Seminal plasma of sterlet Acipenser ruthenus was evaluated using comparative proteomics to characterize its protein fractions and to determine any influence of multiple sperm collections on these proteins. An experimental group of fish was used, in which sperm was collected three times at 5 h intervals. Protein fractions of seminal plasma were determined by SDS-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis high-resolution gels (2D). At all stripping times, five protein bands with molecular weights of 93, 53, 48, 33 and 28 kDa were identified using SDS-PAGE. No significant differences (p > 0.05) in relative mass of protein bands among collections were observed. At the third collection, 20 protein spots were detected from the two-dimensional gels, compared to 17 found at the first and second collections. Ten protein spots, from the third stripping, were analysed. Screening of these spots by mass spectrometric analysis showed positive results for spot 10. Direct comparison across public databases revealed sequence similarity with two hypothetical proteins, MCAG_00854 and IscW_ISCW011489. Differences in the seminal plasma protein fractions were found at the third stripping compared to the first two. It is hypothesized that these extra proteins after the third collection could be involved in some step of intracellular mechanism which is responsible for regulating of spermatozoa motility. However, protein identification revealed no significant distinction for any protein spot and protein sequences available in public databases. These results highlighted the need for a complete genome sequences for sturgeons.
- MeSH
- časové faktory MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- regulace genové exprese fyziologie MeSH
- rybí proteiny fyziologie MeSH
- ryby fyziologie MeSH
- sperma fyziologie MeSH
- spermie fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.
- MeSH
- fosforylace účinky léků MeSH
- kapři * fyziologie MeSH
- kryoprezervace veterinární MeSH
- kryoprotektivní látky farmakologie MeSH
- motilita spermií účinky léků MeSH
- proteinkinasy metabolismus MeSH
- spermie účinky léků fyziologie MeSH
- threonin metabolismus MeSH
- tyrosin metabolismus MeSH
- uchování spermatu metody veterinární MeSH
- upregulace účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH
Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.
- MeSH
- 2D gelová elektroforéza MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fertilizace MeSH
- kapři metabolismus MeSH
- kryoprezervace veterinární MeSH
- motilita spermií MeSH
- ovum fyziologie MeSH
- proteomika MeSH
- rybí proteiny metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The soil actinobacteria Rhodococcus rhodochrous PA-34, Rhodococcus sp. NDB 1165 and Nocardia globerula NHB-2 grown in the presence of isobutyronitrile exhibited nitrilase activities towards benzonitrile (approx. 1.1-1.9 U mg(-1) dry cell weight). The resting cell suspensions eliminated benzonitrile and the benzonitrile analogues chloroxynil (3,5-dichloro-4-hydroxybenzonitrile), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile) (0.5 mM each) from reaction mixtures at 30 degrees C and pH 8.0. The products were isolated and identified as the corresponding substituted benzoic acids. The reaction rates decreased in the order benzonitrile > chloroxynil > bromoxynil > ioxynil in all strains. Depending on the strain, 92-100, 70-90 and 30-51% of chloroxynil, bromoxynil and ioxynil, respectively, was hydrolyzed after 5 h. After a 20-h incubation, almost full conversion of chloroxynil and bromoxynil was observed in all strains, while only about 60% of the added ioxynil was converted into carboxylic acid. The product of ioxynil was not metabolized any further, and those of the other two herbicides very slowly. None of the nitrilase-producing strains hydrolyzed dichlobenil (2,6-dichlorobenzonitrile). 3,5-Dibromo-4-hydroxybenzoic acid exhibited less inhibitory effect than bromoxynil both on luminescent bacteria and germinating seeds of Lactuca sativa. 3,5-Diiodo-4-hydroxybenzoic acid only exhibited lower toxicity than ioxynil in the latter test.
- MeSH
- Actinobacteria účinky léků enzymologie metabolismus MeSH
- amidohydrolasy metabolismus MeSH
- amidy metabolismus MeSH
- aminohydrolasy metabolismus MeSH
- biodegradace účinky léků MeSH
- biotransformace účinky léků MeSH
- herbicidy metabolismus toxicita MeSH
- hydrolýza účinky léků MeSH
- kořeny rostlin účinky léků růst a vývoj MeSH
- nitrily metabolismus toxicita MeSH
- půdní mikrobiologie MeSH
- salát (hlávkový) účinky léků růst a vývoj MeSH
- testy akutní toxicity MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- lipoproteiny genetika metabolismus MeSH
- membránové proteiny genetika chemie metabolismus MeSH
- Neisseria meningitidis genetika MeSH
- proteiny vnější bakteriální membrány genetika metabolismus MeSH
- techniky in vitro MeSH
- vápník farmakologie MeSH
- železo metabolismus MeSH
Vývoj sliznice jícnu a žaludku v oblasti gastroezofageální junkce byl sledován u 61 plodů ve 13. - 41. týdnu gestačního věku. Histologické nálezy ukázaly, že vícevrstevný epitel jícnu je překryt ve 13. -15. týdnu souvislou vrstvou cylindrických hlenových řasinkových buněk, které jsou ložiskově přítomny ještě ve 25. týdnu a později mizí. Zaludeční sliznici tvořily do 15. týdne pouze jamky. Vývoj žlázové části sliznice začínal v 15. týdnu proliferací žlázových tubulů na spodine jamek, které se později diferencovaly do žlázek oxyntického typu. Korporální sliznice žaludku byla kompletně vyvinuta ve 27. týdnu. Sliznice kardie nebyla nalezena u žádného z 10 plodů vyšetřených v rozmezí 27. - 41. týdne. Naše nálezy podporují názor, že kardie žaludku není fyziologickou strukturou, ale žlázovou metaplazií sliznice distábíího jícnu, ke které dochází v důsledku gastroezofágeálního refluxu.
The development of the esophageal and gastric mucosa in the gastroesophageal junction was studied in 61 fetuses of 13 - 41 weeks of the gestational age. During the 13th - 15th week, the esophageal multilayered epithelium was covered by a continuous layer of columnar mucous ciliated cells which were present only focally till the 25th week and disappeared later. Before the 15th week, the gastric mucosa was formed by pits only. The glands started as proliferating tubules in the basal parts of the pits in the 15th week. Further, they differentiated into oxyntic glands. The mucosa of the corpus was fully developed in the 27th week. The cardiac mucosa was absent in all the 10 fetuses exami mined between the 27th and 41st week of gestation. This suppoii;s the view that the gastric cardiac mucosa is not a physiological structure but that it results from glandular metaplasia of the distal esophageal mucosa due to gastroesophageal reflux.
