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.

• Klíčová slova
• cumulus expansion, extracellular matrix, hyaluronan, inter-alpha-trypsin inhibitor,
• 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
• Názvy látek
• alfa-globuliny MeSH
• C-reaktivní protein MeSH
• inter-alpha-inhibitor MeSH Prohlížeč
• kyselina hyaluronová * MeSH
• PTX3 protein MeSH Prohlížeč
• sérový amyloidový protein 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.

• Klíčová slova
• extracellular matrix, hyaluronan, oocyte-cumulus complex, versican,
• 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
• Názvy látek
• epitopy MeSH
• versikany MeSH

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
• lapatinib 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
• Názvy látek
• chinazoliny MeSH
• folikuly stimulující hormon MeSH
• inhibitory růstu MeSH
• lapatinib 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
• Názvy látek
• 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide MeSH Prohlížeč
• 6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride MeSH Prohlížeč
• benzamidy MeSH
• C-reaktivní protein MeSH
• chinazoliny MeSH
• dioxoly MeSH
• epidermální růstový faktor MeSH
• erbB receptory MeSH
• folikuly stimulující hormon MeSH
• glukuronosyltransferasa MeSH
• isochinoliny MeSH
• kyselina hyaluronová MeSH
• molekuly buněčné adheze MeSH
• progesteron MeSH
• protein Smad2 MeSH
• protein Smad3 MeSH
• PTX3 protein MeSH Prohlížeč
• pyridiny MeSH
• pyrroly MeSH
• RTKI cpd MeSH Prohlížeč
• sérový amyloidový protein MeSH
• tyrphostiny MeSH

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre-cultured for 72 hr in a medium supplemented with dbcAMP. The pre-cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP-dependent pathway. The dbcAMP-treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH.

• MeSH
• analýza rozptylu MeSH
• buněčné kultury metody   MeSH
• dibutyryl cyklický AMP farmakologie   MeSH
• exprese genu účinky léků   MeSH
• folikuly stimulující hormon farmakologie   MeSH
• glukuronosyltransferasa biosyntéza   genetika   metabolismus   MeSH
• kumulární buňky cytologie   účinky léků   metabolismus   MeSH
• kyselina hyaluronová metabolismus   MeSH
• luteinizační hormon farmakologie   MeSH
• messenger RNA biosyntéza   genetika   MeSH
• oocyty cytologie   účinky léků   růst a vývoj   MeSH
• ovariální folikul účinky léků   metabolismus   MeSH
• prasata MeSH
• progesteron metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• receptory LH biosyntéza   genetika   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
• folikuly stimulující hormon MeSH
• glukuronosyltransferasa MeSH
• kyselina hyaluronová MeSH
• luteinizační hormon MeSH
• messenger RNA MeSH
• progesteron MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• receptory LH MeSH