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.

To define signaling pathways that drive FSH-and epidermal growth factor (EGF)-like peptide-induced cumulus expansion and oocyte meiotic resumption, in vitro cultured pig cumulus-oocyte complexes were treated with specific protein kinase inhibitors. We found that FSH-induced maturation of oocytes was blocked in germinal vesicle (GV) stage by protein kinase A (PKA), MAPK14, MAPK3/1, and EGF receptor (EGFR) tyrosine kinase inhibitors (H89, SB203580, U0126, and AG1478 respectively) whereas phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog (PI3K/AKT) inhibitor (LY294002) blocked maturation of oocytes in metaphase I (MI). Amphiregulin (AREG)-induced maturation of oocytes was efficiently blocked in GV by U0126, AG1478, and low concentrations of LY294002; H89, SB203580, and high concentrations of LY294002 allowed the oocytes to undergo breakdown of GV and blocked maturation in MI. Both FSH-and AREG-induced cumulus expansion was incompletely inhibited by H89 and completely inhibited by SB203580, U0126, AG1478, and LY294002. The inhibitors partially or completely inhibited expression of expansion-related genes (HAS2, PTGS2, and TNFAIP6) with two exceptions: H89 inhibited only TNFAIP6 expression and LY294002 increased expression of PTGS2. The results of this study are consistent with the idea that PKA and MAPK14 pathways are essential for FSH-induced transactivation of the EGFR, and synthesis of EGF-like peptides in cumulus cells and MAPK3/1 is involved in regulation of transcriptional and posttranscriptional events in cumulus cells required for meiotic resumption and cumulus expansion. PI3K/AKT signaling is important for regulation of cumulus expansion, AREG-induced meiotic resumption, and oocyte MI/MII transition. The present data also indicate the existence of an FSH-activated and PKA-independent pathway involved in regulation of HAS2 and PTGS2 expression in cumulus cells.

BackgroundThe gonadotropin-induced resumption of oocyte meiosis in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. Both the gonadotropins and the EGF-like peptides possess the capacity to stimulate resumption of oocyte meiosis in vitro via activation of a broad signaling network in cumulus cells. To better understand the rapid genomic actions of gonadotropins (FSH) and EGF-like peptides, we analyzed transcriptomes of cumulus cells at 3 h after their stimulation.MethodsWe hybridized aRNA from cumulus cells to a pig oligonucleotide microarray and compared the transcriptomes of FSH- and AREG/EREG-stimulated cumulus cells with untreated control cells and vice versa. The identified over- and underexpressed genes were subjected to functional genomic analysis according to their molecular and cellular functions. The expression pattern of 50 selected genes with a known or potential function in ovarian development was verified by real-time qRT-PCR.ResultsBoth FSH and AREG/EREG increased the expression of genes associated with regulation of cell proliferation, cell migration, blood coagulation and extracellular matrix remodeling. FSH alone induced the expression of genes involved in inflammatory response and in the response to reactive oxygen species. Moreover, FSH stimulated the expression of genes closely related to some ovulatory events either exclusively or significantly more than AREG/EREG (AREG, ADAMTS1, HAS2, TNFAIP6, PLAUR, PLAT, and HSD17B7). In contrast to AREG/EREG, FSH also increased the expression of genes coding for key transcription factors (CEBPB, FOS, ID1/3, and NR5A2), which may contribute to the differing expression profiles of FSH- and AREG/EREG-treated cumulus cells.ConclusionsThe impact of FSH on cumulus cell gene transcription was higher than the impact of EGF-like factors in terms of the number of cell functions affected as well as the number of over- and underexpressed genes. Both FSH and EGF-like factors overexpressed genes involved in the post-ovulatory switch in steroidogenesis and tissue remodelling. However, FSH was remarkably more efficient in the up-regulation of several specific genes essential for ovulation of matured oocytes and also genes that been reported to play an important role in maturation of cumulus-enclosed oocytes in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-015-0112-2) contains supplementary material, which is available to authorized users.

Freshly isolated quail embryonic heart at Hamburger‐Hamilton stage 28, stained with voltage sensitive dye and optically mapped to reveal ventricular activation pattern (indicated by color isochrones in 1 ms intervals). The activation wave sweeps from left to right in the direction of the arrow. Maturation of ventricular activation patterns is accelerated by hypoxic incubation, as described in detail together with other changes in ventricular angio‐ and myoarchitecture. From “Abnormal Myocardial and coronary Vasculature Development in Experimental Hypoxia,” by Ondrej Nanka, et al., on page 1187, in this issue.

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.