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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 oocytes within preantral and early antral/antral follicles of Gpr3+/+ ovaries. (A) Low magnification view of a section of an antral follicle labeled with the Gαs antibody. Inset shows a preantral follicle. The detector gain was the same for both images. (B and C) High magnification view of the oocyte in the preantral (B) and antral (C) follicle shown in A. The confocal microscope settings and bars were the same for B and C. (D) Plasma membrane-to-cytoplasm fluorescence ratios for oocytes in preantral follicles (3.2 ± 0.2) or early antral/antral follicles (3.6 ± 0.2). Mean ± SEM (error bars; n = number of oocytes). The ratios were not significantly different (t test, P = 0.09). Data are from two Gpr3+/+ mice, one of which was injected with eCG 43 h before ovary collection to increase the number of antral follicles. (E) RT-PCR to compare the amount of Gpr3 RNA in oocytes from preantral and early antral/antral follicles. The indicated numbers of microliters of cDNA were used for each RT-PCR; 1 μl cDNA was derived from 0.08 oocytes (supplemental Materials and methods). A mixture of primers for Gpr3 and Rpl19 was included in each reaction. The ratios of band intensities for Gpr3 and Rpl19 PCR products in each lane of the gel were used to obtain a relative measure of the amount of Gpr3 RNA per oocyte. The relative amount of Gpr3 RNA in oocytes from preantral follicles compared with oocytes from early antral/antral follicles was 1.3–1.6 (range for this and another similar experiment).
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fig3: Immunofluorescence localization of Gαs in oocytes within preantral and early antral/antral follicles of Gpr3+/+ ovaries. (A) Low magnification view of a section of an antral follicle labeled with the Gαs antibody. Inset shows a preantral follicle. The detector gain was the same for both images. (B and C) High magnification view of the oocyte in the preantral (B) and antral (C) follicle shown in A. The confocal microscope settings and bars were the same for B and C. (D) Plasma membrane-to-cytoplasm fluorescence ratios for oocytes in preantral follicles (3.2 ± 0.2) or early antral/antral follicles (3.6 ± 0.2). Mean ± SEM (error bars; n = number of oocytes). The ratios were not significantly different (t test, P = 0.09). Data are from two Gpr3+/+ mice, one of which was injected with eCG 43 h before ovary collection to increase the number of antral follicles. (E) RT-PCR to compare the amount of Gpr3 RNA in oocytes from preantral and early antral/antral follicles. The indicated numbers of microliters of cDNA were used for each RT-PCR; 1 μl cDNA was derived from 0.08 oocytes (supplemental Materials and methods). A mixture of primers for Gpr3 and Rpl19 was included in each reaction. The ratios of band intensities for Gpr3 and Rpl19 PCR products in each lane of the gel were used to obtain a relative measure of the amount of Gpr3 RNA per oocyte. The relative amount of Gpr3 RNA in oocytes from preantral follicles compared with oocytes from early antral/antral follicles was 1.3–1.6 (range for this and another similar experiment).

Mentions: 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.


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 oocytes within preantral and early antral/antral follicles of Gpr3+/+ ovaries. (A) Low magnification view of a section of an antral follicle labeled with the Gαs antibody. Inset shows a preantral follicle. The detector gain was the same for both images. (B and C) High magnification view of the oocyte in the preantral (B) and antral (C) follicle shown in A. The confocal microscope settings and bars were the same for B and C. (D) Plasma membrane-to-cytoplasm fluorescence ratios for oocytes in preantral follicles (3.2 ± 0.2) or early antral/antral follicles (3.6 ± 0.2). Mean ± SEM (error bars; n = number of oocytes). The ratios were not significantly different (t test, P = 0.09). Data are from two Gpr3+/+ mice, one of which was injected with eCG 43 h before ovary collection to increase the number of antral follicles. (E) RT-PCR to compare the amount of Gpr3 RNA in oocytes from preantral and early antral/antral follicles. The indicated numbers of microliters of cDNA were used for each RT-PCR; 1 μl cDNA was derived from 0.08 oocytes (supplemental Materials and methods). A mixture of primers for Gpr3 and Rpl19 was included in each reaction. The ratios of band intensities for Gpr3 and Rpl19 PCR products in each lane of the gel were used to obtain a relative measure of the amount of Gpr3 RNA per oocyte. The relative amount of Gpr3 RNA in oocytes from preantral follicles compared with oocytes from early antral/antral follicles was 1.3–1.6 (range for this and another similar experiment).
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Related In: Results  -  Collection

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fig3: Immunofluorescence localization of Gαs in oocytes within preantral and early antral/antral follicles of Gpr3+/+ ovaries. (A) Low magnification view of a section of an antral follicle labeled with the Gαs antibody. Inset shows a preantral follicle. The detector gain was the same for both images. (B and C) High magnification view of the oocyte in the preantral (B) and antral (C) follicle shown in A. The confocal microscope settings and bars were the same for B and C. (D) Plasma membrane-to-cytoplasm fluorescence ratios for oocytes in preantral follicles (3.2 ± 0.2) or early antral/antral follicles (3.6 ± 0.2). Mean ± SEM (error bars; n = number of oocytes). The ratios were not significantly different (t test, P = 0.09). Data are from two Gpr3+/+ mice, one of which was injected with eCG 43 h before ovary collection to increase the number of antral follicles. (E) RT-PCR to compare the amount of Gpr3 RNA in oocytes from preantral and early antral/antral follicles. The indicated numbers of microliters of cDNA were used for each RT-PCR; 1 μl cDNA was derived from 0.08 oocytes (supplemental Materials and methods). A mixture of primers for Gpr3 and Rpl19 was included in each reaction. The ratios of band intensities for Gpr3 and Rpl19 PCR products in each lane of the gel were used to obtain a relative measure of the amount of Gpr3 RNA per oocyte. The relative amount of Gpr3 RNA in oocytes from preantral follicles compared with oocytes from early antral/antral follicles was 1.3–1.6 (range for this and another similar experiment).
Mentions: 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.

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