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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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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).
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fig2: 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).

Mentions: In Gpr3+/+ oocytes, Gαs fluorescence was present in both the plasma membrane and in the cytoplasm; within the cytoplasm, irregularly shaped bright spots up to several micrometers in diameter as well as diffuse fluorescence were visible in most sections (Fig. 2, A and C). In Gpr3−/− oocytes, Gαs was more strongly localized in the plasma membrane (Fig. 2, B and C). Intensity measurements (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1) showed that in Gpr3−/− oocytes, the Gαs fluorescence was higher in the plasma membrane and lower in the cytoplasm than in Gpr3+/+ oocytes (Table I). For each oocyte, we determined the plasma membrane-to-cytoplasm fluorescence intensity ratio and found that the mean ratio was significantly smaller for Gpr3+/+ oocytes than for Gpr3−/− oocytes (Fig. 2 D). These results show that GPR3 causes Gαs to move from the oocyte plasma membrane to the cytoplasm; the simplest interpretation of this observation is that 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)

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).
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fig2: 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).
Mentions: In Gpr3+/+ oocytes, Gαs fluorescence was present in both the plasma membrane and in the cytoplasm; within the cytoplasm, irregularly shaped bright spots up to several micrometers in diameter as well as diffuse fluorescence were visible in most sections (Fig. 2, A and C). In Gpr3−/− oocytes, Gαs was more strongly localized in the plasma membrane (Fig. 2, B and C). Intensity measurements (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506194/DC1) showed that in Gpr3−/− oocytes, the Gαs fluorescence was higher in the plasma membrane and lower in the cytoplasm than in Gpr3+/+ oocytes (Table I). For each oocyte, we determined the plasma membrane-to-cytoplasm fluorescence intensity ratio and found that the mean ratio was significantly smaller for Gpr3+/+ oocytes than for Gpr3−/− oocytes (Fig. 2 D). These results show that GPR3 causes Gαs to move from the oocyte plasma membrane to the cytoplasm; the simplest interpretation of this observation is that 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