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. 2020 Nov 2;18(11):e3000901. doi: 10.1371/journal.pbio.3000901

Fig 4. mPR is internalized through clathrin-dependent endocytosis in response to P4.

Fig 4

(A) Oocyte maturation in response to P4 after 4 hours pre-incubation with incremental concentrations of different compounds as indicated. Con: untreated oocytes (mean ± SEM; n = 3–5 donor females). (B) Activation of the MAPK and Plk cascades in the different indicated conditions. Tub is shown as a loading control. (C) GVBD normalized to untreated oocytes (Con) in oocytes overexpressing WT or DN forms of dynamin, Rab5, caveolin, or ARF6 following overnight treatment with P4. For the overexpression, oocytes were injected with 20 ng RNA/oocytes for all the clones. (mean ± SEM; n = 3–9 donor females per condition). (D) Percent of the total mPR population at the plasma membrane (PM) in oocytes and eggs at the indicated conditions. Oocytes were injected with 40 ng SNAP25Δ20 (mean ± SEM; n = 7–27 oocytes per condition, from 3 donor females). (E-F) Oocyte maturation in response to P4 or SNAP25Δ20 (Δ20) (40 ng/oocyte) following knockdown of mPRβ (mPR-AS) (mean ± SEM; n = 4 donor females) (E) or APPL1 (APPL1-AS) (F) (mean ± SEM; n = 3 donor females). **p < 0.01, ***p < 0.001. Refer to S1 Data file. ARF6, ADP-ribosylation factor 6; DN, dominant-negative; Egg, P4-matured eggs; GVBD, germinal vesicle breakdown; MAPK, mitogen-activated protein kinase; mPR, membrane progesterone receptor; ns, not significant; Ooc, untreated oocytes; Pit-C, Pitstop2 control; Pit2, Pitstop2; P4, progesterone; Rab5, Ras-related protein Rab-5A; SNAP25Δ20, dominant-negative synaptosome associate protein 25; Tub, tubulin; WT, wild-type.