Martoriati A et al. (2023), Follicular cells protect Xenopus oocyte from ab...

XB-ART-59827

J Biol Chem 2023 Jun 22;:104950. doi: 10.1016/j.jbc.2023.104950.

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Follicular cells protect Xenopus oocyte from abnormal maturation via integrin signaling downregulation and O-GlcNAcylation control.

Martoriati A , Molinaro C , Marchand G , Fliniaux I , Marin M , Bodart JF , Takeda-Uchimura Y , Lefebvre T , Dehennaut V , Cailliau K .

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Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the EGF receptor and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin β1 or Grb7 binding to the EGF receptor. Additionally, we determined that oocytes surrounded by their follicular cells expressing EGFR/Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrinβ1/EGF receptor complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase, OGA), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of O-GlcNAc transferase, OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT/OGA duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression.

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Genes referenced: egf grb7 ogt

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	Figure 1. Grb7 triggers an accelerated germinal vesicle breakdown (GVBD) in oocytes deprotected from their layer of surrounding follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR). The human EGFR was expressed in stage VI Xenopus oocytes surrounded by follicular cells (FCs) and stage VI oocytes deprotected from their follicular cells (DEs, defolliculated oocytes). One hour before EGF stimulation (5 nM) or not in control (Ct), Grb7 was microinjected in the equatorial region at concentrations of 5, 10, 20, or 100 ng. A, Hoechst 33258 staining of the surrounding layer of oocytes with follicular cells (left) or defolliculated (right); the scale bars represent 1000 μm. B, EGFR immunoprecipitations followed by Western blots with anti-EGFR and anti–phospho-tyrosine (P-Tyr). C, example of GVBD of stage VI Xenopus oocytes; the scale bars represent 1000 μm. D and E, the GVBD attesting for the progression from prophase I to metaphase II (maturation) was scored 12 h after stimulation by EGF or not (Ct) in FC and DE oocytes. F, the GVBD50 representing the time required to obtain half of the response of FC and DE oocytes to undergo GVBD was determined. Experiments were performed on 10 to 20 oocytes from three different females. Statistical significance was accepted in DE and FC for ∗p < 0.05.
	Figure 2. In oocytes deprotected from their follicular cells, the epidermal growth factor receptor (EGFR)–EGF signaling complex recruits integrin β1, phosphorylated Grb7, but lacks OGT. Stage VI Xenopus oocytes, with surrounding follicular cells (FC) or defolliculated (DE), expressing EGFR were injected with 20 ng of Grb7 or not 1 h before EGF stimulation (5 nM) or left unstimulated. A, after protein normalization, Western blots were performed on FC or DE oocytes, and isolated follicular cells (IFs) with anti-integrin β1, anti-FAK, and anti-actin antibodies. B, immunoprecipitations were performed with anti-EGFR or anti-GST-Grb7 antibodies, and Western blots were performed with antibodies against EGFR, phospho-tyrosine (P-Tyr), O-GlcNAcylation (O-GlcNAc), GST (Grb7-GST), O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), FAK, and integrin β1. Control (Ct) was an immunoprecipitation performed with anti-GST-Grb7 on FC cells expressing EGFR and injected with Grb7. C, sWGA pulldowns were performed with or without competition with 0.5 M free GlcNAc and followed by Western blot analysis with anti-O-GlcNAcylation (O-GlcNAc) or anti-GST-Grb7 antibodies. D, after immunoprecipitations with anti-integrin β1, Western blots were performed with anti-EGFR, -GST (Grb7-GST), -OGT, and -integrin β1 antibodies. Ct: control as in B. E, scheme showing Grb7 domains and competitive Grb7 peptides: proline-rich (PR), RalGEF/AF6, or Ras associated like (RA), Pleckstrin homology (PH), phospho-tyrosine interacting region PIR (BPS, between PH and SH2); and Src homology 2 (SH2). F–H, Grb7 peptides Y188, Y338, both peptides (G7-YY), control mismatched peptide (G7-MP), or anti-FAK antibodies were microinjected with Grb7 (20 ng), 1 h before EGF stimulation. Ct: control as in B. Experiments were performed on 25 oocytes and three different females. FAK, focal adhesion kinase; GST, glutathione-S-transferase; sWGA, succinylated wheat germ agglutinin.
	Figure 3. In defolliculated oocytes (DEs) expressing epidermal growth factor receptor (EGFR)–Grb7, integrin inhibition rescues germinal vesicle breakdown (GVBD). Stage VI Xenopus oocytes deprotected from follicular cells (D), and expressing EGFR were stimulated or not by EGF (5 nM), 1 h after microinjection with Grb7 (20 ng), with or without SH2 domain of Grb7 60 ng, and before an incubation or not with 500 nM of integrin inhibitor echistatin (Echist) for 12 h. A, the GVBD attesting for meiosis progression (maturation) was scored. B, the GVBD50 representing the time of half-responsive DE oocytes to undergo GVBD was determined. C and D, immunoprecipitations were performed with anti-EGFR or anti-GST-Grb7 antibodies. Western blots were performed with antibodies against EGFR, phospho-tyrosine (P-Tyr), O-GlcNAcylation (O-GlcNAc), GST (Grb7-GST), O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), and integrin β1. Experiments were performed on three different females. Statistical significance against DE was accepted for ∗p < 0.05 and ∗∗p < 0.01. GST, glutathione-S-transferase.
	Figure 4. In folliculated oocytes, integrin activation triggers germinal vesicle breakdown (GVBD) acceleration, and neither OGT nor O-GlcNAcylated Grb7 are recruited in the epidermal growth factor receptor (EGFR) complex. Stage VI Xenopus oocytes displaying their follicular cells layer (FC) and expressing EGFR were injected or not 1 h before EGF stimulation (5 nM) with Grb7 (20 ng) and incubated or not with 10 μM of pyrintegrin activator (Int Act) or left unstimulated. A, the GVBD attesting for meiosis progression was scored. B, the GVBD50 (time of half responsive FC oocytes to undergo GVBD) was determined. C, immunodetections were performed after immunoprecipitations with anti-EGFR or anti-GST-Grb7 antibodies and followed by Western blots with antibodies against EGFR, GST (Grb7-GST), O-linked N-acetylglucosamine [GlcNAc] transferase) (OGT), integrin β1, and O-GlcNAcylation (O-GlcNAc). Experiments were repeated on 20 oocytes and three females. Statistical significance against FC was accepted for ∗p < 0.05. GST, glutathione-S-transferase.
	Figure 5. Abnormal spindle formation occurs in defolliculated epidermal growth factor receptor (EGFR)–Grb7-expressing oocytes that display germinal vesicle breakdown (GVBD) acceleration.Xenopus defolliculated oocytes (DEs) and folliculated oocytes (FCs) expressing EGFR were injected or not with Grb7 (20 ng) and incubated or not with 500 nM of integrin inhibitor echistatin (Echist) for DE or with Grb7 (20 ng) with or without 10 μM of pyrintegrin activator (Int Act) for FC, 1 h before EGF stimulation (5 nM); controls were left unstimulated. Naïve oocytes were treated with progesterone (PG). Oocytes were fixed, dehydrated, and paraffin-embedded before sections (7 μm) were stained with nuclear red to detect nuclei and chromosomes and picroindigocarmine to reveal cytoplasmic structures. The normal, abnormal, or absent spindles were scored. Cortical pigment (CP), plasma membrane (PM), and vitelline platelet (VP); the arrow points to the chromosomes on the spindle, and the scale bar represents 35 μm. Statistical significance against DE or FC was accepted for ∗∗∗∗p < 0.001.
	Figure 6. Favoring O-GlcNAcylation by O-GlcNAcase (OGA) inhibition lowers GVBD50in defolliculated oocytes (DEs). Xenopus DE and folliculated oocytes (FCs), expressing epidermal growth factor receptor (EGFR), were microinjected with Grb7 (15 ng) or not, incubated or not with 50 or 100 μM OGA inhibitor Thiamet G (ThG), 1 h before EGF stimulation (5 nM). DE oocytes were submitted to integrin inhibitor echistatin 500 nM (Echist). A and B, the germinal vesicle breakdowns (GVBDs) were scored. C and D, GVBD50 (time of half-responsive FC oocytes to undergo GVBD) were determined. Results were obtained on 20 oocytes from three different females. Statistical significance was accepted for ∗∗p < 0.01 and ∗∗∗∗p < 0.001. GVBD50, half-maximal time attesting for a maturation.
	Figure 7. Favoring O-GlcNAcylation by O-GlcNAcase (OGA) inhibition increases abnormal spindles in defolliculated oocytes (DEs).Xenopus DEs and folliculated oocytes (FCs) expressing epidermal growth factor receptor (EGFR) were microinjected with Grb7 (15 ng) or not, incubated or not with 100 μM OGA inhibitor Thiamet G (ThG), 1 h before epidermal growth factor (EGF) stimulation (5 nM). DEs were submitted to 500 nM of integrin inhibitor echistatin (Echist). A and B, the normal, abnormal, or absent spindles were scored in oocytes fixed, dehydrated, and paraffin-embedded on sections (7 μm) stained with nuclear red (nuclei and chromosome detection) and picroindigocarmine (cytoplasmic structure detection). C, Grb7-GST and EGFR immunoprecipitations were realized and followed by Western blotting with anti-O-GlcNAcylation (O-GlcNAc), anti-GST (Grb7-GST), and anti-OGT antibodies. D, sWGA pulldowns were performed before Western blotting with anti-O-GlcNAcylation (O-GlcNAc) and anti-OGT antibodies. Results were obtained on 10 to 20 oocytes from three different females. Statistical significance was accepted for ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001. GST, glutathione-S-transferase; sWGA, succinylated wheat germ agglutinin.
	Figure 8. Inhibition of O-GlcNAcylation using O-GlcNAc transferase (OGT) inhibitor inhibits oocyte germinal vesicle breakdown (GVBD). Defolliculated (DE) and folliculated oocytes (FCs) expressing epidermal growth factor (EGF) receptor (EGFR) microinjected with Grb7 (15 ng) or not were incubated or not with 15 and 30 μM OGT inhibitor OSMI-4, 1 h before EGF stimulation (5 nM). FCs were treated or not with 10 μM of pyrintegrin activator (Int Act). A and B, the GVBDs were scored during 12 h. C and D, GVBD50 (time of half-responsive FC oocytes to undergo GVBD) were determined. Experiments were repeated on 20 oocytes from two to three Xenopus females. Statistical significance against DE or FC was accepted for ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001.
	Figure 9. Inhibition of O-GlcNAcylation using an O-GlcNAc transferase (OGT) inhibitor increases abnormal oocyte spindles. Defolliculated (DE) and folliculated oocytes (FC) expressing epidermal growth factor (EGF) receptor (EGFR), microinjected with Grb7 (15ng) or not were microinjected or not with 15 μM OGT inhibitor OSMI-4, 1 h before EGF stimulation (5 nM). FCs were treated with or without 10 μM of pyrintegrin activator (Int Act). A and B, normal, abnormal, and absent spindles were scored on semithin sections (7 μm) stained with nuclear red and picroindigocarmine in oocytes. C, Grb7-GST or EGFR immunoprecipitations were realized and followed by Western blots with anti-O-GlcNAcylation (O-GlcNAc), anti-GST (Grb7-GST), and anti-OGT antibodies. D, sWGA pulldowns were followed by Western blots with anti-O-GlcNAcylation (O-GlcNAc) and anti-OGT antibodies. Experiments were repeated on 10 to 20 oocytes from two to three Xenopus females. Statistical significance against DE or FC was accepted for ∗∗∗p < 0.005 and ∗∗∗∗p < 0.001. GST, glutathione-S-transferase; sWGA, succinylated wheat germ agglutinin.
	Figure 10. Model for Xenopus oocyte protection by follicular cells from abnormal maturation (G2–M transition) triggered by an epidermal growth factor (EGF) receptor (EGFR)/integrin–FAK complex and the adaptor Grb7 phosphorylation.A, in defolliculated oocytes (DE), EGF addition to EGFR-expressing oocytes triggers the tyrosine kinase receptors autophosphorylation (P) and the recruitment of effectors including adaptors Grb2 and Grb7. Activated EGFRs form heterocomplexes with integrin–FAK, leading to Grb7 phosphorylation on two tyrosine residues Y188 and Y338. Consequently, these signaling pathways favor a rapid meiotic transition from prophase I to metaphase II (maturation) and abnormal spindle formation that does not localize at the oocyte plasma membrane. B, in follicular cells (FCs), the formation of a multicomplex between EGFR–EGF and integrin–FAK is prevented by the layer of surrounding follicular cells and their tight contact with the oocyte membrane. Phosphorylated EGFRs recruit OGT, Grb7 is O-GlcNAcylated, and abnormal oocyte phenotype is avoided. The oocyte OGT–OGA balance avoids unwanted phenotypic defaults. In A and B, inhibition of protein O-GlcNAcylation (OGT inhibitor) increases phenotypic defaults. Folliculated oocytes are more insensitive toward protein O-GlcNAcylation removal (OGA inhibition). Oocyte endogenous proteins are in red, and heterologous expressed proteins are in blue. FAK, focal adhesion kinase; OGT, O-GlcNAc transferase.

References [+] :

Abbassi, Epidermal growth factor receptor signaling uncouples germ cells from the somatic follicular compartment at ovulation. 2021, Pubmed