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
- Alpha-Globulins metabolism MeSH
- C-Reactive Protein metabolism MeSH
- Extracellular Matrix * metabolism MeSH
- Cumulus Cells * metabolism MeSH
- Hyaluronic Acid * metabolism MeSH
- Humans MeSH
- Mice MeSH
- Oocytes * metabolism MeSH
- Ovarian Follicle * metabolism MeSH
- Serum Amyloid P-Component metabolism genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review 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
- Cell Differentiation MeSH
- Epitopes immunology MeSH
- Cells, Cultured MeSH
- Oocytes cytology immunology metabolism MeSH
- Swine MeSH
- Versicans genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- 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.
- 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
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
- Cell Differentiation drug effects physiology MeSH
- Quinazolines pharmacology MeSH
- Follicle Stimulating Hormone pharmacology MeSH
- Growth Inhibitors pharmacology MeSH
- Cells, Cultured MeSH
- Cumulus Cells cytology drug effects MeSH
- Meiosis drug effects physiology MeSH
- Oocytes cytology drug effects MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Benzamides pharmacology MeSH
- C-Reactive Protein metabolism MeSH
- Quinazolines pharmacology MeSH
- Dioxoles pharmacology MeSH
- Epidermal Growth Factor metabolism MeSH
- ErbB Receptors antagonists & inhibitors metabolism MeSH
- Follicle Stimulating Hormone metabolism MeSH
- Glucuronosyltransferase antagonists & inhibitors metabolism MeSH
- Isoquinolines pharmacology MeSH
- Cumulus Cells metabolism MeSH
- Hyaluronic Acid biosynthesis MeSH
- Meiosis drug effects MeSH
- Cell Adhesion Molecules antagonists & inhibitors metabolism MeSH
- Mice MeSH
- Oocytes enzymology physiology MeSH
- Swine MeSH
- Progesterone biosynthesis MeSH
- Smad2 Protein antagonists & inhibitors metabolism MeSH
- Smad3 Protein antagonists & inhibitors metabolism MeSH
- Pyridines pharmacology MeSH
- Pyrroles pharmacology MeSH
- Serum Amyloid P-Component metabolism MeSH
- Signal Transduction drug effects MeSH
- Tyrphostins pharmacology MeSH
- Gene Expression Regulation, Developmental drug effects MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Cell Differentiation drug effects genetics MeSH
- Embryonic Development drug effects genetics MeSH
- Epidermal Growth Factor chemistry pharmacology MeSH
- Follicle Stimulating Hormone pharmacology MeSH
- Gonadotropins pharmacology MeSH
- Embryo Culture Techniques MeSH
- Cells, Cultured MeSH
- Cumulus Cells drug effects metabolism physiology MeSH
- Oocytes drug effects metabolism physiology MeSH
- Oogenesis drug effects genetics MeSH
- Parthenogenesis drug effects genetics physiology MeSH
- Peptide Fragments chemistry pharmacology MeSH
- Swine genetics metabolism physiology MeSH
- Cell Proliferation drug effects MeSH
- Gene Expression Profiling MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study 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
- Diagnostic Techniques, Cardiovascular instrumentation utilization MeSH
- Electroencephalography methods instrumentation utilization MeSH
- Electrocardiography methods instrumentation utilization MeSH
- Financing, Organized MeSH
- Myocardial Ischemia diagnosis MeSH
- Brain Ischemia diagnosis MeSH
- Humans MeSH
- Body Surface Potential Mapping methods instrumentation utilization MeSH
- Models, Anatomic MeSH
- Software Design MeSH
- Signal Processing, Computer-Assisted instrumentation MeSH
- Imaging, Three-Dimensional methods instrumentation utilization MeSH
- Check Tag
- Humans 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
- Alpha-Globulins metabolism MeSH
- Time Factors MeSH
- Epitopes metabolism MeSH
- Financing, Organized MeSH
- Follicular Phase metabolism MeSH
- Follicular Fluid metabolism MeSH
- Mice, Inbred Strains MeSH
- Cells, Cultured MeSH
- Hyaluronic Acid metabolism MeSH
- Cell Adhesion Molecules immunology metabolism MeSH
- Antibodies, Monoclonal pharmacology immunology MeSH
- Mice MeSH
- Ovarian Follicle cytology metabolism drug effects MeSH
- Swine MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
