Pre-IVM with C-type natriuretic peptide promotes mitochondrial biogenesis of bovine oocytes via activation of CREB

Abstract

The aim of this study was to evaluate the effects of C-type natriuretic peptide (CNP) treatment prior to in vitro maturation (IVM) on mitochondria biogenesis in bovine oocyte matured in vitro and explore the related causes. The results showed that treatment with CNP before IVM significantly improved mitochondrial content, elevated the expression of genes related to mitochondria biogenesis, and increased the protein levels of phosphorylation of cAMP-response element binding protein (p-CREB) in bovine oocytes following IVM. However, further studies revealed that treatment with CNP before IVM could not increased the protein levels of p-CREB in bovine oocytes when natriuretic peptide receptor 2 activities was inhibited using the relative specific inhibitor Gö6976. In addition, treatment with CNP before IVM could not improved mitochondrial content or elevated the expression of genes related to mitochondria biogenesis in bovine oocytes when CREB activities was abolished using the specific inhibitor 666-15. In summary, these results provide evidence that treatment of bovine oocytes with CNP before IVM promotes mitochondrial biogenesis in vitro, possibly by activating CREB.

Keywords: Bovine oocyte; CNP; CREB; Mitochondrial biogenesis.

Figure 1

Effects of CNP treatment before IVM on mitochondria biogenesis during bovine oocytes maturation. Immature cumulus-oocyte complexes (COCs) were subjected to IVM for 24 h (Control no pre-IVM), or cultured in pre-IVM medium supplemented without or with CNP (Control pre-IVM and CNP pre-IVM, respectively) for 6 h, followed by 24 h of IVM. (A) Representative images of the oocyte stained with MitoTracker green in each treatment group after IVM. Scale bar = 50 µm. (B) Relative fluorescence intensities of the mitochondrial content in each treatment group oocytes after IVM (56 oocytes/group). (C–E) Relative mRNA transcript abundance of genes modulating mitochondria biogenesis, mitochondrial proteins genes encoded by both nuclear and mitochondrial genomes in bovine oocytes from each treatment group after IVM (150 oocytes/per group). Compared with the oocytes in the control (no pre-IVM) and pre-IVM (no CNP) group, *P < 0.05. The data are from at least three independent experiments.

Figure 2

Effects of CNP treatment before IVM on abundance of p-CREB protein during bovine oocytes maturation. Immature cumulus-oocyte complexes (COCs) were subjected to IVM for 24 h (Control no pre-IVM), or cultured in pre-IVM medium supplemented without or with CNP (Control pre-IVM and CNP pre-IVM, respectively) for 6 h, followed by 24 h of IVM. (A) Representative immunofluorescence images of the p-CREB in bovine oocytes from each treatment group. Scale bar = 50 µm (B) Quantitative analysis of abundance of bovine oocytes p-CREB protein by immunostaining in each treatment group after IVM. Compared with the oocytes in the control (no pre-IVM) and pre-IVM (no CNP) group, *P < 0.05. The data are from at least three independent experiments.

Figure 3

NPR2 inhibition abrogates CNP-stimulated CREB phosphorylation during bovine oocytes maturation. Immature cumulus-oocyte complexes (COCs) were treated in basal medium in the absence or presence of 200 nM CNP and/or 5 μM Gö6976, a competitive inhibitor of NPR2 (Basal medium, CNP, Gö6976, and CNP + Gö6976, respectively) for 6 h, followed by IVM for 24 h. (A) Representative immunofluorescence images of the p-CREB in bovine oocytes from each treatment group. Scale bar = 50 µm. (B) Quantitative analysis of abundance of bovine oocytes p-CREB protein by immunostaining in each treatment group after IVM. *P < 0.05. The data are from at least three independent experiments.

Figure 4

Inhibition of CREB abolishes the beneficial effect of CNP treatment before IVM on mitochondria biogenesis during bovine oocytes maturation. Immature cumulus-oocyte complexes (COCs) were treated in basal medium in the absence or presence of 200 nM CNP and/or 1 μM 666-15, a specific CREB inhibitor (Basal medium, CNP, 666-15 and CNP+666-15, respectively) for 6 h, followed by 24 h of IVM. (A) Representative images of the oocyte stained with MitoTracker green in each treatment group after IVM. Scale bar = 50 µm. (B) Relative fluorescence intensities of the mitochondrial content in each treatment group oocytes after IVM. (C) Relative mRNA transcript abundance of genes modulating mitochondria biogenesis in bovine oocytes from each treatment group after IVM. *P < 0.05. The data are from at least three independent experiments.