Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures. In addition, the effect of BMP-15 on oocyte maturation- and steroidogenesis-related transcripts after 4, 8, 16, and 24 hours of cultivation was evaluated using real-time RT-PCR. We demonstrated that the FSH/LH-induced cumulus expansion was accompanied by a significant increase in CD44, PTGS2, CYP11A1 (at 4 h) and AREG, HAS2, TNFAIP6, STAR (at 8 h) mRNAs. While FSH/LH-stimulated total HA synthesis by OCC was not affected by BMP-15 in serum-supplemented medium, its retention within the complex was significantly increased after the action of BMP-15 in comparison to FSH/LH alone (P < 0.001; 65% versus 35%, respectively). Moreover, we detected a significant increase in the expression of AREG and TNFAIP6 (both at 16 h), and CYP11A1 (at 24 h) in FSH/LH-stimulated OCC due to the action of BMP-15 compared to complexes cultured only with FSH/LH. In the presence of serum, BMP-15 markedly increased FSH/LH-stimulated progesterone secretion by OCC (about 69%) and induced a significant decrease in FSH/LH-induced progesterone release by GC (about 35%) compared to FSH/LH alone. The present results indicate that the addition of BMP-15 to the gonadotropin-stimulated OCC cultured in serum-supplemented medium might improve oocyte-cumulus maturation.
- MeSH
- gonadotropiny farmakologie MeSH
- kostní morfogenetický protein 15 farmakologie MeSH
- kultivované buňky MeSH
- kyselina hyaluronová biosyntéza MeSH
- ovariální folikul účinky léků metabolismus MeSH
- prasata MeSH
- progesteron biosyntéza MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
It has been previously shown that multimeric pentraxin 3 (PTX3) is a key component of the cumulus oophorus extracellular matrix (ECM) in mice. In response to the ovulatory LH surge, the cumulus cells assemble a unique ECM that envelopes the oocyte and cumulus cell complex. Importantly, cumuli from PTX3(-/-) mice were defective in their ECM organization and their fertility was impaired. It has been demonstrated that tumor necrosis factor alpha-induced protein 6 catalyzes the formation of heavy chains of (inter-alpha-trypsin inhibitor) -hyaluronan complexes and these are then cross-linked via PTX3. This process is tightly regulated and requires the proteins to meet/interact in the correct order. Finally, in this way, the above-listed proteins form the cumulus oophorus ECM. We investigated whether PTX3 is expressed in the porcine preovulatory follicle. Porcine oocyte-cumulus complexes (OCC) and mural granulosa cells (MGC) from gilts were obtained either after stimulation in vivo with eCG/hCG (4, 8, 16, 24, and 32 h) or culture in vitro (4, 24, and 44 h) in FSH/LH-supplemented medium. The methods performed were real-time reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunostaining. The expression of PTX3 transcripts was significantly increased 24 h after either in vivo hCG stimulation or in vitro FSH/LH treatment in both OCC and MGC. Western blot analysis with PTX3 antibody revealed that not only matrix extracts from in vivo-stimulated gilts contain high levels of PTX3 protein but also matrix extracts of FSH/LH-stimulated OCC cultured in medium supplemented either with follicular fluid or with porcine serum. The localization of PTX3 in the cumulus oocyte complex was confirmed by immunostaining. In conclusion, PTX3 is produced by porcine OCC and MGC both in vivo and in vitro with gonadotropin stimuli inducing cumulus expansion.
- MeSH
- C-reaktivní protein analýza genetika MeSH
- choriogonadotropin farmakologie MeSH
- exprese genu účinky léků MeSH
- folikulární buňky metabolismus MeSH
- folikuly stimulující hormon farmakologie MeSH
- gonadotropiny koňské farmakologie MeSH
- gonadotropiny farmakologie MeSH
- kultivační média MeSH
- kultivované buňky MeSH
- kumulární buňky chemie metabolismus MeSH
- luteinizační hormon farmakologie MeSH
- messenger RNA analýza MeSH
- oocyty chemie metabolismus MeSH
- sérový amyloidový protein analýza genetika MeSH
- Sus scrofa metabolismus 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
Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor α-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.
- MeSH
- extracelulární matrix účinky léků metabolismus MeSH
- inhibitory cysteinových proteinas farmakologie MeSH
- inhibitory proteasomu MeSH
- kumulární buňky účinky léků metabolismus MeSH
- kvantitativní polymerázová řetězová reakce veterinární MeSH
- leupeptiny farmakologie MeSH
- messenger RNA biosyntéza genetika MeSH
- molekuly buněčné adheze biosyntéza MeSH
- oocyty účinky léků metabolismus MeSH
- prasata MeSH
- progesteron biosyntéza MeSH
- proteasomový endopeptidasový komplex metabolismus 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
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
Autori vyšetrili 19 bežcov pred a po 100 km behu, ktorý absolvovali v priemere za 8:24:01 hod. Spirometrický záznam bol získaný spirometrom VICATEST pri nastaviteľnej teplote okolia. Zistili, že najväčšie percentuálne poklesy hodnôt po 100-kilometrovom behu boli predovšetkým v prietokových parametroch (PEF, MEF), a to až o 17,2 % a 26,3 %. Pomerne malé zníženie spirometrických parametrov po behu dokazuje vysokú odolnosť dýchacích svalov proti únave. Analýza korelačných vzťahov poukázala na to, že bežci s menším poklesom spirometrických parametrov dosiahli lepšie výsledky v behu na 100 km. Korelácie však neboli štatisticky významné.
Authors examined 19 runners prior and after 100 km run. The runners achieved average performance of 8:24.01 hrs. Spirometric examination was performed on VICATEST spirometer, with adjustable ambient temperature. They observed the greatest percentual drop subsequent to 100 km run in airflow parameters (PEF, MEF) - 17,6 % and 26,3 % respectively. Comparatively small decrease of spirometric parameters subsequent to the run proves that respiratory muscles are highly resistant to fatigue. Analysis of the correlations has shown that the runners with lesser drop in respiratory parameters achieved better results in the 100 km run. These correlations, however, were not significant.
- MeSH
- běh MeSH
- dýchací svaly MeSH
- lehká atletika MeSH
- lidé MeSH
- respirační funkční testy MeSH
- spirometrie MeSH
- únava MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- srovnávací studie MeSH
