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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 effect of SPC and SM on meiotic resumption in porcine oocytes.(A) Porcine oocytes were collected in TCM-199 medium with or without 1 or 10 µM SPC or 10 µM SM and then placed in 200 µl drops under mineral oil. Oocytes were scored for meiotic maturation at 0 h, 12 h, 24 h, 30 h, and 36 h. (B) GVBD rates of porcine oocytes treated with 10 µM SPC, 10 µM SM or without ligand 24 h or 30 h of isolation. The number “n” on top of the bars indicates the total treated oocytes in each group. Letters ‘a’, ‘b’ and the symbol “*” indicate statistically significant difference (P<0.05). Data represent the mean ± SEM of at least three repeated experiments. (C) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after SM or SPC treatment by western blot using 150 oocytes in each sample. The results are shown along with those of control oocytes cultured without any drugs. Samples were collected from culture medium with or without SM or SPC at 0 h, 24 h and 30 h.
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pone-0038807-g006: The effect of SPC and SM on meiotic resumption in porcine oocytes.(A) Porcine oocytes were collected in TCM-199 medium with or without 1 or 10 µM SPC or 10 µM SM and then placed in 200 µl drops under mineral oil. Oocytes were scored for meiotic maturation at 0 h, 12 h, 24 h, 30 h, and 36 h. (B) GVBD rates of porcine oocytes treated with 10 µM SPC, 10 µM SM or without ligand 24 h or 30 h of isolation. The number “n” on top of the bars indicates the total treated oocytes in each group. Letters ‘a’, ‘b’ and the symbol “*” indicate statistically significant difference (P<0.05). Data represent the mean ± SEM of at least three repeated experiments. (C) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after SM or SPC treatment by western blot using 150 oocytes in each sample. The results are shown along with those of control oocytes cultured without any drugs. Samples were collected from culture medium with or without SM or SPC at 0 h, 24 h and 30 h.

Mentions: To detect whether a GPR3 ligand, sphingosylphosphorylcholine (SPC), affects meiotic maturation, porcine oocytes were cultured in the presence or absence of 1 to 10 µM SPC. Negative control was sphingomyelin (SM) (10 µM), a phospholipid (a non-ligand for GPRs) present in the plasma membrane. Oocytes cultured in the presence of 10 µM SPC showed a significant delay in the GVBD (Fig. 6A). However, oocytes cultured in SM showed no difference in GVBD rate at 24 h as compared with the control. In porcine oocytes cultured in the presence of 10 µM SPC, the GVBD rate was only 54.23% at 24 h, whereas 82.96% control oocytes underwent GVBD. Even when cultured for 30 h, the GVBD rate still remained with the same tendency in the three groups: GVBD rates were 60.89% (SPC), 88.38% (SM), and 84.86% (control), respectively (Fig. 6B).


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 effect of SPC and SM on meiotic resumption in porcine oocytes.(A) Porcine oocytes were collected in TCM-199 medium with or without 1 or 10 µM SPC or 10 µM SM and then placed in 200 µl drops under mineral oil. Oocytes were scored for meiotic maturation at 0 h, 12 h, 24 h, 30 h, and 36 h. (B) GVBD rates of porcine oocytes treated with 10 µM SPC, 10 µM SM or without ligand 24 h or 30 h of isolation. The number “n” on top of the bars indicates the total treated oocytes in each group. Letters ‘a’, ‘b’ and the symbol “*” indicate statistically significant difference (P<0.05). Data represent the mean ± SEM of at least three repeated experiments. (C) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after SM or SPC treatment by western blot using 150 oocytes in each sample. The results are shown along with those of control oocytes cultured without any drugs. Samples were collected from culture medium with or without SM or SPC at 0 h, 24 h and 30 h.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038807-g006: The effect of SPC and SM on meiotic resumption in porcine oocytes.(A) Porcine oocytes were collected in TCM-199 medium with or without 1 or 10 µM SPC or 10 µM SM and then placed in 200 µl drops under mineral oil. Oocytes were scored for meiotic maturation at 0 h, 12 h, 24 h, 30 h, and 36 h. (B) GVBD rates of porcine oocytes treated with 10 µM SPC, 10 µM SM or without ligand 24 h or 30 h of isolation. The number “n” on top of the bars indicates the total treated oocytes in each group. Letters ‘a’, ‘b’ and the symbol “*” indicate statistically significant difference (P<0.05). Data represent the mean ± SEM of at least three repeated experiments. (C) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after SM or SPC treatment by western blot using 150 oocytes in each sample. The results are shown along with those of control oocytes cultured without any drugs. Samples were collected from culture medium with or without SM or SPC at 0 h, 24 h and 30 h.
Mentions: To detect whether a GPR3 ligand, sphingosylphosphorylcholine (SPC), affects meiotic maturation, porcine oocytes were cultured in the presence or absence of 1 to 10 µM SPC. Negative control was sphingomyelin (SM) (10 µM), a phospholipid (a non-ligand for GPRs) present in the plasma membrane. Oocytes cultured in the presence of 10 µM SPC showed a significant delay in the GVBD (Fig. 6A). However, oocytes cultured in SM showed no difference in GVBD rate at 24 h as compared with the control. In porcine oocytes cultured in the presence of 10 µM SPC, the GVBD rate was only 54.23% at 24 h, whereas 82.96% control oocytes underwent GVBD. Even when cultured for 30 h, the GVBD rate still remained with the same tendency in the three groups: GVBD rates were 60.89% (SPC), 88.38% (SM), and 84.86% (control), respectively (Fig. 6B).

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