BACKGROUND: The full maturational capability of mammalian oocytes is accompanied by nuclear and cytoplasmic modifications, which are associated with proliferation and differentiation of surrounding cumulus cells. These events are regulated on molecular level by the expression of target genes involved in signal transduction pathways crucial for folliculogenesis and oogenesis. Transforming growth factor beta signaling includes several molecules that are involved in the regulation of oogenesis and embryo growth, including bone morphogenetic protein (BMP). However, the BMP-related gene expression profile in oocytes at different maturational stages requires further investigation. METHODS: Oocytes were isolated from pubertal crossbred Landrace gilts follicles, selected with a use of BCB staining test and analyzed before and after in vitro maturation. Gene expression profiles were examined using an Affymetrix microarray approach and validated by RT-qPCR. Database for Annotation, Visualization, and Integrated Discovery (DAVID) software was used for the extraction of the genes belonging to a BMP-signaling pathway ontology group. RESULTS: The assay revealed 12,258 different transcripts in porcine oocytes, among which 379 genes were down-regulated and 40 were up-regulated. The DAVID database indicated a "BMP signaling pathway" ontology group, which was significantly regulated in both groups of oocytes. We discovered five up-regulated genes in oocytes before versus after in vitro maturation (IVM): chordin-like 1 (CHRDL1), follistatin (FST), transforming growth factor-beta receptor-type III (TGFβR3), decapentaplegic homolog 4 (SMAD4), and inhibitor of DNA binding 1 (ID1). CONCLUSIONS: Increased expression of CHRDL1, FST, TGFβR3, SMAD4, and ID1 transcripts before IVM suggested a subordinate role of the BMP signaling pathway in porcine oocyte maturational competence. Conversely, it is postulated that these genes are involved in early stages of folliculogenesis and oogenesis regulation in pigs, since in oocytes before IVM increased expression was observed.
- MeSH
- IVM techniky * MeSH
- kostní morfogenetické proteiny genetika metabolismus MeSH
- kumulární buňky cytologie metabolismus fyziologie MeSH
- mikročipová analýza MeSH
- oocyty cytologie metabolismus fyziologie MeSH
- oogeneze genetika MeSH
- prasata genetika metabolismus MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In recent years, molecular techniques have brought about new solutions that focus on the developmental capacity of female oocytes and reproductive performance in the mammalian species. The developmental potency is the ability of oocytes to reach the MII stage following the long stages of folliculo- and oogenesis. The main proteins involved in this process belong to the connexin (Cx) family, which are responsible for the formation of gap junction (GJC) connections between the female gamete and surrounding somatic cells. The Cx are involved in bi-directional transport of small molecules and are therefore responsible for correct oocyte-somatic cell nutrition, proliferation, and differentiation. However, the application of certain molecular techniques often leads to destabilization or destruction of the materials of interest, such as cells or whole tissues. Therefore, the applications of microfluidic methods, which are non-invasive and quantitative, give new opportunities to further this area of biomedical research. Microfluidic research is based on real-time experiments that allow for control and/ or observation of the results during each step. The purpose of this review is to present both positive and negative aspects of molecular-microfluidic methods while describing the role of connexins in oocyte developmental capacity.
- MeSH
- biologický transport MeSH
- konexiny analýza genetika fyziologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kumulární buňky chemie fyziologie MeSH
- laboratoř na čipu MeSH
- messenger RNA analýza MeSH
- mezerový spoj MeSH
- mezibuněčná komunikace MeSH
- mikrofluidní analytické techniky * MeSH
- molekulární biologie metody MeSH
- oocyty chemie fyziologie MeSH
- oogeneze * MeSH
- savci fyziologie MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.
- MeSH
- IVM techniky metody MeSH
- konfokální mikroskopie metody MeSH
- kumulární buňky cytologie fyziologie MeSH
- membránové glykoproteiny genetika metabolismus MeSH
- oocyty fyziologie MeSH
- psi MeSH
- receptory buněčného povrchu genetika metabolismus MeSH
- regulace genové exprese MeSH
- vaječné proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
It has been shown that following endogenous gonadotropin surge, oocyte-cumulus complexes (OCC) synthesize hyaluronan (HA) in a process called cumulus expansion. During this process, HA associates with proteins and proteoglycans to form the expanded HA-rich oocyte-cumulus extracellular matrix (ECM), where the heavy chains of the serum derived inter-α-trypsin inhibitor family (IαI) bind covalently to HA. No study has been performed on the occurrence and regulation of this process during oocyte maturation in species other than mouse and pig, although, the heavy chains (of IαI)-HA complex was purified from human amniotic membrane. The present review pointing out that: 1/ formation of expanded HA-rich oocyte-cumulus ECM is dependent on the presence of IαI molecules, 2/ the heavy chains of IαI molecules identified in the serum are covalently linked to HA during cumulus expansion in mouse and pig, 3/ the family of IαI molecules can freely cross the blood-follicle barrier, and the follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum to form expanded cumulus ECM in pig, and 4/ proteins of the IαI family can affect reproductive process by modulating the expression of a large number of cellular genes during a preovulatory period. Finally, this review provides clear evidence that IαI family members present in the serum or follicular fluid become responsible for cumulus expansion, as without these proteins, expanded cumulus HA-rich ECM is not formed and HA is released into medium.
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
This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, (3)H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.
- MeSH
- antagonisté hormonů farmakologie MeSH
- C-reaktivní protein genetika metabolismus MeSH
- extracelulární matrix metabolismus MeSH
- folikuly stimulující hormon MeSH
- indomethacin farmakologie MeSH
- inhibitory cyklooxygenasy farmakologie MeSH
- IVM techniky veterinární MeSH
- kumulární buňky účinky léků fyziologie MeSH
- kyselina hyaluronová MeSH
- luteinizační hormon MeSH
- mifepriston farmakologie MeSH
- molekuly buněčné adheze genetika metabolismus MeSH
- oocyty účinky léků fyziologie MeSH
- prasata * MeSH
- progesteron metabolismus MeSH
- regulace genové exprese účinky léků MeSH
- sérový amyloidový protein genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This review deals with molecular mechanisms controlling three important ovarian follicular processes: 1) expansion of the cumulus, 2) synthesis of the hyaluronan (HA), and 3) production of the progesterone in oocyte cumulus complexes (OCCs). The expansion of the mice cumuli induced by FSH or 8-bromo cAMP is dependent upon a specific factor(s) secreted by the oocyte (called "cumulus expansion enabling factor", CEEF). The porcine oocytes produce at least two factors that have influence on the formation and stability of the preovulatory extracellular cumulus matrix (ECM), although oocytectomy does not alter the ability of the cumulus cells to respond to FSH and forskolin by increased cAMP content, HA synthesis, and subsequent cumulus expansion, The net synthesis of HA, during the FSH-stimulated expansion of the OCCs in the presence of serum, correlates directly with accumulation of glycosaminoglycans in the ECM. In pig, insulin growth factor 1 (IGF1) is a component of the serum that promotes the FSH-stimulated synthesis and retention of HA within the expanded ECM by phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)- and mitogen-activated kinase 3 and 1 (MAPK3/1)-dependent mechanisms. Mouse, porcine, bovine, and rat oocytes produce CEEF(s). Possible candidate for the CEEF in the mouse is growth differentiation factor 9 (GDF9) secreted by oocytes. In pig, GDF9 mRNA is expressed not only in the oocytes but also in the cumulus and mural granulosa cells of the growing and preovulatory follicles, although the relative abundance of the GDF9 in the somatic cells is approximately 4 times lower than in the oocytes. Cross talk between FSH/ epidermal growth factor receptor (EGFR) and transforming growth factor β (TGFβ)/GDF9 signaling pathways is essential for functional activities of the porcine OCCs, since FSH enhances EGF-induced tyrosine phosphorylation of EGFR, indicating that FSH signaling pathway may stimulate specific EGFR-regulating proteins. Also, FSH-induced synthesis of both HA and progesterone is reduced but not abolished by AG1478 (EGFR tyrosine kinase inhibitor), indicating that other signaling pathways elicited by FSH are operating in parallel. Furthermore, SMAD2/3 signaling pathway is involved in the control of both cumulus expansion and steroidogenesis in porcine OCCs, since SMAD2/3 activation by GDF9/ TGFβ produced by oocyte and/or cumulus cells, significantly affects gonadotropin-induced HA and progesterone synthesis by porcine cumulus cells. Keywords: oocyte-cumulus complex, hyaluronan, progesterone, cumulus expansion.
- MeSH
- biologické modely MeSH
- IVM techniky * metody veterinární MeSH
- krysa rodu rattus MeSH
- kumulární buňky metabolismus fyziologie MeSH
- kyselina hyaluronová biosyntéza MeSH
- myši MeSH
- oocyty metabolismus fyziologie MeSH
- prasata * metabolismus fyziologie MeSH
- proliferace buněk MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
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.
- MeSH
- cyklooxygenasa 2 genetika MeSH
- erbB receptory fyziologie MeSH
- folikuly stimulující hormon farmakologie MeSH
- glukuronosyltransferasa genetika MeSH
- glykoproteiny farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- kumulární buňky fyziologie MeSH
- meióza účinky léků fyziologie MeSH
- mezibuněčné signální peptidy a proteiny farmakologie MeSH
- mitogenem aktivovaná proteinkinasa 14 antagonisté a inhibitory fyziologie MeSH
- oocyty účinky léků fyziologie MeSH
- proteinkinasy závislé na cyklickém AMP antagonisté a inhibitory fyziologie MeSH
- regulace genové exprese fyziologie MeSH
- signální transdukce účinky léků fyziologie MeSH
- Sus scrofa fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
