Abstract
BackgroundRecent results indicate a key role for cyclic guanosine monophosphate (cGMP) in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles. The aim of our study was to determine whether the resumption of oocyte meiosis and expansion of cumulus cells in isolated pig cumulus-oocyte complexes (COCs) can be blocked by a high intracellular concentration of cGMP, and whether this effect is mediated by a cGMP-dependent inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1).MethodsThe COCs were isolated from ovaries of slaughtered gilts and cultured in vitro in M199 supplemented with 5% fetal calf serum. The expression levels of the C-type natriuretic peptide (CNP) precursor (NPPC) and its receptor (NPR2) mRNAs during the culture of COCs were determined by real-time RT-PCR. To control the intracellular concentration of cGMP in the COCs, the culture medium was further supplemented with CNP or various concentrations of synthetic cGMP analogues; the concentration of cGMP in COCs was then assessed by ELISA. The effect of the drugs on oocyte maturation was assessed after 24 and 44 h of culture by determining nuclear maturation. The expansion of cumulus cells was assessed by light microscopy and the expression of cumulus expansion-related genes by real-time RT-PCR. A possible effect of cGMP on FSH-induced activation of MAPK3/1 was assessed by immunoblotting the COC proteins with phospho-specific and total anti-Erk1/2 antibodies.ResultsThe COCs expressed NPPC and NPR2, the key components of cGMP synthesis, and produced a large amount of cGMP upon stimulation with exogenous CNP, which lead to a significant (P < 0.05) delay in oocyte meiotic resumption. The COCs also responded to cGMP analogues by inhibiting the resumption of oocyte meiosis. The inhibitory effect of cGMP on meiotic resumption was reversed by stimulating the COCs with FSH. However, high concentration of intracellular cGMP was not able to suppress FSH-induced activation of MAPK3/1 in cumulus cells, cumulus expansion and expression of expansion-related genes (P > 0.05).ConclusionsThe findings of this study indicate that high cGMP concentrations inhibit the maturation of pig oocytes in vitro but the inhibitory mechanism does not involve the suppression of MAPK3/1 activation in cumulus cells.
Highlights
Recent results indicate a key role for cyclic guanosine monophosphate in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles
We found that the expression of natriuretic peptide receptor 2 (NPR2) in the follicle-stimulating hormone (FSH)-stimulated Cumulus-oocyte complex (COC) significantly (P < 0.05) decreased after 4 h of culture and remained low until the end of the experiment at 20 h
The high level of cyclic guanosine monophosphate (cGMP) induced by 1 h pre-culture of the COCs with C-type natriuretic peptide (CNP) was not reduced by FSH for 8 h of culture, but it was significantly (P < 0.01) lowered at 12 h of culture (Figure 2b)
Summary
Recent results indicate a key role for cyclic guanosine monophosphate (cGMP) in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles. The first pathway consists of a reduction in cGMP transport from somatic follicular cells to the oocyte This pathway is dependent on the kinase activity of the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases 3/1 (MAPK3/1) that are required for the phosphorylation of connexin 43 and gap junction closure [9,10,11]. In response to LH stimulus, EGF-like factors amphiregulin (AREG) and epiregulin (EREG) are expressed and released from mural granulosa cells in mice [12]. These peptides trigger the resumption of meiosis and cumulus expansion in mice [12], pigs [13] and humans [14]. The cGMP level may be reduced in somatic cells of the follicle even if the EGFR activity is inhibited or absent [10]
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