A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development.

• Keywords
• Akt kinase, early embryo, mRNA translation, mTORC1, meiosis, mitosis, oocyte, spindle,
• MeSH
• Phosphatidylinositol 3-Kinase metabolism   MeSH
• Embryonic Development MeSH
• Phosphatidylinositol 3-Kinases * metabolism   MeSH
• Phosphoproteins metabolism   MeSH
• Phosphorylation genetics   MeSH
• Oocytes metabolism   MeSH
• Oogenesis MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Proto-Oncogene Proteins c-akt * metabolism   MeSH
• Mammals metabolism   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Phosphatidylinositol 3-Kinase MeSH
• Phosphatidylinositol 3-Kinases * MeSH
• Phosphoproteins MeSH
• Protein Serine-Threonine Kinases MeSH
• Proto-Oncogene Proteins c-akt * MeSH

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.

• Keywords
• AKT kinase, FGF2, IGF1, LIF, MAP kinase 3/1, gene expression, oocyte competence, oocyte maturation,
• MeSH
• In Vitro Oocyte Maturation Techniques methods   veterinary   MeSH
• Culture Media chemistry   pharmacology   MeSH
• Cells, Cultured MeSH
• Meiosis drug effects   physiology   MeSH
• Mitogen-Activated Protein Kinase 1 physiology   MeSH
• Mitogen-Activated Protein Kinase 3 physiology   MeSH
• Oocytes cytology   drug effects   physiology   MeSH
• Oogenesis drug effects   physiology   MeSH
• Swine MeSH
• Proto-Oncogene Proteins c-akt physiology   MeSH
• Signal Transduction drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Culture Media MeSH
• Mitogen-Activated Protein Kinase 1 MeSH
• Mitogen-Activated Protein Kinase 3 MeSH
• Proto-Oncogene Proteins c-akt MeSH

Traditional methods for the evaluation of oocyte quality are based on morphological classification of the follicle, cumulus-oocyte complex, polar body and meiotic spindle. This study is focused on the differences between the morphological assessment of oocyte quality, the assessment based on Lissamine Green B (LB) staining and the analysis of oocytes using a proteomic approach. We evaluated the effectiveness of electrochemical and chemical parthenogenetic activation under our laboratory conditions and evaluated the applicability of Lissamine Green B staining of cumulus-oocyte complexes (COCs) as a non-invasive method for predicting the maturational and developmental competence of porcine oocytes cultured in vitro. We determined that chemical parthenogenetic activation using ionomycin and 6-dimethylaminopurine was slightly more effective than electrochemical activation. After oocyte selection according to LB staining, we found significant differences (P<0.05) between the LB- group and LB+ group and the control group in their maturation, cleavage rate and rate of blastocysts. Proteomic analyses identified a selection of proteins that were differentially expressed in each group of analysed oocytes. Oocytes of the LB- group exhibited an increased variability of proteins involved in transcription regulation, proteosynthesis and the protein folding crucial for oocyte maturation and further embryonic development. These results found a better competence of LB- oocytes in maturation, cleavage and ability to reach the blastocyst stage.

• Keywords
• Lissamine Green B, mass spectrometry, oocyte quality, parthenogenetic activation, pig,
• Publication type
• Journal Article MeSH

Fertilization of the mammalian oocyte requires interactions between spermatozoa and expanded cumulus extracellular matrix (ECM) that surrounds the oocyte. This review focuses on key molecules that play an important role in the formation of the cumulus ECM, generated by the oocyte-cumulus complex. In particular, the specific inhibitors (AG1478, lapatinib, indomethacin and MG132) and progesterone receptor antagonist (RU486) exerting their effects through the remodeling of the ECM of the cumulus cells surrounding the oocyte have been described. After gonadotropin stimulus, cumulus cells expand and form hyaluronan (HA)-rich cumulus ECM. In pigs, the proper structure of the cumulus ECM depends on the interaction between HA and serum-derived proteins of the inter-alpha-trypsin inhibitor (IαI) protein family. We have demonstrated the synthesis of HA by cumulus cells, and the presence of the IαI, tumor necrosis factor-alpha-induced protein 6 and pentraxin 3 in expanding oocyte-cumulus complexes (OCC). We have evaluated the covalent linkage of heavy chains of IαI proteins to HA, as the principal component of the expanded HA-rich cumulus ECM, in porcine OCC cultured in medium with specific inhibitors: AG1478 and lapatinib (both inhibitors of epidermal growth factor receptor tyrosine kinase activity); MG132 (a specific proteasomal inhibitor), indomethacin (cyclooxygenase inhibitor); and progesterone receptor antagonist (RU486). We have found that both RU486 and indomethacin does not disrupt the formation of the covalent linkage between the heavy chains of IαI to HA in the expanded OCC. In contrast, the inhibitors AG1478 and lapatinib prevent gonadotropin-induced cumulus expansion. Finally, the formation of oocyte-cumulus ECM relying on the covalent transfer of heavy chains of IαI molecules to HA has been inhibited in the presence of MG132.

• Keywords
• extracellular matrix, hyaluronan, inter-alpha-trypsin inhibitor, oocyte-cumulus complexes, pentraxin 3, tumor necrosis factor-alpha-induced protein 6,
• MeSH
• C-Reactive Protein metabolism   MeSH
• Extracellular Matrix drug effects   metabolism   MeSH
• Cumulus Cells cytology   drug effects   metabolism   MeSH
• Hyaluronic Acid metabolism   MeSH
• Mifepristone pharmacology   MeSH
• Cell Adhesion Molecules metabolism   MeSH
• Oocytes cytology   metabolism   MeSH
• Reproduction drug effects   MeSH
• Serum Amyloid P-Component metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• C-Reactive Protein MeSH
• Hyaluronic Acid MeSH
• Mifepristone MeSH
• Cell Adhesion Molecules MeSH
• PTX3 protein MeSH Browser
• Serum Amyloid P-Component MeSH
• Tnfaip6 protein, mouse MeSH Browser

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
• Granulosa Cells metabolism   MeSH
• MAP Kinase Signaling System genetics   MeSH
• Meiosis physiology   MeSH
• Mitogen-Activated Protein Kinase 1 metabolism   MeSH
• Mitogen-Activated Protein Kinase 3 metabolism   MeSH
• Mutation MeSH
• Mice MeSH
• Oocytes physiology   MeSH
• Steroids biosynthesis   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Mitogen-Activated Protein Kinase 1 MeSH
• Mitogen-Activated Protein Kinase 3 MeSH
• Steroids 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
• Amphiregulin pharmacology   physiology   MeSH
• Epidermal Growth Factor pharmacology   physiology   MeSH
• Epiregulin pharmacology   physiology   MeSH
• Follicle Stimulating Hormone pharmacology   physiology   MeSH
• Cells, Cultured MeSH
• Cumulus Cells drug effects   metabolism   MeSH
• Oocytes drug effects   metabolism   MeSH
• Swine MeSH
• Gene Expression Regulation MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amphiregulin MeSH
• Epidermal Growth Factor MeSH
• Epiregulin MeSH
• Follicle Stimulating Hormone 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
• Enzyme Activation drug effects   MeSH
• Gonadotropins pharmacology   MeSH
• Cyclic GMP pharmacology   MeSH
• Cells, Cultured MeSH
• Cumulus Cells drug effects   physiology   MeSH
• Meiosis drug effects   MeSH
• Mitogen-Activated Protein Kinase 3 metabolism   MeSH
• Oocytes drug effects   physiology   MeSH
• Oogenesis drug effects   MeSH
• Sus scrofa MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gonadotropins MeSH
• Cyclic GMP MeSH
• Mitogen-Activated Protein Kinase 3 MeSH