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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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Expression and subcellular localization of GPR3 during porcine oocyte meiotic maturation.(A) Subcellular localization of GPR3 as revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated at the inner cytoplasm and plasma membrane at the pro-MI stage. From MI to MII, GPR3 aggregated at the plasma membrane. (B) Expression of GPR3 protein was measured by western blotting. Samples were collected at 0 h, 24 h, 30 h, and 44 h of culture which were the time points when most oocytes had reached the GV, GVBD, MI and MII stages, respectively. Each sample was derived from 150 oocytes. Green, GPR3; Blue, chromatin. Bar = 40 µm.
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pone-0038807-g001: Expression and subcellular localization of GPR3 during porcine oocyte meiotic maturation.(A) Subcellular localization of GPR3 as revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated at the inner cytoplasm and plasma membrane at the pro-MI stage. From MI to MII, GPR3 aggregated at the plasma membrane. (B) Expression of GPR3 protein was measured by western blotting. Samples were collected at 0 h, 24 h, 30 h, and 44 h of culture which were the time points when most oocytes had reached the GV, GVBD, MI and MII stages, respectively. Each sample was derived from 150 oocytes. Green, GPR3; Blue, chromatin. Bar = 40 µm.

Mentions: To determine the function of GPR3 during meiosis, subcellular localization and expression were examined. The localization of GPR3 in porcine oocytes is shown in Fig. 1A. In GV oocytes, the prominent staining of GPR3 was mainly concentrated at the nuclear membrane, and the remaining GPR3 accumulated at the plasma membrane. After germinal vesicle breakdown (GVBD), the positive signal of GPR3 was observed evenly in the inner cytoplasm and plasma membrane. In the MI and MII stages, GPR3 was distributed at the plasma membrane. In control group, we did not detect any green positive signal. For affirming the subcellular localization of GPR3 in GV stage, we scanned the oocytes thoroughly in numerous planes and obtained the same result with the single plane (see Fig. S1). Western blot analysis showed that GPR3 was expressed in all porcine oocyte stages. As shown in Fig. 1B, the GPR3 expression displayed a maximal level at the GV stage, and remained stable from GVBD to the MII stages.


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)

Expression and subcellular localization of GPR3 during porcine oocyte meiotic maturation.(A) Subcellular localization of GPR3 as revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated at the inner cytoplasm and plasma membrane at the pro-MI stage. From MI to MII, GPR3 aggregated at the plasma membrane. (B) Expression of GPR3 protein was measured by western blotting. Samples were collected at 0 h, 24 h, 30 h, and 44 h of culture which were the time points when most oocytes had reached the GV, GVBD, MI and MII stages, respectively. Each sample was derived from 150 oocytes. Green, GPR3; Blue, chromatin. Bar = 40 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038807-g001: Expression and subcellular localization of GPR3 during porcine oocyte meiotic maturation.(A) Subcellular localization of GPR3 as revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated at the inner cytoplasm and plasma membrane at the pro-MI stage. From MI to MII, GPR3 aggregated at the plasma membrane. (B) Expression of GPR3 protein was measured by western blotting. Samples were collected at 0 h, 24 h, 30 h, and 44 h of culture which were the time points when most oocytes had reached the GV, GVBD, MI and MII stages, respectively. Each sample was derived from 150 oocytes. Green, GPR3; Blue, chromatin. Bar = 40 µm.
Mentions: To determine the function of GPR3 during meiosis, subcellular localization and expression were examined. The localization of GPR3 in porcine oocytes is shown in Fig. 1A. In GV oocytes, the prominent staining of GPR3 was mainly concentrated at the nuclear membrane, and the remaining GPR3 accumulated at the plasma membrane. After germinal vesicle breakdown (GVBD), the positive signal of GPR3 was observed evenly in the inner cytoplasm and plasma membrane. In the MI and MII stages, GPR3 was distributed at the plasma membrane. In control group, we did not detect any green positive signal. For affirming the subcellular localization of GPR3 in GV stage, we scanned the oocytes thoroughly in numerous planes and obtained the same result with the single plane (see Fig. S1). Western blot analysis showed that GPR3 was expressed in all porcine oocyte stages. As shown in Fig. 1B, the GPR3 expression displayed a maximal level at the GV stage, and remained stable from GVBD to the MII stages.

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