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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 GPR3 RNAi on meiotic resumption in porcine oocytes.(A) Samples from control and RNAi groups were collected to test the efficiency of GPR3-RNAi. Control: 150 oocytes injected with 25 µM control siRNA; RNAi: 150 oocytes injected with 25 µM GPR3 siRNA. Oocytes were then incubated for 24 h or 30 h in the TCM-199 medium with or without 4 mM HX before collection for western blotting. (B) Oocytes cultured with normal medium without HX were injected with 25 µM GPR3 siRNA or control siRNA and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (C) Oocytes treated with 4 mM HX were injected with the GPR3 siRNA or control siRNA and then cultured in the presence of HX for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (D) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after siRNA injection by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected from culture medium with or without HX at 0 h, 24 h and 30 h. (E) cAMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (F) cGMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).
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pone-0038807-g004: The effect of GPR3 RNAi on meiotic resumption in porcine oocytes.(A) Samples from control and RNAi groups were collected to test the efficiency of GPR3-RNAi. Control: 150 oocytes injected with 25 µM control siRNA; RNAi: 150 oocytes injected with 25 µM GPR3 siRNA. Oocytes were then incubated for 24 h or 30 h in the TCM-199 medium with or without 4 mM HX before collection for western blotting. (B) Oocytes cultured with normal medium without HX were injected with 25 µM GPR3 siRNA or control siRNA and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (C) Oocytes treated with 4 mM HX were injected with the GPR3 siRNA or control siRNA and then cultured in the presence of HX for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (D) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after siRNA injection by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected from culture medium with or without HX at 0 h, 24 h and 30 h. (E) cAMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (F) cGMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).

Mentions: We further used RNAi to investigate the effects of GPR3 downregulation on meiosis progression. The GV stage oocytes were maintained in meiotic arrest by incubation in medium containing HX; they were injected with GPR3 siRNA or negative control siRNA, and the meiotic progression was followed up to 48 h. RNAi efficiency was detected by western blot. Compared with the control groups (control siRNA injection), the GPR3 expression in siRNA-injected oocytes was decreased to 27% (Fig. 4A), revealing successful downregulation.


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 GPR3 RNAi on meiotic resumption in porcine oocytes.(A) Samples from control and RNAi groups were collected to test the efficiency of GPR3-RNAi. Control: 150 oocytes injected with 25 µM control siRNA; RNAi: 150 oocytes injected with 25 µM GPR3 siRNA. Oocytes were then incubated for 24 h or 30 h in the TCM-199 medium with or without 4 mM HX before collection for western blotting. (B) Oocytes cultured with normal medium without HX were injected with 25 µM GPR3 siRNA or control siRNA and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (C) Oocytes treated with 4 mM HX were injected with the GPR3 siRNA or control siRNA and then cultured in the presence of HX for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (D) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after siRNA injection by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected from culture medium with or without HX at 0 h, 24 h and 30 h. (E) cAMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (F) cGMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).
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pone-0038807-g004: The effect of GPR3 RNAi on meiotic resumption in porcine oocytes.(A) Samples from control and RNAi groups were collected to test the efficiency of GPR3-RNAi. Control: 150 oocytes injected with 25 µM control siRNA; RNAi: 150 oocytes injected with 25 µM GPR3 siRNA. Oocytes were then incubated for 24 h or 30 h in the TCM-199 medium with or without 4 mM HX before collection for western blotting. (B) Oocytes cultured with normal medium without HX were injected with 25 µM GPR3 siRNA or control siRNA and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (C) Oocytes treated with 4 mM HX were injected with the GPR3 siRNA or control siRNA and then cultured in the presence of HX for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (D) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after siRNA injection by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected from culture medium with or without HX at 0 h, 24 h and 30 h. (E) cAMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (F) cGMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).
Mentions: We further used RNAi to investigate the effects of GPR3 downregulation on meiosis progression. The GV stage oocytes were maintained in meiotic arrest by incubation in medium containing HX; they were injected with GPR3 siRNA or negative control siRNA, and the meiotic progression was followed up to 48 h. RNAi efficiency was detected by western blot. Compared with the control groups (control siRNA injection), the GPR3 expression in siRNA-injected oocytes was decreased to 27% (Fig. 4A), revealing successful downregulation.

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