells (movie S7). (L) Quantification of video recordings (_n_cotransfected = 17 and _n_nontransfected = 14) of bound sperm (yellow cross) assessed by CASA, ****P ≤ 0.0001.

Fig. 4.

Fig. 4.. Sperm binding/fusion with MAIA and JUNO cotransfected cells.

(A to C) Membrane disruption and sperm head fusion (white arrows) with cotransfected HEK293T (HEK) by MAIA (purple), JUNO (red), and cell membrane–incorporated GFP-tagged farnesylated K-Ras (green) (movie S8). (D) Quantification of sperm binding to cotransfected HEK293T (HEK) with MAIA, JUNO, and MAIA + JUNO. (E to G) Immunofluorescence data postprocessing. (E) Acrosome-reacted sperm (white arrow) equatorial segment stained with PNA (yellow) fused with cell membrane (green); the apical part of the sperm head within the cell cytoplasm is shown in the top right corner (visualized by Imaris). (F and G) Sperm-HEK penetration and fusion 3D model (Imaris), MAIA (purple), JUNO (red), and AF488-phalloidine–stained F-actin (green) (movie S9). (H) Sperm-HEK binding decrease after sperm-cAMWNEDc coincubation. (H and D) Image Xpress screening system; ImageJ/FIJI; error bars (SEM); EV, empty vector; J, JUNO; and M, MAIA. (I1 and I2) 3D visualization of the sperm head within the cytoplasm of MAIA-JUNO cotransfected HEK cell using FIB-scanning electron microscopy. (I1) Consecutive FIB-scanning electron microscopy sections showing fusion (red arrows) between the plasma membrane of MAIA-JUNO cotransfected HEK cell and the inner acrosomal membrane (IAM) of the sperm head around the equatorial segment. Overview of an internalized sperm head (top) and detail of the membrane fusion (bottom) demarcated by dotted rectangle (above). Spacing between frames, 250 nm. (I2) 3D volume rendering of the whole FIB-scanning electron microscopy dataset (_x_-_y_-z projection side view) showing partial exposure of the sperm head to the cytoplasm of the HEK cell and the site of membrane fusion (red arrows) (movie S10).

Fig. 5.

Fig. 5.. MAIA-IZUMO1 protein-protein interaction and sperm-cell GFP fusion assay.

(A) Recombinant MAIA and JUNO protein localization in HEK plasmatic membrane; both proteins are shown with two isoforms (two bands) because of protein glycosylation; α-tubulin was used as a loading control for cytosolic fraction; Na/K adenosine triphosphatase (ATPase) was used as a reference protein for membrane fraction. (B and C) Co-IP of (B) MAIA-JUNO and (C) MAIA-IZUMO1 using cotransfected HEK (B) without or (C) with sperm; ISO control, isotype IgG control antibody (IgG light chain, 25 kDa; IgG heavy chain, 50 kDa). (D to G) PLA assay for MAIA-IZUMO1 on JUNO + MAIA cotransfected HEK with sperm; the positive signal is designated exclusively to the point of sperm head–cell attachment (white asterisks) (fig. S5). (D and E) MAIA-IZUMO1; (F) positive control: α-tubulin–β-tubulin; (G) negative control: MAIA–β-tubulin. (H to J) GFP fusion assay of sperm with HEK cells. (H) Number of GFP-positive cells (n) per well, nontransfected (NT), MAIA, and MAIA + JUNO cotransfected HEK cells with fused GFP-transfected sperm; *P ≤ 0.05. (I) Polymerase chain reaction (PCR) showing GFP in transfected ejaculated (Ej) and acrosome-reacted (AR) sperm, nontransfected, and marker (M). (J) Representative fluorescent images showing analyzed groups (H and I), positively transfected cells (white arrows) fused with sperm with visible tail are shown in the bottom right corner, and fluorescent signal is merged with bright field. ns, not significant.

Fig. 6.

Fig. 6.. Outline of human gamete binding.

(A) Fertilizing sperm display oscillatory motility tethered to the oolemma due to the initial JUNO/IZUMO1 interaction. (B) IZUMO1 dimerization triggers the transfer of JUNO from the egg into the sperm membrane. (C) Tight binding of MAIA in the created JUNO/IZUMO1 binding pocket. (D) MAIA conformational change to extracellular Fc domains, leading to close membrane proximity enabling gamete fusion with the loss of sperm motility (figs. S8 and S9). The question mark (C and D) may represent an unknown or CD9 protein, as proposed in fig. S9 (A1 and A2).

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