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.

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro.

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 µM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 µM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 µM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).

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.

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.

Short review of some properties of muscular proteins.

Proteins are the basic material of tissue structure. They are the most important component of striated skeletal muscle. Their classification is correlated with the histological structure of muscle tissue. The muscle proteins can be divided in to contratile, regulatory, sarcoplasmic and extracellular forms. The most important are the contractile proteins actin and myosin. Among the regulatory proteins, troponin, tropomyosin, M-protein, beta-actin, gamma-actin and C-protein are great importance. Sarcolemma is also a protein component. Proteins such as myoglobin, myogen, myoalbumin and x-globulin are found in the sarcoplasm and proteins such as elastin, collagen and reticulin are also found in the muscle. The remaining of proteins includes myofibrillar proteins of the Z-disc as well as small quantities of other proteins. This work is a short review of some properties of the muscular proteins.

In vitro ageing of pig oocytes: effects of the histone deacetylase inhibitor trichostatin A.

After in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.

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.

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.

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.

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.

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 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.

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.

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.

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 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.

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.

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.

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.