The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

• Klíčová slova
• AKT kinase, FGF2, IGF1, LIF, MAP kinase 3/1, gene expression, oocyte competence, oocyte maturation,
• MeSH
• IVM techniky metody   veterinární   MeSH
• kultivační média chemie   farmakologie   MeSH
• kultivované buňky MeSH
• meióza účinky léků   fyziologie   MeSH
• mitogenem aktivovaná proteinkinasa 1 fyziologie   MeSH
• mitogenem aktivovaná proteinkinasa 3 fyziologie   MeSH
• oocyty cytologie   účinky léků   fyziologie   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• prasata MeSH
• protoonkogenní proteiny c-akt fyziologie   MeSH
• signální transdukce účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kultivační média MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivovaná proteinkinasa 3 MeSH
• protoonkogenní proteiny c-akt MeSH

The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.

• Klíčová slova
• FSH, amphiregulin, cumulus cells, epidermal growth factor receptor, mitogen-activated protein kinase 3/1, signal transduction,
• MeSH
• aktivace transkripce MeSH
• amfiregulin metabolismus   MeSH
• erbB receptory metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy genetika   MeSH
• folikuly stimulující hormon farmakologie   MeSH
• IVM techniky MeSH
• kultivované buňky MeSH
• kumulární buňky cytologie   účinky léků   metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 1 genetika   MeSH
• mitogenem aktivovaná proteinkinasa 3 genetika   MeSH
• oocyty cytologie   účinky léků   metabolismus   MeSH
• prasata MeSH
• signální transdukce účinky léků   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amfiregulin MeSH
• erbB receptory MeSH
• extracelulárním signálem regulované MAP kinasy MeSH
• folikuly stimulující hormon MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivovaná proteinkinasa 3 MeSH
• skupina kinas odvozených od src-genu MeSH

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.

• MeSH
• folikulární buňky metabolismus   MeSH
• MAP kinasový signální systém genetika   MeSH
• meióza fyziologie   MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 3 metabolismus   MeSH
• mutace MeSH
• myši MeSH
• oocyty fyziologie   MeSH
• steroidy biosyntéza   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivovaná proteinkinasa 3 MeSH
• steroidy MeSH

BACKGROUND: The 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. METHODS: We 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. RESULTS: Both 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. CONCLUSIONS: The 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.

• MeSH
• amfiregulin farmakologie   fyziologie   MeSH
• epidermální růstový faktor farmakologie   fyziologie   MeSH
• epiregulin farmakologie   fyziologie   MeSH
• folikuly stimulující hormon farmakologie   fyziologie   MeSH
• kultivované buňky MeSH
• kumulární buňky účinky léků   metabolismus   MeSH
• oocyty účinky léků   metabolismus   MeSH
• prasata MeSH
• regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfiregulin MeSH
• epidermální růstový faktor MeSH
• epiregulin MeSH
• folikuly stimulující hormon MeSH

BACKGROUND: Recent 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). METHODS: The 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. RESULTS: The 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). CONCLUSIONS: The 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.

• MeSH
• aktivace enzymů účinky léků   MeSH
• gonadotropiny farmakologie   MeSH
• guanosinmonofosfát cyklický farmakologie   MeSH
• kultivované buňky MeSH
• kumulární buňky účinky léků   fyziologie   MeSH
• meióza účinky léků   MeSH
• mitogenem aktivovaná proteinkinasa 3 metabolismus   MeSH
• oocyty účinky léků   fyziologie   MeSH
• oogeneze účinky léků   MeSH
• Sus scrofa MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• gonadotropiny MeSH
• guanosinmonofosfát cyklický MeSH
• mitogenem aktivovaná proteinkinasa 3 MeSH