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The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes.

Yang CR, Wei Y, Qi ST, Chen L, Zhang QH, Ma JY, Luo YB, Wang YP, Hou Y, Schatten H, Liu ZH, Sun QY - PLoS ONE (2012)

Bottom Line: The results showed that GPR3 was expressed at various stages during porcine oocyte maturation.On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation.Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Northeast Agricultural University of China, Harbin, China.

ABSTRACT
The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.

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The efficiency of GPR3 overexpression.(A) Immunofluorescence detection of GPR3 overexpression. Control: oocytes were injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: oocyets were injected with 2.5 mg/ml Myc6-GPR3 mRNA solution. Green, Myc6-GPR3; Blue, chromatin. Bar = 40 µm. (B) Samples from control and overexpression groups were collected to test the expression of Myc6-GPR3. Control: 150 oocytes injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: 150 oocytes injected with 2.5 mg/ml Myc6- GPR3 mRNA solution. Oocytes were then incubated for 15 h in TCM-199 medium containing 2.5 µM Milrinone before collection or culture for western blotting.
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pone-0038807-g002: The efficiency of GPR3 overexpression.(A) Immunofluorescence detection of GPR3 overexpression. Control: oocytes were injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: oocyets were injected with 2.5 mg/ml Myc6-GPR3 mRNA solution. Green, Myc6-GPR3; Blue, chromatin. Bar = 40 µm. (B) Samples from control and overexpression groups were collected to test the expression of Myc6-GPR3. Control: 150 oocytes injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: 150 oocytes injected with 2.5 mg/ml Myc6- GPR3 mRNA solution. Oocytes were then incubated for 15 h in TCM-199 medium containing 2.5 µM Milrinone before collection or culture for western blotting.

Mentions: We next examined the effect of GPR3 overexpression on meiotic resumption. Immunofluorescence was used to confirm the overexpression of Myc6-GPR3. Oocytes injected with the same amount of Myc6 mRNA were used as control, which did not result in a specific signal (Fig. 2A). Successful overexpression was observed in the Myc6-GPR3 mRNA injection group (Fig. 2A). Myc6-GPR overexpression was also confirmed by western blot. A clear band was detected after Myc6-GPR3 mRNA injection, whereas injection of Myc6 mRNA in the control group did not result in specific band detection (Fig. 2B).


The G protein coupled receptor 3 is involved in cAMP and cGMP signaling and maintenance of meiotic arrest in porcine oocytes.

Yang CR, Wei Y, Qi ST, Chen L, Zhang QH, Ma JY, Luo YB, Wang YP, Hou Y, Schatten H, Liu ZH, Sun QY - PLoS ONE (2012)

The efficiency of GPR3 overexpression.(A) Immunofluorescence detection of GPR3 overexpression. Control: oocytes were injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: oocyets were injected with 2.5 mg/ml Myc6-GPR3 mRNA solution. Green, Myc6-GPR3; Blue, chromatin. Bar = 40 µm. (B) Samples from control and overexpression groups were collected to test the expression of Myc6-GPR3. Control: 150 oocytes injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: 150 oocytes injected with 2.5 mg/ml Myc6- GPR3 mRNA solution. Oocytes were then incubated for 15 h in TCM-199 medium containing 2.5 µM Milrinone before collection or culture for western blotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369857&req=5

pone-0038807-g002: The efficiency of GPR3 overexpression.(A) Immunofluorescence detection of GPR3 overexpression. Control: oocytes were injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: oocyets were injected with 2.5 mg/ml Myc6-GPR3 mRNA solution. Green, Myc6-GPR3; Blue, chromatin. Bar = 40 µm. (B) Samples from control and overexpression groups were collected to test the expression of Myc6-GPR3. Control: 150 oocytes injected with 2.5 mg/ml Myc6 mRNA solution; Overexpression: 150 oocytes injected with 2.5 mg/ml Myc6- GPR3 mRNA solution. Oocytes were then incubated for 15 h in TCM-199 medium containing 2.5 µM Milrinone before collection or culture for western blotting.
Mentions: We next examined the effect of GPR3 overexpression on meiotic resumption. Immunofluorescence was used to confirm the overexpression of Myc6-GPR3. Oocytes injected with the same amount of Myc6 mRNA were used as control, which did not result in a specific signal (Fig. 2A). Successful overexpression was observed in the Myc6-GPR3 mRNA injection group (Fig. 2A). Myc6-GPR overexpression was also confirmed by western blot. A clear band was detected after Myc6-GPR3 mRNA injection, whereas injection of Myc6 mRNA in the control group did not result in specific band detection (Fig. 2B).

Bottom Line: The results showed that GPR3 was expressed at various stages during porcine oocyte maturation.On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation.Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation.

View Article: PubMed Central - PubMed

Affiliation: College of Life Science, Northeast Agricultural University of China, Harbin, China.

ABSTRACT
The arrest of meiotic prophase in mammalian oocytes within fully grown follicles is dependent on cyclic adenosine monophosphate (cAMP) regulation. A large part of cAMP is produced by the Gs-linked G-protein-coupled receptor (GPR) pathway. In the present study, we examined whether GPR3 is involved in the maintenance of meiotic arrest in porcine oocytes. Expression and distribution of GPR3 were examined by western blot and immunofluorescence microscopy, respectively. The results showed that GPR3 was expressed at various stages during porcine oocyte maturation. At the germinal vesicle (GV) stage, GPR3 displayed a maximal expression level, and its expression remained stable from pro-metaphase I (MI) to metaphase II (MII). Immunofluorescence staining showed that GPR3 was mainly distributed at the nuclear envelope during the GV stage and localized to the plasma membrane at pro-MI, MI and MII stages. RNA interference (RNAi) was used to knock down the GPR3 expression within oocytes. Injection of small interfering double-stranded RNA (siRNA) targeting GPR3 stimulated meiotic resumption of oocytes. On the other hand, overexpression of GPR3 inhibited meiotic maturation of porcine oocytes, which was caused by increase of cGMP and cAMP levels and inhibition of cyclin B accumulation. Furthermore, incubation of porcine oocytes with the GPR3 ligand sphingosylphosphorylcholine (SPC) inhibited oocyte maturation. We propose that GPR3 is required for maintenance of meiotic arrest in porcine oocytes through pathways involved in the regulation of cAMP and cGMP.

Show MeSH
Related in: MedlinePlus