Jaroslav Petr

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 by combined treatment of ethanol and cycloheximide

Abstract Activation of meiosis in oocytes by artificial means is important in studies of oocyte function. In pigs, it seems that treatment with ethanol alone is inadequate for efficient activation of oocytes. Data collected in cattle, suggested that addition of a protein synthesis inhibitor increased the effectivness of ethanol for oocyte activation. We investigated the combined effects of exposure to ethanol and to the protein synthesis inhibitor cycloheximide, on activation of in vitro-matured pig oocytes. Treatment with ethanol alone (concentrations 0, 5, 7 and 10 %) for intervals of up to 3 minutes resulted in very limited activation rates (max. 15%). A culture of IVM pig oocytes with cycloheximide alone (10 μg/ml) for 24 hours did not induce oocyte activation either. However, exposure of IVM pig oocytes to 7 and 10 % ethanol followed by culture with cyloheximide substantially increased the activation rate. A maximal activation rate (over 80%) was observed when oocytes were treated with 10% ethanol for 1 min and subsequently cultured with cycloheximide.

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.

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.

Nitric oxide-dependent activation of pig oocytes: role of calcium.

Pig oocytes matured in vitro are parthenogenetically activated after treatment with nitric oxide (NO)-donor SNAP. The chelation of intracellular calcium ions with BAPTA-AM suppressed the SNAP-induced activation in a dose-dependent manner. Activation by a NO-donor is dependent on the influx of calcium from extracellular spaces, because the blockage of calcium channels by verapamil had significantly reduced the activation rate in SNAP-treated oocytes. The blockage of inositol triphosphate receptors had no effect on the activation of oocytes by a NO-donor. On the other hand, the blockers of ryanodine receptors, procaine and ruthenium red, inhibited the activation of oocytes induced by a NO-donor. These data indicate that the activation of pig oocytes by a NO-donor is calcium-dependent. The calcium for the activation is mobilized from extracellular and intracellular spaces. For the mobilization of intracellular calcium stores, it is the ryanodine receptors and not the inositol triphosphate receptors that play a key role.

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 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. Mol. Reprod. Dev. 58:196–204, 2001.

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.

Bisphenol S negatively affects the meotic maturation of pig oocytes

Bisphenol A (BPA), a chemical component of plastics, is a widely distributed environmental pollutant and contaminant of water, air, and food that negatively impacts human health. Concerns regarding BPA have led to the use of BPA-free alternatives, one of which is bisphenol S (BPS). However, the effects of BPS are not well characterized, and its specific effects on reproduction and fertility remain unknown. It is therefore necessary to evaluate any effects of BPS on mammalian oocytes. The present study is the first to demonstrate the markedly negative effects of BPS on pig oocyte maturation in vitro, even at doses lower than those humans are exposed to in the environment. Our results demonstrate (1) an effect of BPS on the course of the meiotic cell cycle; (2) the failure of tubulin fibre formation, which controls proper chromosome movement; (3) changes in the supply of maternal mRNA; (4) changes in the protein amounts and distribution of oestrogen receptors α and β and of aromatase; and (5) disrupted cumulus cell expansion. Thus, these results confirm that BPS is an example of regrettable substitution because this substance exerts similar or even worse negative effects than those of the material it replaced.

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.