Biomedical subjects

J Petr

Publications and source records attributed to J Petr.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway.

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.

Aminoquinolines↗

PubMed2003

Ultrastructural localisation of calcium deposits in pig oocytes maturing in vitro: effects of verapamil.

The culture of pig oocytes in the presence of the calcium channel blocker verapamil (0.02 mM) resulted in the blocking of meiosis at the metaphase I stage, and only a small fraction (about 28%) of the oocytes were able to continue their maturation to the stage of metaphase II. Hence, meiotic maturation in pig oocytes is a calcium-dependent process. After isolation of the pig oocytes from their follicles, the intracellular calcium deposits in the oocyte and granulosa cells, detectable using the combined oxalate-pyroantimonate method, are depleted. The amount of calcium deposits in the oocyte and granulosa cells increased during oocyte meiotic maturation in vitro, especially in the nucleus, mitochondria, vacuoles and cytoplasm. The replenishment of calcium deposits is significantly changed under the effect of verapamil. The increase in calcium deposits in the oocyte nucleus was delayed, a much larger amount of deposits was formed in the mitochondria, and the amount of deposits in the vacuoles was demonstrably smaller. A significant peak in the accumulation of calcium deposits was observed in the cytoplasm of verapamil-treated oocytes after 16 h of in vitro culture. We propose that an altered pattern in the replenishment of calcium deposits can disturb intracellular signalling and prevent the exit of oocytes from the metaphase I stage.

Animals↗

PubMed2003

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.

Animals↗

PubMed2002

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.

Adenosine Diphosphate Ribose↗

PubMed2001

Ultrastructural localization of calcium deposits during in vitro culture of pig oocytes.

Calcium deposits were localized using the combined oxalate-pyroantimonate technique in follicle-enclosed oocytes fixed in situ. These deposits can be observed within vacuoles, mitochondria, and on the surface of yolk granules as well as in the caryoplasm, but are absent from the endoplasmic reticulum. Isolation of the oocyte from the follicle resulted in the immediate depletion of these calcium deposits. Replenishment of these deposits started during the first 8 hr of in vitro culture of the oocyte and they were gradually replenished to the levels observed before the liberation of oocytes during in vitro maturation to the stage of metaphase II.

Animals↗

PubMed2001

Enrichment of bifidobacteria in the hen caeca by dietary inulin.

Caecal bifidobacterial concentration was increased more than 3-fold in inulin-treated laying hens. The counts of bifidobacteria in birds fed as the control were 9.64, in inulin-diet fed ones 10.17 log CFU/g of caecal content, respectively. Dietary inulin had no effect on caecal microbial metabolite concentration. The proportion of inulin-fermenting bifidobacteria in the total bifidobacteria increased 2-fold in inulin-treated birds.

Animal Feed↗

PubMed2001

Bifidobacteria are obligate inhabitants of the crop of adult laying hens.

Modified TPY agar (MTPY; with addition of glacial acetic acid and mupirocin) was used in the enumeration of bifidobacteria from hen crop and faeces. The colonies on MTPY medium inoculated with crop and faeces samples were Gram-stained, screened for presence of fructose-6-phosphate phosphoketolase activity and tested for fermentation patterns using ANAEROtest kits. It was revealed that bifidobacteria are obligate inhabitants of the hen crop, reaching counts of 10(7)/g of crop content. The occurrence of bifidobacteria in the hen crop was evidently not the consequence of ingestion of faeces as all the bifidobacteria strains isolated from the hen crop fermented glucose and fructose, while most strains isolated from hen faeces did not ferment either glucose or fructose. The results suggest that bifidobacteria are common and may even be one of the predominant parts of the hen crop flora.

Animals↗

PubMed2001

Activation of pig oocytes using calcium ionophore: effect of the protein kinase inhibitor 6-dimethyl aminopurine.

The aim of our study was to investigate the parthenogenetic activation of in vitro matured pig oocytes after their combined treatment with calcium ionophore A 23187 and the inhibitor of protein kinases, 6-dimethylaminopurine (6-DMAP) and to study the further embryonic development of oocytes activated using this treatment. The oocytes were exposed to ionophore (10, 25 or 50 microM) for 0.5, 1, 3, 5 or 7 min and then cultured with 6-DMAP (0 or 2 microM) The highest activation rate (up to 88% of the activated eggs reached the pronuclear stage) was observed after combined treatment of the oocytes with 50 microM ionophore and 6-DMAP. The highest rate of embryonic development was observed after treatment with 25 microM ionophore without 6-DMAP, when up to 51% of the eggs developed beyond two-cell stage, 2% of the eggs developed up to the stage of morula and up to 3% of the eggs reached the stage of blastocyst. When 50 microM ionophore was used, the embryonic development of the activated eggs was arrested before the morula and blastocyst stage. After treatment of the activated eggs with 6-DMAP, we did not observe any development beyond the stage of 16 blastomeres. We can conclude that combined treatment with calcium ionophore A 23187 and 6-DMAP increases the activation rate in pig oocytes matured in vitro, but this combined treatment exerts a detrimental effect on further embryonic development of the activated eggs.

Adenine↗

PubMed2000

A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca.

Avian caeca contain a large and diverse population of bacteria. Certain genera, including Bifidobacterium, are thought to exert health-promoting effects. Two media were evaluated to determine their sensitivity and selectivity for bifidobacteria in the hen caecal samples: modified Wilkins-Chalgren agar (MW; Oxoid) with the addition of glacial acetic acid (1 ml/l) and mupirocin (100 mg/l) and modified TPY agar (MTPY; ADSA, Spain) with glacial acetic acid (1 ml/l) and mupirocin (100 mg/l). The colonies arising on the plates inoculated with caecal samples were Gram stained, screened for the presence of fructose-6-phosphate phosphoketolase activity, and tested for fermentation patterns using ANAEROtest (Lachema, Czech Republic) and API 50 CHL (BioMérieux, France) kits. Both agars were selective for bifidobacteria, however, MTPY agar showed higher cfu/g than MW agar. Bifidobacterial counts were higher than 10(10) cfu/g of caecal contents using MTPY agar. Most of strains isolated from this medium fermented melibiose, sucrose, and raffinose, but not glucose. Soya peptone (5 g/l; Oxoid) stimulated the growth of glucose non-fermenting strains in complex liquid media. The results suggest that the media for selective enumeration and isolation of bifidobacteria in poultry caecal samples should not contain glucose as the sole carbon source. It can be concluded that MTPY medium is highly selective and permits the growth of both glucose fermenting and glucose non-fermenting bifidobacteria.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

Activation of porcine oocytes using cyclopiazonic acid, an inhibitor of calcium-dependent ATPases.

Cyclopiazonic acid (CPA), a potent inhibitor of endogenous calcium-dependent ATPases, is able to induce parthenogenetic activation in pig oocytes matured in vitro. Sixty-four percent of matured pig eggs cultured with 100 nM CPA for 4 hr were activated. A similar activation rate was observed in oocytes treated with thapsigargin, another inhibitor of calcium-dependent ATPases. The parthenogenetic development of CPA-activated eggs did not proceed beyond the 8-cell stage. The blockage of calcium channels by verapamil only slightly decreased the proportion of CPA-activated pig oocytes. This indicates that the release of calcium from intracellular stores is sufficient for oocyte activation and calcium influx from extracellular sources has no significant role. The significant decrease in CPA-activated oocytes (100 nM of CPA for 4 hr) after a microinjection of heparin indicated that the mobilization of intracellular calcium stores is mediated through inositol trisphosphate receptors. On the other hand, the only slightly depressed activation rate in oocytes microinjected with ruthenium red and procaine indicates that CPA mobilizes a much smaller amount of calcium through the ryanodine receptors. The marked inhibitory effect of ophiobolin A and W7 on the activation of CPA-treated pig oocytes suggests that the calcium signal, as the second messenger, acts downstream through calmodulin. J. Exp. Zool. 287:304-315, 2000.

Animals↗

PubMed1999

Evaluation of selective media for bifidobacteria in poultry and rabbit caecal samples.

Five media were evaluated to determine their selectivity for Bifidobacterium sp. in hen and rabbit caecal samples. The colonies arising on the plates inoculated with the caecal samples were Gram stained and screened for the presence of fructose-6-phosphate phosphoketolase activity. Rogosa agar modified by the addition of cysteine-hydrochloride (0.05% w/v), Beeren's agar (with 5 ml/l of propionic acid as a selective agent), BS 2 agar (containing per one litre sodium propionate 15 g, lithium chloride 3 g, paromomycin sulphate 50 mg, neomycin sulphate 200 mg), and Wilkins-Chalgren agar (MW) modified by the addition of acetic acid (1 ml/l) and mupirocin (100 mg/l) were selective for Bifidobacterium sp. from rabbit caecal samples. In contrast, only MW medium was suitable for the isolation and enumeration of bifidobacteria in hen caecal samples. In conclusion, the results suggest that MW agar showed the greatest selectivity. A further advantage of this medium is its case of preparation. Therefore this agar could contribute to the study of the effects of the ingestion both probiotics and prebiotics. Finally, it could be noted that the bifidobacteria selective media should be chosen in respect of the animal species origin of the sample tested.

Aldehyde-Lyases↗

PubMed1999

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.

Animals↗

PubMed1997

The protein phosphatase inhibitor okadaic acid inhibits exit from metaphase II in parthenogenetically activated pig oocytes.

The exposure of in vitro matured pig oocytes to the calcium ionophore A 23187 (50 microM, 7 min) resulted in parthenogenetic activation in 67% of the oocytes. When the activated oocytes were cultured, they formed pronuclei. In these oocytes, tubulin labelling revealed a rearrangement of the microtubules into an interphase meshwork. The activated oocytes also lost their ability to form cytoplasmic asters after short-term taxol treatment. The activation rate of the oocytes was further increased when they were cultured with a protein synthesis inhibitor, cycloheximide, after ionophore treatment. A culture of ionophore-treated oocytes with okadaic acid, the inhibitor of protein phosphatases 1 and 2A, prevents the events characterizing oocyte activation. In oocytes cultured with okadaic acid, chromatin remained condensed, and cytoplasm retained its ability to respond to taxol treatment by the formation of cytoplasmic asters. This effect of okadaic acid was observed even in oocytes in which the activating stimulus was followed by a culture with cycloheximide. This data allows us to conclude that protein phosphatases 1 and 2A play an important role during the transition from metaphase II to interphase after activation of the pig oocyte.

Animals↗

PubMed1997

Cyclopiazonic acid induces accelerated progress of meiosis in pig oocytes.

In mammalian oocytes, calcium plays an important role in the regulation of meiotic maturation. In our study, we used the mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, to mobilise intracellular calcium deposits during in vitro maturation of pig oocytes. The CPA treatment of maturing oocytes significantly accelerated the progress of their maturation. Oocytes entered the CPA-sensitive period after 21 h of in vitro culture. A very short (5 min) exposure to CPA (100 mM) is sufficient to accelerate maturation and it seems that accelerated maturation can be triggered by a transient elevation of intracellular calcium levels. The effect of CPA is not mediated through the cumulus cells, because maturation is accelerated by CPA treatment even in oocytes devoid of cumulus cells. Culture of oocytes with the calcium channel blocker verapamil (concentrations ranging from 0.01 to 0.04 mM) blocked the progress of oocyte maturation beyond the stage of metaphase I. This block can be overcome by the mobilisation of intracellular calcium deposits after CPA treatment (100 nM). The microinjection of heparin (20 pl, 0.1 mg/ml), the inhibitor of inositol triphosphate receptors, before CPA treatment prevented the acceleration of oocyte maturation. This indicates that CPA mobilises the release of calcium deposits through inositol trisphosphate receptors. On the other hand, the microinjection of procaine (20 pl, 200 nM) or the microinjection of ruthenium red (20 pl, 50 mM), both inhibitors of ryanodine receptors, did not prevent accelerated maturation in CPA-treated oocytes. If present in pig oocytes, ryanodine receptors evidently play no part in the liberation of calcium from intracellular stores after CPA treatment.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

Beneficial influence of Vero cells on in vitro maturation and fertilization of bovine oocytes.

The aim of this study was to examine the effects of Vero cells and other somatic cells on in vitro maturation of bovine oocytes. Both denuded oocytes and oocytes with intact cumuli (COCs) were cultured on monolayer of Vero cells, cumulus cells and granulosa cells. The effect of gonadotropins was investigated after the addition of gonadotropins to the culture medium. The evaluation using analysis of variance revealed that removal of cumulus cells generally reduced the percentage of oocytes completing their maturation in vitro and that this effect could not be overcome by the addition of gonadotropins to the culture medium. However, in individual experiments, when oocytes were co-cultured with different monolayers of somatic cells, Vero cells were able significantly support the maturation of denuded oocytes, and their beneficial effect was further enhanced by the addition of gonadotropins (76 vs 80.9%). We did not observe a similar effect after the co-culture of oocytes with a monolayer of cumulus cells (65.3 and 53%, respectively). Granulosa cell monolayer delayed maturation in the both COCs and denuded oocytes (10.5 and 16.5%, respectively). In vitro fertilization was successful in most of the experimental groups. However, when denuded oocytes were cultured without any somatic cell support, they did not decondense the penetrated sperm head after in vitro fertilization. This study demonstrates that 1) Vero cells beneficially affect the in vitro maturation of bovine oocytes; 2) cumulus cells in the form of monolayer lose their beneficial influence on in vitro maturation of bovine oocytes; and 3) granulosa cells and FSH and LH alone (without somatic cells) do not show positive effects on in vitro maturation of bovine oocytes.

Journal Article↗