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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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Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc6-GPR3 or control Myc6 and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected 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 after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without 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-g003: Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc6-GPR3 or control Myc6 and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected 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 after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without 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: To further study the effect of GPR3 overexpression on arrest of meiotic resumption, we examined the GVBD rate after overexpression. In our culture system without HX, 81.42% of the oocytes underwent GVBD at 24 h of culture and the rate reached 85.87% at 30 h (Fig. 3A). Injection of Myc6 mRNA did not significantly change these rates (72.95% and 78.95%, respectively). However, when oocytes were injected with Myc6-GPR3 mRNA, the GVBD rates were significantly decreased to 44.34% and 48.37% at 24 h and 30 h, respectively. These results suggested that overexpression of GPR3 inhibited meiotic resumption.


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)

Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc6-GPR3 or control Myc6 and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected 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 after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038807-g003: Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc6-GPR3 or control Myc6 and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected 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 after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc6-GPR3 injection group and control Myc6 injection group after culture without 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: To further study the effect of GPR3 overexpression on arrest of meiotic resumption, we examined the GVBD rate after overexpression. In our culture system without HX, 81.42% of the oocytes underwent GVBD at 24 h of culture and the rate reached 85.87% at 30 h (Fig. 3A). Injection of Myc6 mRNA did not significantly change these rates (72.95% and 78.95%, respectively). However, when oocytes were injected with Myc6-GPR3 mRNA, the GVBD rates were significantly decreased to 44.34% and 48.37% at 24 h and 30 h, respectively. These results suggested that overexpression of GPR3 inhibited meiotic resumption.

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