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Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein.

Freudzon L, Norris RP, Hand AR, Tanaka S, Saeki Y, Jones TL, Rasenick MM, Berlot CH, Mehlmann LM, Jaffe LA - J. Cell Biol. (2005)

Bottom Line: GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte.However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes.Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032.

ABSTRACT
The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

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GαsGFP fluorescence in Gpr3−/− preantral follicle-enclosed oocytes that had been coinjected with GαsGFP RNA and Gpr3 RNA. Oocytes were injected with 15 pg GαsGFP RNA and the indicated amounts of Gpr3 or a control RNA encoding β-globin, and the follicles were cultured overnight before imaging. (A–C) 7 pg β-globin (A), Gpr3 (B), and 7 fg Gpr3 (C) RNA. The confocal microscope settings and bars were the same for all images. (D) Plasma membrane-to-cytoplasm GαsGFP fluorescence ratios for Gpr3−/− oocytes that were injected with the indicated RNAs. Mean ± SEM (error bars; n = number of oocytes). Data are from three mice. For the images of oocytes that had been injected with 7 pg Gpr3 RNA (n = 10), no plasma membrane fluorescence could be identified, so no ratio was calculated. Ratios for oocytes that were injected with 7 pg β-globin RNA (7.4 ± 0.5) or 7 fg Gpr3 RNA (2.6 ± 0.2) were significantly different (t test, P < 0.0001). The ratio for Gpr3−/− oocytes + 7 fg Gpr3 RNA was not different from that for Gpr3+/+ oocytes (Fig. 4 E; P = 0.14), and the ratio for Gpr3−/− oocytes + 7 pg β-globin RNA was not different from that for Gpr3−/− oocytes (Fig. 4 E; P = 0.69).
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fig5: GαsGFP fluorescence in Gpr3−/− preantral follicle-enclosed oocytes that had been coinjected with GαsGFP RNA and Gpr3 RNA. Oocytes were injected with 15 pg GαsGFP RNA and the indicated amounts of Gpr3 or a control RNA encoding β-globin, and the follicles were cultured overnight before imaging. (A–C) 7 pg β-globin (A), Gpr3 (B), and 7 fg Gpr3 (C) RNA. The confocal microscope settings and bars were the same for all images. (D) Plasma membrane-to-cytoplasm GαsGFP fluorescence ratios for Gpr3−/− oocytes that were injected with the indicated RNAs. Mean ± SEM (error bars; n = number of oocytes). Data are from three mice. For the images of oocytes that had been injected with 7 pg Gpr3 RNA (n = 10), no plasma membrane fluorescence could be identified, so no ratio was calculated. Ratios for oocytes that were injected with 7 pg β-globin RNA (7.4 ± 0.5) or 7 fg Gpr3 RNA (2.6 ± 0.2) were significantly different (t test, P < 0.0001). The ratio for Gpr3−/− oocytes + 7 fg Gpr3 RNA was not different from that for Gpr3+/+ oocytes (Fig. 4 E; P = 0.14), and the ratio for Gpr3−/− oocytes + 7 pg β-globin RNA was not different from that for Gpr3−/− oocytes (Fig. 4 E; P = 0.69).

Mentions: In Gpr3−/− oocytes that had been injected with 7 pg of a control RNA (β-globin), GαsGFP was highly localized in the plasma membrane (Fig. 5, A and D), as in Gpr3−/− oocytes that were injected with GαsGFP RNA alone (Fig. 4, D and E). In contrast, in Gpr3−/− oocytes that had been injected with 7 pg of Gpr3 RNA, GαsGFP fluorescence was present in the cytoplasm and was not localized in the plasma membrane (Fig. 5 B). As the amount of Gpr3 RNA was reduced while keeping the amount of GαsGFP RNA constant, the fraction of GαsGFP in the plasma membrane increased. The injection of 7 fg Gpr3 RNA resulted in a plasma membrane-to-cytoplasm fluorescence ratio that was comparable with that in Gpr3+/+ oocytes (compare Fig. 5, C and D with Fig. 4, C and E). Similar results were obtained by using a GPR3-RFP construct, which allowed us to monitor the level of GPR3 expression as a function of the amount of RNA injected and to show that the amount of GPR3 protein correlates with the amount of GαsGFP internalization (supplemental Results and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1). Because an increase in the relative amount of Gαs in the cytoplasm versus the plasma membrane is an indicator of Gs activation, these findings confirm that within the oocyte, GPR3 activates Gs.


Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein.

Freudzon L, Norris RP, Hand AR, Tanaka S, Saeki Y, Jones TL, Rasenick MM, Berlot CH, Mehlmann LM, Jaffe LA - J. Cell Biol. (2005)

GαsGFP fluorescence in Gpr3−/− preantral follicle-enclosed oocytes that had been coinjected with GαsGFP RNA and Gpr3 RNA. Oocytes were injected with 15 pg GαsGFP RNA and the indicated amounts of Gpr3 or a control RNA encoding β-globin, and the follicles were cultured overnight before imaging. (A–C) 7 pg β-globin (A), Gpr3 (B), and 7 fg Gpr3 (C) RNA. The confocal microscope settings and bars were the same for all images. (D) Plasma membrane-to-cytoplasm GαsGFP fluorescence ratios for Gpr3−/− oocytes that were injected with the indicated RNAs. Mean ± SEM (error bars; n = number of oocytes). Data are from three mice. For the images of oocytes that had been injected with 7 pg Gpr3 RNA (n = 10), no plasma membrane fluorescence could be identified, so no ratio was calculated. Ratios for oocytes that were injected with 7 pg β-globin RNA (7.4 ± 0.5) or 7 fg Gpr3 RNA (2.6 ± 0.2) were significantly different (t test, P < 0.0001). The ratio for Gpr3−/− oocytes + 7 fg Gpr3 RNA was not different from that for Gpr3+/+ oocytes (Fig. 4 E; P = 0.14), and the ratio for Gpr3−/− oocytes + 7 pg β-globin RNA was not different from that for Gpr3−/− oocytes (Fig. 4 E; P = 0.69).
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fig5: GαsGFP fluorescence in Gpr3−/− preantral follicle-enclosed oocytes that had been coinjected with GαsGFP RNA and Gpr3 RNA. Oocytes were injected with 15 pg GαsGFP RNA and the indicated amounts of Gpr3 or a control RNA encoding β-globin, and the follicles were cultured overnight before imaging. (A–C) 7 pg β-globin (A), Gpr3 (B), and 7 fg Gpr3 (C) RNA. The confocal microscope settings and bars were the same for all images. (D) Plasma membrane-to-cytoplasm GαsGFP fluorescence ratios for Gpr3−/− oocytes that were injected with the indicated RNAs. Mean ± SEM (error bars; n = number of oocytes). Data are from three mice. For the images of oocytes that had been injected with 7 pg Gpr3 RNA (n = 10), no plasma membrane fluorescence could be identified, so no ratio was calculated. Ratios for oocytes that were injected with 7 pg β-globin RNA (7.4 ± 0.5) or 7 fg Gpr3 RNA (2.6 ± 0.2) were significantly different (t test, P < 0.0001). The ratio for Gpr3−/− oocytes + 7 fg Gpr3 RNA was not different from that for Gpr3+/+ oocytes (Fig. 4 E; P = 0.14), and the ratio for Gpr3−/− oocytes + 7 pg β-globin RNA was not different from that for Gpr3−/− oocytes (Fig. 4 E; P = 0.69).
Mentions: In Gpr3−/− oocytes that had been injected with 7 pg of a control RNA (β-globin), GαsGFP was highly localized in the plasma membrane (Fig. 5, A and D), as in Gpr3−/− oocytes that were injected with GαsGFP RNA alone (Fig. 4, D and E). In contrast, in Gpr3−/− oocytes that had been injected with 7 pg of Gpr3 RNA, GαsGFP fluorescence was present in the cytoplasm and was not localized in the plasma membrane (Fig. 5 B). As the amount of Gpr3 RNA was reduced while keeping the amount of GαsGFP RNA constant, the fraction of GαsGFP in the plasma membrane increased. The injection of 7 fg Gpr3 RNA resulted in a plasma membrane-to-cytoplasm fluorescence ratio that was comparable with that in Gpr3+/+ oocytes (compare Fig. 5, C and D with Fig. 4, C and E). Similar results were obtained by using a GPR3-RFP construct, which allowed us to monitor the level of GPR3 expression as a function of the amount of RNA injected and to show that the amount of GPR3 protein correlates with the amount of GαsGFP internalization (supplemental Results and Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1). Because an increase in the relative amount of Gαs in the cytoplasm versus the plasma membrane is an indicator of Gs activation, these findings confirm that within the oocyte, GPR3 activates Gs.

Bottom Line: GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte.However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes.Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032.

ABSTRACT
The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.

Show MeSH
Related in: MedlinePlus