259 PRELIMINARY INVESTIGATIONS OF THE EFFECT OF NITRIC OXIDE ON THE EXPRESSION OF GENES INVOLVED IN THE NITRIC OXIDE/CYCLIC GUANOSINE MONOPHOSPHATE/CYCLIC ADENOSINE MONOPHOSPHATE PATHWAY DURING MEIOSIS RESUMPTION IN BOVINE OOCYTES

Abstract

Nitric oxide is a chemical messenger generated by the activity of the enzyme NO synthase (NOS) and has been implicated in the control of oocyte maturation. Nitric oxide stimulates guanylate cyclase (GC) to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases (PDE) that may interfere with cyclic adenosine monophosphate (cAMP) levels, a nucleotide also involved in meiosis resumption. In a previous study, we found that increasing NO levels in the presence of a NO donor (S-nitroso-N-acetylpenicillamine, SNAP) resulted in a delayed resumption of meiosis and a lower rate of germinal vesicle breakdown after 9 h of in vitro maturation. A temporary increase in cGMP levels was also observed with the same treatment, which was reversed by inhibiting GC activity with oxadiazolo-quinoxaline-one (ODQ; unpublished data). These results suggest that NO acted via GC/cGMP and that even a temporary increase in the cGMP level led to a delay of meiosis resumption. The aim of the present study was to determine the role played by NO on the expression of genes encoding for enzymes of the NO/GC/cGMP and cAMP pathways during the first 9 h of oocyte maturation. Cumulus–oocyte complexes were in vitro matured for 9 h in a semi-defined medium (TCM-199 + 3 mg mL–1 of BSA) with 10 to 7 M SNAP associated or not associated with 100 µM ODQ, a GC inhibitor. A group of oocytes incubated in the absence of inhibitors was considered the control. Total RNA was extracted from pools of 20 denuded oocytes with TRIzol (Life Technologies, Grand Island, NY, USA) and reverse transcribed into complementary DNA using a high-capacity reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed by real-time PCR using SYBR Green (Applied Biosystems). The genes that had their expression measured pertained to one of the following groups: 1) genes encoding for enzymes that synthesise NO (NOS2 and NOS3); 2) genes involved in the control of cGMP levels (GUCY1B3 and PDE5A) or the enzymes activated by it (PKG1 and PKG2); or 3) genes involved in the control of cAMP levels (ADCY3, ADCY6, ADCY9, PDE3A, and PDE8A) or one of the enzymes activated by it (PKA1). GAPDH and PPIA were selected as housekeeping genes using qbasePLUS version 2.3 (Biogazelle, Zwijnaarde, Belgium). Data from 5 replicates were analysed using LinRegPCR version 11.1 and SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). All genes were found to be expressed in the three experimental groups; however, a significant difference in gene expression levels was not found among groups. Results suggest that NO does not act on oocyte maturation by affecting the expression of the investigated genes in oocytes. To our knowledge, this is the first report to demonstrate the expression of the ADCY3, ADCY6, and ADCY9 genes in bovine oocytes. Further research is in progress to study the effect of the SNAP treatment on the expression of these genes in cumulus cells. Financial support from FAPESP 2010/18023-9.