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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+/+ and Gpr3−/− preantral follicle-enclosed oocytes. (A) Scanning transmission and fluorescence images of a Gpr3+/+ preantral follicle in which the oocyte had been injected with GαsGFP RNA 20 h before. In the transmitted light image of the oocyte, the bright sphere on the left is an oil drop that was introduced by microinjection; the nucleus and nucleolus are visible on the top right. In the fluorescence image, the oil drop and nucleus appear as dark areas where GαsGFP is excluded. (B) Immunoblot comparing the amount of GαsGFP and endogenous Gαs in GαsGFP RNA-injected and noninjected oocytes from a Gpr3+/+ mouse. Oocytes were injected with the RNA while inside their preantral follicles; after culturing the follicles overnight, the oocytes were isolated, and gel samples were prepared (14 oocytes per lane). The blot was probed with an antibody against Gαs (RM). The ratio of GαsGFP protein in the injected oocytes to Gαs in uninjected control oocytes was 1.1–1.5 (range for two experiments). The expression of GαsGFP caused some reduction of endogenous Gαs protein. (C and D) GαsGFP fluorescence in Gpr3+/+ (C) and Gpr3−/− (D) oocytes. (C) Fluorescence is present in the plasma membrane, in spots in the cytoplasm, and diffusely within the cytoplasm. (D) Fluorescence is present primarily in the plasma membrane. The confocal microscope settings and bars were the same for C and D. (E) Plasma membrane-to-cytoplasm fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes. Mean ± SEM (error bars; n = number of oocytes). Ratios for Gpr3+/+ oocytes (3.1 ± 0.2) and Gpr3−/− oocytes (7.1 ± 0.4) were significantly different (t test, P < 0.0001). Data are from three Gpr3+/+ and three Gpr3−/− mice.
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fig4: GαsGFP fluorescence in Gpr3+/+ and Gpr3−/− preantral follicle-enclosed oocytes. (A) Scanning transmission and fluorescence images of a Gpr3+/+ preantral follicle in which the oocyte had been injected with GαsGFP RNA 20 h before. In the transmitted light image of the oocyte, the bright sphere on the left is an oil drop that was introduced by microinjection; the nucleus and nucleolus are visible on the top right. In the fluorescence image, the oil drop and nucleus appear as dark areas where GαsGFP is excluded. (B) Immunoblot comparing the amount of GαsGFP and endogenous Gαs in GαsGFP RNA-injected and noninjected oocytes from a Gpr3+/+ mouse. Oocytes were injected with the RNA while inside their preantral follicles; after culturing the follicles overnight, the oocytes were isolated, and gel samples were prepared (14 oocytes per lane). The blot was probed with an antibody against Gαs (RM). The ratio of GαsGFP protein in the injected oocytes to Gαs in uninjected control oocytes was 1.1–1.5 (range for two experiments). The expression of GαsGFP caused some reduction of endogenous Gαs protein. (C and D) GαsGFP fluorescence in Gpr3+/+ (C) and Gpr3−/− (D) oocytes. (C) Fluorescence is present in the plasma membrane, in spots in the cytoplasm, and diffusely within the cytoplasm. (D) Fluorescence is present primarily in the plasma membrane. The confocal microscope settings and bars were the same for C and D. (E) Plasma membrane-to-cytoplasm fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes. Mean ± SEM (error bars; n = number of oocytes). Ratios for Gpr3+/+ oocytes (3.1 ± 0.2) and Gpr3−/− oocytes (7.1 ± 0.4) were significantly different (t test, P < 0.0001). Data are from three Gpr3+/+ and three Gpr3−/− mice.

Mentions: Immunofluorescence localization and immunoblotting of Gαs in oocytes in Gpr3+/+ and Gpr3−/− ovaries. (A and B) Gαs in Gpr3+/+ (A) and Gpr3−/− (B) oocytes in a preantral follicle. The confocal microscope settings and bars were the same for A and B; the dark region in each oocyte is the nucleus. (C) Pixel intensity on a scale of 0–255 along a line drawn through each oocyte image, choosing a line that avoided the nucleus. (D) Plasma membrane-to-cytoplasm Gαs fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes, which were determined as described in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1). Mean ± SEM (error bars; n = number of oocytes). The fluorescence ratios for Gpr3+/+ oocytes (3.6 ± 0.3) and Gpr3−/− oocytes (13.4 ± 1.3) were significantly different (t test, P = 0.0001). Data are from two Gpr3+/+ and two Gpr3−/− mice; ovaries from mice of the two genotypes were processed in parallel. (E) Immunoblot comparing the amount of Gαs protein in Gpr3+/+ and Gpr3−/− oocytes that were isolated from preantral follicles. 15 oocytes per lane. The 52-kD splice variant of Gαs is predominant in mouse oocytes, although a small amount of the 45-kD form is present (Fig. 4 B; Mehlmann et al., 2002).


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+/+ and Gpr3−/− preantral follicle-enclosed oocytes. (A) Scanning transmission and fluorescence images of a Gpr3+/+ preantral follicle in which the oocyte had been injected with GαsGFP RNA 20 h before. In the transmitted light image of the oocyte, the bright sphere on the left is an oil drop that was introduced by microinjection; the nucleus and nucleolus are visible on the top right. In the fluorescence image, the oil drop and nucleus appear as dark areas where GαsGFP is excluded. (B) Immunoblot comparing the amount of GαsGFP and endogenous Gαs in GαsGFP RNA-injected and noninjected oocytes from a Gpr3+/+ mouse. Oocytes were injected with the RNA while inside their preantral follicles; after culturing the follicles overnight, the oocytes were isolated, and gel samples were prepared (14 oocytes per lane). The blot was probed with an antibody against Gαs (RM). The ratio of GαsGFP protein in the injected oocytes to Gαs in uninjected control oocytes was 1.1–1.5 (range for two experiments). The expression of GαsGFP caused some reduction of endogenous Gαs protein. (C and D) GαsGFP fluorescence in Gpr3+/+ (C) and Gpr3−/− (D) oocytes. (C) Fluorescence is present in the plasma membrane, in spots in the cytoplasm, and diffusely within the cytoplasm. (D) Fluorescence is present primarily in the plasma membrane. The confocal microscope settings and bars were the same for C and D. (E) Plasma membrane-to-cytoplasm fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes. Mean ± SEM (error bars; n = number of oocytes). Ratios for Gpr3+/+ oocytes (3.1 ± 0.2) and Gpr3−/− oocytes (7.1 ± 0.4) were significantly different (t test, P < 0.0001). Data are from three Gpr3+/+ and three Gpr3−/− mice.
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fig4: GαsGFP fluorescence in Gpr3+/+ and Gpr3−/− preantral follicle-enclosed oocytes. (A) Scanning transmission and fluorescence images of a Gpr3+/+ preantral follicle in which the oocyte had been injected with GαsGFP RNA 20 h before. In the transmitted light image of the oocyte, the bright sphere on the left is an oil drop that was introduced by microinjection; the nucleus and nucleolus are visible on the top right. In the fluorescence image, the oil drop and nucleus appear as dark areas where GαsGFP is excluded. (B) Immunoblot comparing the amount of GαsGFP and endogenous Gαs in GαsGFP RNA-injected and noninjected oocytes from a Gpr3+/+ mouse. Oocytes were injected with the RNA while inside their preantral follicles; after culturing the follicles overnight, the oocytes were isolated, and gel samples were prepared (14 oocytes per lane). The blot was probed with an antibody against Gαs (RM). The ratio of GαsGFP protein in the injected oocytes to Gαs in uninjected control oocytes was 1.1–1.5 (range for two experiments). The expression of GαsGFP caused some reduction of endogenous Gαs protein. (C and D) GαsGFP fluorescence in Gpr3+/+ (C) and Gpr3−/− (D) oocytes. (C) Fluorescence is present in the plasma membrane, in spots in the cytoplasm, and diffusely within the cytoplasm. (D) Fluorescence is present primarily in the plasma membrane. The confocal microscope settings and bars were the same for C and D. (E) Plasma membrane-to-cytoplasm fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes. Mean ± SEM (error bars; n = number of oocytes). Ratios for Gpr3+/+ oocytes (3.1 ± 0.2) and Gpr3−/− oocytes (7.1 ± 0.4) were significantly different (t test, P < 0.0001). Data are from three Gpr3+/+ and three Gpr3−/− mice.
Mentions: Immunofluorescence localization and immunoblotting of Gαs in oocytes in Gpr3+/+ and Gpr3−/− ovaries. (A and B) Gαs in Gpr3+/+ (A) and Gpr3−/− (B) oocytes in a preantral follicle. The confocal microscope settings and bars were the same for A and B; the dark region in each oocyte is the nucleus. (C) Pixel intensity on a scale of 0–255 along a line drawn through each oocyte image, choosing a line that avoided the nucleus. (D) Plasma membrane-to-cytoplasm Gαs fluorescence ratios for Gpr3+/+ and Gpr3−/− oocytes, which were determined as described in Fig. S1 (available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1). Mean ± SEM (error bars; n = number of oocytes). The fluorescence ratios for Gpr3+/+ oocytes (3.6 ± 0.3) and Gpr3−/− oocytes (13.4 ± 1.3) were significantly different (t test, P = 0.0001). Data are from two Gpr3+/+ and two Gpr3−/− mice; ovaries from mice of the two genotypes were processed in parallel. (E) Immunoblot comparing the amount of Gαs protein in Gpr3+/+ and Gpr3−/− oocytes that were isolated from preantral follicles. 15 oocytes per lane. The 52-kD splice variant of Gαs is predominant in mouse oocytes, although a small amount of the 45-kD form is present (Fig. 4 B; Mehlmann et al., 2002).

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