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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 of Gαs in a mouse ovary. (A) Immunoblot demonstrating Gαs antibody specificity. 1 μg of ovary lysate (Gpr3+/+) was loaded on the gel; both the 52- and 45-kD splice variants of Gαs (Robishaw et al., 1986) were seen. (B) A section of a Gpr3+/+ ovary labeled with an antibody against Gαs. This ovary, which was obtained from a mouse that had not been injected with eCG to stimulate antral follicle formation, contained mostly preantral follicles and some early antral follicles. Follicles with 120–190-μm diam were classified as preantral, whereas those with ≥200-μm diam were classified as early antral (with multiple small antral spaces) or antral (with a single large antral space; Fig. 3 A). These categories were not completely distinct because some follicles with <200-μm diam showed a formation of antral spaces. (C) A control section labeled with nonimmune IgG. Confocal microscope settings and bars were the same for B and C.
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fig1: Immunofluorescence localization of Gαs in a mouse ovary. (A) Immunoblot demonstrating Gαs antibody specificity. 1 μg of ovary lysate (Gpr3+/+) was loaded on the gel; both the 52- and 45-kD splice variants of Gαs (Robishaw et al., 1986) were seen. (B) A section of a Gpr3+/+ ovary labeled with an antibody against Gαs. This ovary, which was obtained from a mouse that had not been injected with eCG to stimulate antral follicle formation, contained mostly preantral follicles and some early antral follicles. Follicles with 120–190-μm diam were classified as preantral, whereas those with ≥200-μm diam were classified as early antral (with multiple small antral spaces) or antral (with a single large antral space; Fig. 3 A). These categories were not completely distinct because some follicles with <200-μm diam showed a formation of antral spaces. (C) A control section labeled with nonimmune IgG. Confocal microscope settings and bars were the same for B and C.

Mentions: By using an antibody against the COOH-terminal decapeptide of Gαs (Simonds et al., 1989), which specifically recognized Gαs in mouse ovary (Fig. 1 A), we examined the distribution of Gαs immunofluorescence in preantral follicle-enclosed oocytes in frozen sections of ovaries (Fig. 1, B and C) from Gpr3+/+ and Gpr3−/− mice. These sections showed that Gαs was present at a high level in theca/interstitial cells that surround each follicle and in the oocyte. Gαs was also present at lower levels in the follicular somatic cells.


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 of Gαs in a mouse ovary. (A) Immunoblot demonstrating Gαs antibody specificity. 1 μg of ovary lysate (Gpr3+/+) was loaded on the gel; both the 52- and 45-kD splice variants of Gαs (Robishaw et al., 1986) were seen. (B) A section of a Gpr3+/+ ovary labeled with an antibody against Gαs. This ovary, which was obtained from a mouse that had not been injected with eCG to stimulate antral follicle formation, contained mostly preantral follicles and some early antral follicles. Follicles with 120–190-μm diam were classified as preantral, whereas those with ≥200-μm diam were classified as early antral (with multiple small antral spaces) or antral (with a single large antral space; Fig. 3 A). These categories were not completely distinct because some follicles with <200-μm diam showed a formation of antral spaces. (C) A control section labeled with nonimmune IgG. Confocal microscope settings and bars were the same for B and C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171177&req=5

fig1: Immunofluorescence localization of Gαs in a mouse ovary. (A) Immunoblot demonstrating Gαs antibody specificity. 1 μg of ovary lysate (Gpr3+/+) was loaded on the gel; both the 52- and 45-kD splice variants of Gαs (Robishaw et al., 1986) were seen. (B) A section of a Gpr3+/+ ovary labeled with an antibody against Gαs. This ovary, which was obtained from a mouse that had not been injected with eCG to stimulate antral follicle formation, contained mostly preantral follicles and some early antral follicles. Follicles with 120–190-μm diam were classified as preantral, whereas those with ≥200-μm diam were classified as early antral (with multiple small antral spaces) or antral (with a single large antral space; Fig. 3 A). These categories were not completely distinct because some follicles with <200-μm diam showed a formation of antral spaces. (C) A control section labeled with nonimmune IgG. Confocal microscope settings and bars were the same for B and C.
Mentions: By using an antibody against the COOH-terminal decapeptide of Gαs (Simonds et al., 1989), which specifically recognized Gαs in mouse ovary (Fig. 1 A), we examined the distribution of Gαs immunofluorescence in preantral follicle-enclosed oocytes in frozen sections of ovaries (Fig. 1, B and C) from Gpr3+/+ and Gpr3−/− mice. These sections showed that Gαs was present at a high level in theca/interstitial cells that surround each follicle and in the oocyte. Gαs was also present at lower levels in the follicular somatic cells.

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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