In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure.
- MeSH
- alfa-globuliny metabolismus MeSH
- C-reaktivní protein metabolismus MeSH
- extracelulární matrix * metabolismus MeSH
- kumulární buňky * metabolismus MeSH
- kyselina hyaluronová * metabolismus MeSH
- lidé MeSH
- myši MeSH
- oocyty * metabolismus MeSH
- ovariální folikul * metabolismus MeSH
- sérový amyloidový protein metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM.
- MeSH
- buněčná diferenciace MeSH
- epitopy imunologie MeSH
- kultivované buňky MeSH
- oocyty cytologie imunologie metabolismus MeSH
- prasata MeSH
- versikany genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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.
- MeSH
- C-reaktivní protein metabolismus MeSH
- extracelulární matrix účinky léků metabolismus MeSH
- kumulární buňky cytologie účinky léků metabolismus MeSH
- kyselina hyaluronová metabolismus MeSH
- mifepriston farmakologie MeSH
- molekuly buněčné adheze metabolismus MeSH
- oocyty cytologie metabolismus MeSH
- rozmnožování účinky léků MeSH
- sérový amyloidový protein metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy 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.
OBJECTIVE: To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). DESIGN: Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. SETTING: A reproductive biology laboratory. PATIENT(S): Not applicable. INTERVENTION(S): Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. MAIN OUTCOME MEASURE(S): Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. RESULT(S): In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM). CONCLUSION(S): Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.
- MeSH
- buněčná diferenciace účinky léků fyziologie MeSH
- chinazoliny farmakologie MeSH
- folikuly stimulující hormon farmakologie MeSH
- inhibitory růstu farmakologie MeSH
- kultivované buňky MeSH
- kumulární buňky cytologie účinky léků MeSH
- meióza účinky léků fyziologie MeSH
- oocyty cytologie účinky léků MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis.
- MeSH
- benzamidy farmakologie MeSH
- C-reaktivní protein metabolismus MeSH
- chinazoliny farmakologie MeSH
- dioxoly farmakologie MeSH
- epidermální růstový faktor metabolismus MeSH
- erbB receptory antagonisté a inhibitory metabolismus MeSH
- folikuly stimulující hormon metabolismus MeSH
- glukuronosyltransferasa antagonisté a inhibitory metabolismus MeSH
- isochinoliny farmakologie MeSH
- kumulární buňky metabolismus MeSH
- kyselina hyaluronová biosyntéza MeSH
- meióza účinky léků MeSH
- molekuly buněčné adheze antagonisté a inhibitory metabolismus MeSH
- myši MeSH
- oocyty enzymologie fyziologie MeSH
- prasata MeSH
- progesteron biosyntéza MeSH
- protein Smad2 antagonisté a inhibitory metabolismus MeSH
- protein Smad3 antagonisté a inhibitory metabolismus MeSH
- pyridiny farmakologie MeSH
- pyrroly farmakologie MeSH
- sérový amyloidový protein metabolismus MeSH
- signální transdukce účinky léků MeSH
- tyrphostiny farmakologie MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- 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
Obsahem článku je popis měřicího systému určeného k záznamu biopotenciálů. Záznam biopotenciálů je možný pomocí až 128 snímacích povrchových elektrod. Vysoká kvalita měřeného signálu je dosažena díky aktivním elektrodám a programovatelné záznamové jednotce. Vysokou ochranu pacienta zaručuje jednotka díky napájení pomocí Li-ion baterie a propojením s USB portem řídicího počítače přes optický kabel. Systém dále zahrnuje software pro zpracování a vizualizaci signálu v reálném čase a řešení inverzní úlohy pro neinvazivní lokalizaci bioelektrických zdrojů v srdci nebo v mozku. Získaný signál je v počítači zpracován a vizualizován pomocí softwaru LiveMap. Tento modulární, open-source software určený k záznamu, zpracování a vizualizaci multikanálového EKG a EEG signálu umožňuje 2D a 3D vizualizaci dat přímo na model lidského hrudníku nebo hlavy. Software je schopen počítat potenciálové a integrální povrchové mapy stejně jako rozdílové mapy použitelné pro diagnostiku srdce nebo mozku. V systému je obsažena také softwarová podpora řešení inverzní úlohy pro zjišťování lokálních ischemických ložisek v myokardu. K nalezení odpovídajícího dipólu představujícího ischemické ložisko používá systém rozdíly v časových integrálech povrchových potenciálů způsobených změnami repolarizace ischemických buněk myokardu spolu s informacemi o rozměrech hrudníku pacienta. Výsledky měření u pacientů s podezřením na ischemii dokazují, že mapování biopotenciálů spolu s inverzní úlohou by mohlo být využíváno k neinvazivnímu odhalení změn srdeční aktivity již v počátcích ischemie.
Portable measuring system with real-time visualization and signal processing software and an inverse-problem-solving method enabling non-invasive location of bioelectric sources in the heart or brain are presented. Measuring system enables simultaneous recording of biopotentials measured in up to 128 body surface nodes relatively to a chosen reference potential. Active electrodes and intelligent data acquisition unit powered by a Li-ion cell enable to achieve high quality of measured signals. Connection to the USB port of a host computer over an optical cable minimizes capacitive coupling and guaranties high level of patient safety. On the computer side, acquired signals are processed and visualized by LiveMap real-time windows-based software. This modular, open source software package for acquisition, processing and visualization of multichannel ECG and EEG signals provides real-time 2D and 3D visualization of various types of data mapped directly to a human chest model. It can compute isopotential and isointegral surface maps, as well as difference and departure maps applicable for direct heart or brain diagnostics. A simple inverse method for identification of local ischemia of myocardial cells was implemented in the system. It uses alterations in time integrals of surface potentials connected with changed repolarization of ischemic myocardial cells together with information on torso volume conductor to find an equivalent dipole representing the ischemic lesion. Results on patients with suspected ischemia suggest that mapping of biopotentials combined with simple inverse solution might be a useful tool for non-invasive identification of changed heart activities during starting ischemia.
- MeSH
- diagnostické techniky kardiovaskulární přístrojové vybavení využití MeSH
- elektroencefalografie metody přístrojové vybavení využití MeSH
- elektrokardiografie metody přístrojové vybavení využití MeSH
- financování organizované MeSH
- ischemická choroba srdeční diagnóza MeSH
- ischemie mozku diagnóza MeSH
- lidé MeSH
- mapování potenciálů tělesného povrchu metody přístrojové vybavení využití MeSH
- modely anatomické MeSH
- navrhování softwaru MeSH
- počítačové zpracování signálu přístrojové vybavení MeSH
- zobrazování trojrozměrné metody přístrojové vybavení využití MeSH
- Check Tag
- lidé MeSH
We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.
- MeSH
- alfa-globuliny metabolismus MeSH
- časové faktory MeSH
- epitopy metabolismus MeSH
- financování organizované MeSH
- folikulární fáze metabolismus MeSH
- folikulární tekutina metabolismus MeSH
- inbrední kmeny myší MeSH
- kultivované buňky MeSH
- kyselina hyaluronová metabolismus MeSH
- molekuly buněčné adheze imunologie metabolismus MeSH
- monoklonální protilátky farmakologie imunologie MeSH
- myši MeSH
- ovariální folikul cytologie metabolismus účinky léků MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
