Parthenogenetic activation of pig oocytes using pulsatile treatment with a nitric oxide donor.

The nitric oxide donor (+)-S-nitroso-N-acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short-term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2-h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short-term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4-cell stage. Thirty-minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.

Histone deacetylase inhibition improves meiotic competence but not developmental competence in growing pig oocytes.

SummaryIn fully grown pig oocytes, meiotic maturation in vitro is retarded by inhibition of histone deacetylases by trichostatin A (TSA). In growing oocytes with partial meiotic competence, culture with TSA has no significant effect on the meiotic maturation. Growing oocytes treated with TSA mature mainly to metaphase I. The ratio of oocytes that mature to metaphase II is very limited. After transient exposure to TSA, the maturation of growing oocytes with partial meiotic competence takes a different course. When these oocytes are first cultured in a TSA-free medium, then cultured for another 24 h with 100 nM TSA and finally again in a TSA-free medium for 24 h, the ratio of oocytes that mature to metaphase II significantly increases reaching 59%. When oocytes were cultured for the same length of time without transient exposure to TSA, only 19% matured to metaphase II. Those oocytes that matured to metaphase II after transient exposure to TSA were successfully activated using calcium ionophore. However, the subsequent cleavage was very limited. We can conclude that transient exposure of growing pig oocytes with partial meiotic competence to TSA increases oocyte meiotic competence, but it does not enhance developmental competence after parthenogenetic activation.

[From historical data and opinions to present challenges in the field of celiac disease]. / Od historických poznatkov a názorov az po súcasné ulohy na poli celiakie.

Celiac disease is a lifelong autoimmune intestinal disorder caused by sensitivity to gluten. In clinical practice, no causal treatment is currently available and the disease can only be treated by strict adherence to a gluten-free diet. The disease that was known in ancient Greece continues to draw attention of the professional public worldwide and has been studied by many researchers. As a result, a range of theoretical and practical knowledge has been obtained and thus further aspects of the disease can be addressed. At present the discussion is open not only to doctors, pharmacists, scientists and researchers at various posts, but also to economists, lawyers and managers. In the spectrum of disease manifestations, some new forms were diagnosed, including associations with other diseases. Therefore, a higher prevalence of this disease has been reported. Multiple review articles appeared in both the local and international literature to familiarize the professional public with the symptoms and signs of the disease. Their common aim was to contribute to the detection of the disease in children and adults. Despite that, the disease remains underdiagnosed. In this context, new challenges emerge in the field of celiac disease. The current practice in diagnosing celiac disease and the available and candidate dietary interventions for effective protection and support therapy are described. The importance of early screening and advanced approaches to disease management is underlined.

Multiplex analysis of cytokines involved in tumour growth and spontaneous regression in a rat sarcoma model.

The aim of our study was to examine in vivo and in vitro cytokines produced by Lewis ratderived R5-28 sarcoma cells. These cells produce rapidly growing tumours in approximately two weeks after subcutaneous inoculation. However, spontaneous tumour regression was noted in about 40% of animals. For an explanation of this phenomenon, we evaluated the profile of 19 cytokines during tumour growth and spontaneous regression by the use of "antibody array". To detect cytokines directly originated by the sarcoma, the R5-28 cells were cultivated in vitro and then both the supernatants and the cell lysates were analysed. Our experiments showed three cytokines (MCP-1, TIMP-1 and VEGF) to be produced by R5-28 cells in vitro. Moreover, in vivo, another three cytokines (TNF-alpha, beta-NGF and LIX) were detected both in blood sera and tumour lysates, probably produced by immune and stromal cells during tumour growth. Changes in their expression after spontaneous regression are discussed.

Ultrastructural localisation of calcium deposits in pig ovarian follicles.

Calcium intracellular signaling regulates many intracellular events including oocyte maturation. This signaling is strongly dependent on the influx of calcium ions from extracellular spaces and on the state of intracellular calcium stores. In this study, intracellular calcium deposits were detected in follicle-enclosed pig oocytes using the combined oxalate-pyroantimonate method. These deposits were observed in the nucleus, the mitochondria, the cytoplasm, and on the surface of lipid droplets. The amount of calcium deposits was expressed as a percentage of the area of the respective cellular compartment, which is covered with calcium deposits on ultrathin sections. The distribution of calcium deposits in oocytes changed during folliculogenesis. The amount of calcium deposits in nuclei (1.11% of the area of oocyte nuclei) and cytoplasm (1.02%) in oocytes from secondary and early antral follicles (0.90% nuclei; 0.99% cytoplasm) is significantly lower (P < 0.05) than the amount of calcium deposits in these compartments in oocytes from primary follicles (2.51% nuclei; 2.34% cytoplasm) or antral follicles with growing oocyte (2.91% nuclei; 2.21% cytoplasm). The amount of calcium deposits in mitochondria of oocytes from primary follicles (1.27%) or antral follicles with growing oocyte (1.14%) is significantly lower (P < 0.05) than in the nucleus (2.51% in oocytes from primary follicles; 2.91% in growing oocytes from antral follicles) or cytoplasm (2.34% in oocytes from primary follicles; 2.21% in growing oocytes from antral follicles). The amount of calcium deposits in the cytoplasm of fully-grown oocytes (1.46%) dropped to levels significantly lower (P < 0.05) than those observed in the oocyte nucleus (2.29%). On the basis of these data, we can conclude that the population of follicles on pig ovaries differs in the distribution and concentration of calcium deposits in oocytes, and these changes may be involved in the regulation of the meiotic competence of oocytes.

Similarity of antigelatin factor and cold insoluble globulin.

Antigelatin factor, a protein capable of complexing denatured collagen, was separated from human serum by adsorption onto immobilized collagen. Antiserum raised against the material binding to denatured collagen permitted the development of a radioassay for the determination of antigelatin factor in which the complex of antigelatin factor and denatured 125I-labeled collagen is precipitated with this antiserum. Further purification of antigelatin factor was achieved by chromatography on DEAE-cellulose yielding an electrophoretically homogeneous protein. Its migration rate in dodecyl sulfate-polyacrylamide gel electrophoresis was identical with that of cold insoluble globulin (molecular weight approx. 440 000) prepared from human plasma by a published procedure amended by DEAE-cellulose chromatography. Reduction of disulfide bonds yielded subunits of molecular weight approx. 220 000, indistinguishable from those of cold insoluble globulin. The amino acid composition of both proteins was very similar. Immunological identity of both proteins was demonstrated by gel diffusion against monospecific anti-cold insoluble globulin antiserum. Closely related binding curves were obtained if denatured 125I-labeled collagen was reacted with increasing amounts of either cold insoluble globulin or antigelatin factor and the complexes formed were precipitated with anti-cold insoluble globulin antiserum. In addition, antigelatin factor and cold insoluble globulin mediated the fixation of denatured 125I-labeled collagen to trypsinized macrophages in the same way. Therefore, it is concluded that antigelatin factor and cold insoluble globulin are identical or very closely related proteins.

Interphase-like chromatin configuration induced by cycloheximide in maturing pig oocytes: effects of protein phosphatase inhibitors.

Embryo cloning methods could greatly benefit from the manipulation of cell cycle in oocytes from large domestic mammals. The present study was undertaken to examine the effects of the protein synthesis inhibitor cycloheximide and the inhibitors of protein phosphatases 1 and 2A okadaic acid and calyculin A on maturing pig oocytes. Cycloheximide treatment (10 micrograms/ml) induced an interphase-like chromatin configuration (ICC) in maturing oocytes. Up to 69% of the oocytes exhibited ICC when treated with cycloheximide after 24 h of in vitro culture. ICC starts to appear after a 4 h exposure to cycloheximide and the ICC percentage reached its plateau after 12 h of cycloheximide treatment. ICC is fully reversible. The addition of okadaic acid (0.5 microM) inhibited the ICC in cycloheximide-treated maturing oocytes and allowed the completion of maturation in 55% of them. In oocytes with ICC, the immunocytochemistry for tubulin revealed the rearrangement of microtubule into an interphase meshwork and these oocytes lost their ability to induce tubulin assembly, as shown after short-time taxol treatment. The addition of okadaic acid prevented this microtubule rearrangement and preserved a certain level of tubulin assembly. Calyculin appeared to be more effective than okadaic acid in the prevention of ICC. It is concluded that de novo protein synthesis is necessary during a certain period of meiotic maturation for the maintenance of metaphase chromatin configuration in pig oocytes. This protein (or proteins) acts through the inhibition of endogenous protein phosphatases, probably protein phosphatase of 2A type.

Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases, induces exit from metaphase I arrest in growing pig oocytes.

Calcium plays an important role in the regulation of meiotic maturation in mammalian oocytes. In the present study, mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, was used to mobilize intracellular calcium deposits in growing pig oocytes, which had not attained full meiotic competence and in which maturation is thus spontaneously blocked at the metaphase I stage. CPA treatment significantly increased the ratio of growing oocytes that are able to overcome the spontaneously occurring metaphase I block to complete their maturation at the metaphase II stage. CPA treatment of a least 2 hours' duration is necessary to overcome the metaphase I block in growing oocytes. A similar effect upon release from the spontaneous meiotic block at the metaphase I stage was observed after treatment of growing pig oocytes with thapsigargin, another inhibitor of endogenous calcium-dependent ATPases. Numerous calcium deposits in vacuoles, the mitochondria and on the surface of yolk granules in growing pig oocytes were observed. CPA treatment is able to mobilize calcium from the mitochondria, but deposits in vacuoles and deposits on the surface of yolk granules seem to remain intact after CPA treatment. A microinjection of heparin, which is known to bind with the inositol trisphosphate receptors, significantly decreased the ratio of CPA-treated growing oocytes overcoming the block at the metaphase I stage. This indicates that CPA might mobilize calcium in growing pig oocytes through inositol trisphosphate receptors. On the other hand, a microinjection of procaine or a microinjection of ruthenium red, both inhibitors of ryanodine receptors, did not prevent the overcoming of the metaphase I block, induced by CPA treatment. The calcium channel blocker, verapamil, significantly reduces the proportion of CPA-treated growing oocytes that overcome the metaphase I block. This indicates that the influx of calcium from extracellular sources is necessary to overcome the metaphase I block. The calmodulin inhibitors ophiobolin A and W7 also reduce the proportion of CPA-treated growing oocytes overcoming the metaphase I block.

Ultrastructural localisation of calcium deposits in the mouse ovary.

Follicle-enclosed mouse oocytes contain numerous calcium deposits. The ultrastructural distribution of calcium deposits in the nuclei, mitochondria and cytoplasm of mouse oocytes and granulosa cells of primary, secondary and antral follicles was examined using the combined oxalate-pyroantimonate method. The mitochondria of oocytes from all types of follicles had the highest levels of calcium deposits of all oocyte compartments, with the exception of primary follicles, in which oocyte nuclei contained the same level of calcium deposits as the mitochondria. Calcium deposits in the cytoplasm of oocytes from primary follicles were significantly lower than those in the cytoplasm of oocytes from secondary and antral follicles. Calcium deposits in the cytoplasm of granulosa cells were significantly lower than calcium deposits in the mitochondria of granulosa cells and this difference persisted throughout all categories of follicles. Calcium deposits in the nuclei of granulosa cells did not differ from levels in the mitochondria in primary and secondary follicles. In contrast, the nuclei of granulosa cells from antral follicles had lower levels of calcium deposits than the mitochondria. The differences observed in calcium deposits in various cellular compartments in oocytes and granulosa cells in the follicles of ovaries of adult mice can be attributed to their acquisition of meiotic competence and follicular development.

Activation of pig oocytes using calcium ionophore: effect of protein synthesis inhibitor cycloheximide.

In vitro matured pig oocytes were activated using a combined treatment of calcium ionophore A 23187 with cycloheximide. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with cycloheximide (0 or 10 microg/ml) for 6 h. Cycloheximide treatment significantly increased the activation rate of oocytes and the percentage of oocytes that were able to develop after activation. The highest activation rate was observed after treatment with 50 microM ionophore. The highest percentage of developing eggs was observed after combined treatment of ionophore (25 microM) with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.

Activation of in vitro matured pig oocytes using activators of inositol triphosphate or ryanodine receptors.

In our study, we observed the activation of in vitro matured pig oocytes and their subsequent parthenogenetic cleavage after stimulation of ryanodine receptors (RyR) using ryanodine (Ry), caffeine or cyclic adenosine diphosphate ribose (cADPri) or after stimulation of inositol triphosphate receptors (IP(3)R) using D-myo-inositol 1,4,5-triphosphate (IP(3)). Heparin, a potent blocker of IP(3)R, prevented the activation of porcine oocytes using IP(3), but blockers of RyR (ruthenium red or procaine) prevented activation after stimulation by RyR and stimulation by IP(3)R using IP(3). The drugs were injected into oocytes matured to the stage of metaphase II and activation was determined by assessment of pronuclear formation. The activity of H1 kinase was determined and our results demonstrated a significant drop in H1 activity in the activated oocytes. The cleavage of parthenogenetic embryos progresses to more advanced stages after stimulation by IP(3)R than after stimulation by RyR. Our results could indicate that, in pig oocytes, the calcium released from IP(3)-sensitive stores triggers the calcium release from ryanodine-sensitive intracellular stores, which is necessary for oocyte activation. The calmodulin inhibitors ophiobolin A and W7 reduce the activation of oocytes induced by stimulation of RyR or IP(3)R.

The effect of PMSG-priming on subsequent superovulatory response in dairy cows.

Superovulation was induced in 56 dairy cows to evaluate the effect of two different regimens using pregnant mare serum gonadotropin (PMSG). Thirty-two cows (controls) were superovulated between Days 9 and 12 of the estrous cycle with a single dose of PMSG (2 800 IU), while remaining 24 cows (PMSG-primed) received 200 IU of PMSG on Day 4 of the estrous cycle and subsequently a single dose of PMSG (2 800 IU) between Days 8 and 12. The cows in both treatments were each given 0,5 mg of cloprostenol at 48 h after the superovulatory PMSG treatment. They were then artifically inseminated twice, 48 h and 72 h later. Embryos were recovered at sloughter between Days 2 and 5 of the cycle and morphologically evaluated. The number of corpora lutea (CL) in the ovaries of the cows was recorded. The mean number of CL (7.2 vs 17.8) was significantly higher (P 0.01) for PMSG-primed cows. The percentage of recovered ova (60.5 vs 70.2 %) and good embryos (79.3 vs 70.7%) were not significantly different between groups. The percentage of fertilized ova (91.4 vs 83.8%) was significantly (P 0.025) greater for the controls. Results of the study indicate that PMSG-priming increased the ovulation rate in the cows superovulated with PMSG.

Ultrastructural localization of basic lysine-rich proteins during the nucleologenesis in preimplantation bovine embryos.

The process of nucleologenesis was examined in preimplantation bovine embryos by using ethanolic phosphotungstic acid (E-PTA) at the electron microscopic level. E-PTA binds basic lysine-rich proteins and thus makes possible detection of their localization and distribution. In blastomere nuclei of 2-, 4- and early 8-cell embryos, only nucleolus precursor body/bodies (NPBs) appear, being formed from a mass which is homogeneously stained with E-PTA. In cow embryos, the whole nucleologenesis is situated in the 8-cell stage. During the following step of nucleologenesis, the NPB with a big central area (named NPB vacuole) is formed. Fibrillar mass around the central vacuole is intensively stained, especially in the regions in close vicinity to the central vacuole. Many clumps of E-PTA-positive, regularly dispersed material are found inside the vacuole. The next step of nucleologenesis is characterized by the presence of NPB with secondary vacuoles that are also filled with clumps of E-PTA-positive material. Small, intensively stained areas are visible at sites that are probably identical with those where a dense fibrillar component is formed. Until the end of the 8-cell stage, as well as in the morula and early blastocyst, typical fibrillogranular nucleoli are present. These nucleoli have 3 basic components--fibrillar centres (FC), dense fibrillar components (DFC) and granular components (GC) which stain with different increasing intensity. FC and DFC show a strong (particular) reaction while the GC are stained to a lesser degree. In all examined stages of embryonal development, the E-PTA positivity was found within the perinucleolar chromatin and on the clumps of heterochromatin. An analogical localization of basic and acidic regulatory proteins in the examined developmental stages is discussed.

[Functional reaction of a fibroblast surface protein (author's transl)]. / Funktionelle Reaktionen eines Oberflächenproteins von Fibroblasten.

Cold-insoluble globulin (CIG) of plasma represents the soluble form of a fibroblast surface protein. As shown by affinity chromatography it is selectively adsorbed from plasma on immobilized collagen of various types. The following sequence of binding strength between CIG and collagen was evaluated by means of fractionated elution: collagen type III native less than type I native less than type I denatured. The affinity to native collagen is important for the capability of CIG to mediate the attachement of fibroplasts on collagen fibrils. In addition, CIG is a substrate of activated coagulation factor XIII which plays an important, but as yet unclarified role in wound healing.

[Characterization of a serum protein with selective affinity for denatured collagen (author's transl)]. / Charakterisierung eines Serumproteins mit selektiver Affinität zu denaturiertem Kollagen.

A protein known as antigelatin factor (AGF) was isolated from human serum by affinity chromatography with immobilized denatured collagen. In biochemical and immunological assays AGF showed specificity to denatured, but not to native collagen of the types I, II and III. A close relationship to Cell Attachment Protein and Cold Insoluble Globulin was found in comparative studies.

[Blood proteins of calves and their 1st-calver mothers during the early post-natal period]. / Bílkoviny krevního séra telat a jejich matek--prvotelek v raném postnatálním období.

The calves of the Red Spotted breed and their first-calver mothers were subject to the study of total proteinemia and of the changes in protein fractions before parturition and in a ten-day period of post-partal development. The relations between these parameters were also studied. Calves up to the 10th day of life show a rise in total proteinemia whereas cows show insignificant changes. The level of albumins in calves exhibits a slight fluctuation, whereas in mothers it increased before parturition, followed by a drop which lasted until the end of the test period. alpha-globulins show a decreasing tendency in calves from the fourth day of age and have higher values than in cows. beta-globulins in cow blood serum before parturition increase. The level of gamma-globulins in calves rises until the end of the first week of life. A linear correlation dependence was found between the values of beta-globulins in cows and calves in the first two post-partal days and between the value of beta-globulin level of mothers prior to parturition and of calves at birth. In gamma-globulins the correlation dependence is always significant, starting the first day after birth.

[Testing of vitamin E in the newly developed Czechoslovak preparation Trivitamin]. / Testace vitamínu E v cs. vývojovém preparátu Trivitamin.

Experiments were performed to test the newly developed Czechoslovak preparation Trivitamin, including the comparison of its effects at intubation onto the tongue root and at muscular implantation; these effects were compared with the i. m. administration of Axetocal. The levels of vitamin E in the blood of pigs were examined as influenced by the above treatments. The best utilization was obtained in the Trivitamin preparation given to the pigs intramuscularly. Peroral administration ranked second, whereas the lowest reaction was obtained from Axetocal at intramuscular administration. We hope that a good aqueous medium in which the vitamin is suspended in the main prerequisite for a high utilization rate of vitamin E is the model test with the Trivitamin preparation.

[DNA fingerprinting in horses]. / DNA fingerprinting u koní.

Using a multilocus DNA probe, individual - specific hybridization patterns, the so-called DNA fingerprints (TAB) were determined in six horse families by the DNA fingerprinting method. The probe with evolutionally preserved nucleotide sequence from bacteriophage M13 determines hypervariable regions placed in genomic minisatellite DNA. The use of this probe permits an identification of an individual and execution of paternity relationships with a probability over 99.99 per cent.

Pyrethroids cypermethrin, deltamethrin and fenvalerate have different effects on in vitro maturation of pig oocytes at different stages of growth.

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate - as potent specific inhibitors of protein phosphatase calcineurin - can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin - cyclosporin A or hymenistatin I - on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin - allethrin or permethrin - had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.