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

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

• Klíčová slova
• cytoplasmic maturation, egg quality, electron microscopy, human oocyte, in vitro maturation, oocyte ultrastructure,
• MeSH
• cytoplazma účinky léků   ultrastruktura   MeSH
• dospělí MeSH
• endoplazmatické retikulum účinky léků   ultrastruktura   MeSH
• folikuly stimulující hormon farmakologie   MeSH
• indukce ovulace MeSH
• lidé MeSH
• mitochondrie účinky léků   ultrastruktura   MeSH
• mladý dospělý MeSH
• oocyty účinky léků   ultrastruktura   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• segregace chromozomů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladý dospělý MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• folikuly stimulující hormon MeSH

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

• Klíčová slova
• MLN4924, SCF complexes, cumulus cells, early development, oocyte, ubiquitin–proteasome system,
• MeSH
• blastocysta cytologie   účinky léků   fyziologie   MeSH
• časové faktory MeSH
• cyklopentany farmakologie   MeSH
• embryo savčí MeSH
• embryonální vývoj účinky léků   MeSH
• IVM techniky metody   veterinární   MeSH
• kultivované buňky MeSH
• multiproteinové komplexy antagonisté a inhibitory   metabolismus   MeSH
• oocyty cytologie   účinky léků   fyziologie   MeSH
• oogeneze účinky léků   MeSH
• proteinligasy komplexu SCF antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• pyrimidiny farmakologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklopentany MeSH
• multiproteinové komplexy MeSH
• pevonedistat MeSH Prohlížeč
• proteinligasy komplexu SCF MeSH
• pyrimidiny MeSH

The key mechanisms responsible for achievement of full reproductive and developmental capability in mammals are the differentiation and transformation of granulosa cells (GCs) during folliculogenesis, oogenesis, and oocyte maturation. Although the role of 17 beta-estradiol (E2) in ovarian activity is widely known, its effect on proliferative capacity, gap junction connection (GJC) formation, and GCs-luteal cells transformation requires further research. Therefore, the goal of this study was to assess the real-time proliferative activity of porcine GCs in vitro in relation to connexin (Cx), luteinizing hormone receptor (LHR), follicle stimulating hormone receptor (FSHR), and aromatase (CYP19A1) expression during short-term (168 h) primary culture. The cultured GCs were exposed to acute (at 96 h of culture) and/or prolonged (between 0 and 168 h of culture) administration of 1.8 and 3.6 μM E2. The relative abundance of Cx36, Cx37, Cx40, Cx43, LHR, FSHR, and CYP19A1 mRNA was measured. We conclude that the proliferation capability of GCs in vitro is substantially associated with expression of Cxs, LHR, FSHR, and CYP19A1. Furthermore, the GC-luteal cell transformation in vitro may be significantly accompanied by the proliferative activity of GCs in pigs.

• MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• estradiol aplikace a dávkování   MeSH
• folikulární buňky účinky léků   metabolismus   MeSH
• IVM techniky MeSH
• lidé MeSH
• oocyty účinky léků   růst a vývoj   MeSH
• oogeneze účinky léků   genetika   MeSH
• prasata MeSH
• proliferace buněk účinky léků   MeSH
• receptory FSH biosyntéza   genetika   MeSH
• receptory LH biosyntéza   genetika   MeSH
• rodina 19 cytochromů P450 biosyntéza   MeSH
• vývojová regulace genové exprese účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• estradiol MeSH
• receptory FSH MeSH
• receptory LH MeSH
• rodina 19 cytochromů P450 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

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.

• MeSH
• aktivace enzymů účinky léků   MeSH
• aurora kinasa A MeSH
• choriogonadotropin farmakologie   MeSH
• HeLa buňky MeSH
• kinasy aurora MeSH
• kultivované buňky MeSH
• lidé MeSH
• meióza účinky léků   genetika   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• oocyty enzymologie   metabolismus   fyziologie   MeSH
• oogeneze účinky léků   genetika   fyziologie   MeSH
• organizační centrum mikrotubulů účinky léků   metabolismus   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• AURKA protein, human MeSH Prohlížeč
• Aurka protein, mouse MeSH Prohlížeč
• aurora kinasa A MeSH
• choriogonadotropin MeSH
• kinasy aurora MeSH
• protein-serin-threoninkinasy MeSH

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.

• MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• embryonální vývoj účinky léků   genetika   MeSH
• epidermální růstový faktor chemie   farmakologie   MeSH
• folikuly stimulující hormon farmakologie   MeSH
• gonadotropiny farmakologie   MeSH
• kultivace embrya MeSH
• kultivované buňky MeSH
• kumulární buňky účinky léků   metabolismus   fyziologie   MeSH
• oocyty účinky léků   metabolismus   fyziologie   MeSH
• oogeneze účinky léků   genetika   MeSH
• partenogeneze účinky léků   genetika   fyziologie   MeSH
• peptidové fragmenty chemie   farmakologie   MeSH
• prasata * genetika   metabolismus   fyziologie   MeSH
• proliferace buněk účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• epidermální růstový faktor MeSH
• folikuly stimulující hormon MeSH
• gonadotropiny MeSH
• peptidové fragmenty MeSH

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

• MeSH
• aktivace enzymů účinky léků   fyziologie   MeSH
• buněčné kultury MeSH
• dibutyryl cyklický AMP farmakologie   MeSH
• fosfatidylinositoly farmakologie   MeSH
• fosforylace účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• kultivované buňky MeSH
• meióza * fyziologie   MeSH
• onkogenní protein v-akt antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• oocyty účinky léků   metabolismus   fyziologie   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• prasata * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dibutyryl cyklický AMP MeSH
• fosfatidylinositoly MeSH
• inhibitory enzymů MeSH
• onkogenní protein v-akt MeSH
• SH-6 compound MeSH Prohlížeč

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless(1) (ecd(1)) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd(1) conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd(-) clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.

• MeSH
• Drosophila genetika   růst a vývoj   MeSH
• ekdyson farmakologie   MeSH
• endokrinní žlázy růst a vývoj   metabolismus   MeSH
• larva MeSH
• letální geny MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• oogeneze účinky léků   genetika   MeSH
• ovarium růst a vývoj   metabolismus   MeSH
• proteiny Drosophily genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• steroidy metabolismus   MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• ecd protein, Drosophila MeSH Prohlížeč
• ekdyson MeSH
• proteiny Drosophily MeSH
• steroidy MeSH

The present study aimed to demonstrate the dependence of meiotic maturation in pig oocytes on the activity of the protease complex proteasome. The proteasome inhibitor MG132 blocked the exit of maturing pig oocytes from metaphase I stage. Seventy-five per cent of the oocytes were blocked at metaphase I when they were cultured with 10 microM MG132. The blocking effect of MG132 was expressed only when the oocytes were exposed to an inhibitor before the 18th hour of in vitro culture. The effects of MG132 are fully reversible. However, a significant proportion of oocytes (46%) cultured for 48 h in MG132-supplemented medium and then for 24 h in MG132-free medium did not block meiosis at the stage of metaphase II and underwent spontaneous parthenogenetic activation. On the basis of our data we can conclude that exit from the metaphase I stage of meiosis is proteasome-dependent in pig oocytes matured in vitro. On the other hand, our data also indicate that other proteasome-independent events are involved in regulating the exit from metaphase I.

• MeSH
• inhibitory cysteinových proteinas farmakologie   MeSH
• inhibitory proteasomu MeSH
• leupeptiny farmakologie   MeSH
• oocyty účinky léků   enzymologie   růst a vývoj   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• Sus scrofa MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzyloxycarbonylleucyl-leucyl-leucine aldehyde MeSH Prohlížeč
• inhibitory cysteinových proteinas MeSH
• inhibitory proteasomu MeSH
• leupeptiny MeSH