ABHD2 is required for P4-induced oocyte maturation.

A. Schematic representation of the signaling events downstream of mPRβ after progesterone (P4), leading to oocyte maturation. The implication of GPR185 is also depicted. B. mRNAs transcripts levels of mPRβ, ABHD2.S, ABHD2.LS and Xenopus Ornithine decarboxylase (xODC) in oocytes using the Cycle threshold (Ct) generated from real time PCR. C. ABHD2 knockdown experiments. Oocytes were injected with specific ABHD2 antisense (AS) oligonucleotides and incubated at 18 °C for 24 hours. RNAs were prepared and analyzed by RT-PCR to determine the efficacy of ABHD2 knockdown as compared to naïve oocytes and mPRβ RNAs levels. Data are expressed as relative RNAs levels of ANHD2 and mPRβ mRNA transcripts after normalizing to xODC mRNA levels as a house keeping gene. D. Representative WB of ABHD2 in naive and oocytes over-expressing ABHD2.L or ABHD2.S, following ABDH2 antisense (AS) oligonucleotide injection. Tubulin is shown as a loading control. E. Oocyte maturation measured as the levels of % GVBD in oocytes injected with control antisense (Ctrl AS) or ABHD2 antisense (AS) with or without mRNA to overexpress ABHD2.S (AS+WT) and normalized to P4-treated naïve oocytes condition (Naive), (mean + SEM; n = 7, ordinary one-way ANOVA). F. Representative WB of MAPK, Plk1 and Cdc2 phosphorylation from untreated oocytes, eggs matured by overnight (O/N) treatment with P4, oocytes injected with control antisense (Ctrl AS) or ABHD2 antisense (AS) with or without mRNA to overexpress mPRβ. Tubulin is shown as a loading control. G. Oocyte maturation measured as the levels of % GVBD in oocytes injected with control antisense (Ctrl AS) or ABHD2 antisense (AS) with or without mRNA to overexpress mPRβ and normalized to P4-treated naïve oocytes condition (Naive) (mean + SEM; n = 3, ordinary one-way ANOVA). H. Schematic representation of ABHD2.S different domains, including the acyltransferase and lipase motifs, aligned and compared to ABHD2.L. I/K. Oocyte maturation measured as the levels of % GVBD in oocytes injected with control antisense (Ctrl AS) or ABHD2 antisense (AS) with or without mRNA to overexpress ABHD2.S wild type (AS+WT) and the different ABHD2.S mutants as indicated on the panel, and normalized to P4-treated naïve oocytes condition (Naive) (mean + SEM; n = 3, ordinary one-way ANOVA). J/L. Representative WB of MAPK, Plk1 and Cdc2 phosphorylation from untreated oocytes, eggs matured by overnight (O/N) treatment with P4, oocytes injected with control antisense (Ctrl AS) or ABHD2 antisense (AS) with or without mRNA to overexpress the different ABHD2.S clones as indicated on the panel. Tubulin is shown as a loading control.

Metabolomics

A/B. Naïve oocytes and oocytes injected with either mPR or ABHD2 antisense were treated for 5 or 30 min with P4. Per each condition, 5 samples replicates were collected containing 10 pooled oocytes. the experiment was repeated 3 times using 3 different female frogs. The heatmaps data show changes in sphingolipids and glycerophospholipids (total for sub-pathway when available, otherwise the average of individual metabolites) at 5 min (A) and 30 min (B) after treatment with progesterone. C. Heatmap of metabolites exhibiting significant changes in single naïve oocyte where 10 individual oocytes per condition were analyzed, before and 30 min after P4. D. Levels of EETs and HETEs before and after P4 treatment in single oocytes. Naïve oocytes were incubated with either ethanol or P4 10-5M for 30 min. 20 single oocytes per condition were collected and used for analysis. 5-oxoETE and 8(9)-EET were detected in 1 or 2 samples respectively, so they were not included in the statistical analyses although both were lower following P4 treatment. E. Levels of prostaglandins before and after P4 treatment at 5 min and 30 min time point in naïve oocytes. Per each condition, 10 samples replicates were collected containing 10 pooled oocytes each. The following eicosanoids were not detected in either group (D and E): 6kPGF1α, PGF2α, PGE2, TXB2, PGD2, PGA2, PGJ2, 15-deoxyPGJ2, 12-HHTrE, 11-dehydro TXB2, LTB4, LTC4, LTD4, LTE4, 20-hydroxy LTB4, 20-carboxy LTB4, 5(6)-DiHETEs, LXA4, LXB4, 5(6)-EET, 5(6)-DiHET, 8(9)-DiHET, 11(12)-DiHET, 14(15)-DHET, 20-HETE. Similar results were obtained from individual oocyte samples (see Table 4). For Fig. 3C the individual metabolites are listed in Supplemental Fig. 2E. For Fig. 3E the data are normalized to the PG levels at time zero. The raw abundance of individual PG species is shown in Supplemental Fig. 2F. F. Cartoon summarizing the changes in sphingolipids and glycerophospholipids metabolites after progesterone treatment. Increase or decrease in metabolites levels is noted with an upward or a downward red arrow respectively. Chemical inhibitors used for pharmacological studies are also highlighted on the cartoon by a red blunt arrow.

Pharmacological inhibitors potency

Pharmacological validation of the metabolomics findings – the glycerophospholipids pathway.

A/E/I. Concentration-dependent effect of different inhibitors on oocytes maturation. Oocytes were pre-treated for 2 hours with vehicle or with increasing concentrations of specific chemicals inhibitors (as stated on top of each graph), followed by overnight treatment with P4 at 10-5M. Oocyte maturation was measured as the levels of % GVBD normalized to vehicle P4-treated oocytes and plotted in function of the different chemicals’ concentrations. IC50 of each chemical compound was calculated using the nonlinear regression curve fit method. (mean + SEM; n = 3 for each chemical compound experiment). D/F/H/J/K. Effect of chemicals compounds on oocytes maturation at low P4 concentration. Oocytes were pre-treated for 2 hours with the vehicle or with the highest possible concentrations for each specific chemicals’ inhibitor (as stated on the graph) or activator (mastoparan, panel D), followed by overnight treatment with P4 at 10-7M. Oocyte maturation was measured as the levels of % GVBD normalized to the control oocytes condition (treated with vehicle) (mean + SEM; n = 3 for each chemical compound experiment, unpaired t-test). B/C/G/L. Representative WB of MAPK, Plk1 and Cdc2 phosphorylation from untreated oocytes, oocytes pretreated with vehicle for 2 hours then matured overnight (O/N) by P4 at 10-7M (eggs), and oocytes pretreated for 2 hours with the highest possible concentrations for each specific chemicals’ inhibitor then treated O/N with P4 10-7M. Tubulin is shown as a loading control.

S1P signaling and oocyte maturation.

A. mRNAs transcripts levels of S1PR2, S1PR3, and ODC in oocytes and spleen in oocytes measured as the Cycle threshold (Ct) generated from real time PCR. B-C. S1PR3 knockdown experiments. Oocytes were injected with control antisense (Ctrl AS) or specific S1PR3 antisense oligonucleotides and incubated at 18 °C for 24 hours. RNAs and proteins extracts were prepared and analyzed by RT-PCR and WB to determine the efficacy of S1PR3 knockdown as compared to control oocytes (Ctrl AS). B. Histogram showing the relative RNAs levels of S1PR3 mRNA transcripts after normalizing to Xenopus Ornithine decarboxylase (xODC) mRNA levels as a house keeping gene. C. left panel, representative WB comparing S1PR3 protein levels between naïve, Ctrl AS and S1PR3 AS injected oocytes. Tubulin is shown as a loading control. Reft panel, Quantification of S1PR3 protein levels in Ctrl AS and S1PR3 AS injected oocytes. For each condition, the ratio of S1PR3 protein levels over tubulin was first calculated then the ratios were normalized to the Ctrl AS condition (mean + SEM; n = 6). D. Oocyte maturation measured as the levels of % GVBD in oocytes injected with S1PR3 antisense and normalized to P4 or Org OD 02-0 (OD)-treated oocytes injected with control antisense (Ctrl AS) (mean + SEM; n = 7, ordinary one-way ANOVA). E. Representative WB of MAPK, Plk1 and Cdc2 phosphorylation from untreated oocytes, eggs matured by overnight (O/N) treatment with P4 or OD, oocytes injected with control antisense (Ctrl AS) or S1PR3 antisense (S1PR3 AS) and treated O/N with P4 or OD. Tubulin is shown as a loading control. F. Representative WB of MAPK, Plk1, p-Cdc25c (Ser216) and Cdc2 phosphorylation from untreated oocytes, eggs matured by overnight (O/N) treatment with P4 or OD, oocytes injected with S1PR3 antisense (S1PR3 AS) and treated O/N with P4. Tubulin is shown as a loading control. G. GVBD-time course after treatment with P4 10-7M (left panel), 10-5M (middle panel), and 10-4M (right panel), in oocytes injected with water (Con) or with S1PR3 antisense (S1PR3 AS). Percentage of oocytes maturation is normalized to the Con condition (mean + SEM; n = 2 independent female frogs).

mPRβ-ABHD2 co-receptor complex is a P4-dependent PLA2.

A. Time-dependent production of lysothiophospholipid metabolite in reticulocytes overexpressing mPR, ABHD2.S and ABHD2.S+mPR complex in the presence of ethanol as vehicle or P4 10-5M (mean + SEM; n= 3). B. PLA2 specific activity measured at 5 min after P4 treatment in reticulocytes overexpressing mPR, ABHD2.S and ABHD2.S+mPR (mean + SEM; n = 3, ordinary one-way ANOVA). C. Time course of P4-dependent PLA2 activity in reticulocyte lysates expressing mPRβ with either wild-type ABHD2 (ABHD2) or the ABHD2 S207A/D345A/H376A mutant (S/D/H). P4-dependent PLA2 is plotted as the difference in activity in the presence and absence of P4 (Mean + SEM; n = 3). D. Representative WB of the immunoprecipitation (IP) of mPR-GFP using oocytes lysates from un-injected oocytes (Naive) and oocytes over-expressing mPR-GFP and ABHD2.S treated for 40 min with Ethanol (EtOH) or P4. Left panel, the representative WB membrane is probed for ABHD2 and GFP. Right panel, Quantification of the IP experiments as the ratio of ABHD2/mPR-GFP normalized to the ethanol condition (mean + SEM; n = 3, unpaired t-test).

ABHD2 and PLA2 activity are required for mPRβ endocytosis and signaling.

A. Effect of ABHD2 knockdown on mPR-P4 mediated endocytosis. Oocytes were injected with mRNA to overexpress mPR-GFP (Naïve). 48 hours later, some mPR-GFP overexpressed oocytes were also injected with ABHD2 antisense (ABHD2 AS). The following day, naïve oocytes and ABHD2 AS oocytes were imaged, before and 45 min after P4 treatment. Left panel, representative confocal image of mPR-GFP vesicles in naïve and ABHD2 AS oocytes, before and after the progesterone treatment. Right panel, histogram showing the number of mPR-GFP vesicles before and after P4 in naïve and ABHD2 AS oocytes (mean + SEM; n = 20 to 21 oocytes per condition, from 3 independent female frogs, ordinary one-way ANOVA). B. the role of PLA2 in mPR-P4 mediated endocytosis. Oocytes were injected with mRNA to overexpress mPR-GFP. 72 hours later, oocytes were pre-treated with vehicle (naïve) or ACA for 2 hours, followed by imaging, before and 45 min after P4 treatment. The histogram shows the number of mPR-GFP vesicles before and after P4 in naïve and ACA pre-treated oocytes (mean + SEM; n = 16 to 18 oocytes per condition, from 2 independent female frogs, ordinary one-way ANOVA). C. Cartoon depicting the role of SNAP25Δ20 in blocking exocytosis. D. SNAP25Δ20-induced oocyte maturation requires clathrin-dependent endocytosis. Naïve oocytes were pre-treated with vehicle or Pitstop, followed by overnight treatment with P4 or SNAP25Δ20-mRNA injection. Oocyte maturation was measured as the levels of % GVBD in P4 or SNAP25Δ20 injected oocytes normalized to the vehicle condition (mean + SEM; n = 4, ordinary one-way ANOVA). E. SNAP25Δ20-induced maturation requires ABHD2. Oocytes were injected with mPR antisense (mPR AS) or ABHD2 antisense (AS) with or without mRNA to overexpress mPR (AS+mPR). 48 hours later, oocytes were injected with mRNA to overexpress SNAP25Δ20. The following day, oocyte maturation was measured as the levels of % GVBD in mPR, ABHD2 AS and ABHD2 AS+mPR oocytes normalized to naive oocytes injected with SNAP25Δ20, (mean + SEM; n = 3, ordinary one-way ANOVA). F. Representative WB of mPR and SNAP25Δ20 proteins expression levels in naïve, ABHD2 AS and ABHD2 AS+mPR oocytes. Tubulin is shown as a loading control. G. Representative WB of MAPK, Plk1 and Cdc2 phosphorylation from untreated oocytes, eggs matured by overnight (O/N) treatment with P4 or SNAP25Δ20 mRNA injection, and oocytes injected with mPR (mPR AS) or ABHD2 antisense (ABHD2 AS) and treated O/N with P4 or SNAP25Δ20 mRNA injection. Tubulin is shown as a loading control. H. Quantification of p-Plk as the ratio of p-PLK/Tubulin and p-MAPK as the ratio of p-MAPK/Tubulin normalized to the ratios in naive eggs (mean + SEM; n = 4, ordinary one-way ANOVA). I. Oocyte maturation measured as the levels of % GVBD in oocytes pretreated for 2 hours with ACA, NS398 and MP-A08 followed by SNAP25Δ20-mRNA injection and normalized to percentage GVBD in oocytes pre-treated with vehicle followed by SNAP25Δ20-mRNA injection (Veh.) (mean + SEM; n = 3, ordinary one-way ANOVA). J. Model of the signaling cascade triggered in response to P4.