Activation of in vitro matured pig oocytes by combined treatment of ethanol and cycloheximide

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Parthenogenetic activation of mammalian oocytes with artificial stimuli is commonly applied in various reproductive biotechniques, e.g. cloning using nuclear transfer. For this reason, many studies focus on oocyte activation in vitro. Recently we have described the activation of pig oocytes using nitric oxide. This activating stimulus is very specific in many aspects. However, it does not provide an adequate stimulus for parthenogenetic development. It was shown that nitric oxide stimulated some signalling pathways which are inactive in conventional treatments for parthenogenetic activation, e.g. the cGMP-dependent signalling cascade. On the other hand, nitric oxide does not stimulate certain signalling pathways involved in oocyte activation after calcium ionophore, e.g. the PKC signalling cascade. The aim of this review is to characterize the complex processes induced in oocytes after treatment with nitric oxide. Perspectives for further research and the application of nitric oxide for parthenogenetic activation of oocytes are outlined.

“…Such a combined treatment improved the results after oocyte activation with ionophore Ito et al, 2003) or ethanol (Petr et al, 1996).…”

Section: Does Nitric Oxide Activate Mammalian Oocytes?mentioning

confidence: 93%

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confidence: 99%

The role of nitric oxide in parthenogenetic activation of pig oocytes: A review

Petr¹,

Eva²,

Tůmová³

et al. 2007

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“…Generally, the combination of activation stimuli (ethanol, calcium ionophore, electrical pulses, ultrasonic waves) with cycloheximide or 6-DMAP increases the activation rate of oocytes and also increases the portion of oocytes developing to the blastocyst stage (Pressice and Yang, 1994;Nussbaum and Prather, 1995;Petr et al, 1996;Jílek et al, 2000Jílek et al, , 2001Mori et al, 2008). It is not clear why under our experimental design the effect of both inhibi-tors increased the activation rate of oocytes but did not increase the proportion of oocytes developing to the blastocyst stage.…”

Section: Discussionmentioning

confidence: 40%

“…treatment using the protein synthesis inhibitor cycloheximide (Petr et al, 1996;Jílek et al, 2000;Mori et al, 2008) or treatment using the inhibitor of protein kinases 6-dimethyl aminopurine (Nussbaum and Prather, 1995;Jílek et al, 2001). The objective of this study was to enhance the efficiency of pig oocyte activation using pulsatile treatment with NO-donor combined with the inhibition of protein synthesis or inhibition of protein kinases.…”

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confidence: 99%

Effects of cycloheximide or 6-dimethyl aminopurine on the parthenogenetic activation of pig oocytes using pulsatile treatment with nitric oxide donor

Krejcova¹,

Petr²,

Krejčová³

et al. 2009

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Pig oocytes matured in vitro were parthenogenetically activated using nitric oxide donor SNAP (2mM). Continuous treatment successfully activated the oocytes only after more than 12 hours of exposure. Pulsatile treatments during which oocytes were repeatedly exposed to 2mM SNAP for a short time (10, 20 or 30 minutes) were more efficient with regard to the activation rate, even when the total exposure time did not exceed 4 hours. Parthenogenetic development was very limited after continuous treatment with 2mM SNAP. A significantly higher proportion of developing parthenogenetic embryos was observed after the pulsatile treatment (development to the morula stage 0 vs. 18%; development to the blastocyst 0 vs. 7%; P < 0.05). However, this developmental rate was significantly lower (P < 0.05) than the development induced by conventional activation treatment with calcium ionophore (development to the morula stage, 23%; development to the blastocyst stage, 18%). When we combined pulsatile SNAP-treatment with the effect of protein kinase inhibitor 6-dimethyl aminopurine (6-DMAP) (2mM 6-DMAP for 2 hours) or with the inhibitor of protein synthesis cycloheximide (CHX) (10 µM CHX for 2 hours), we observed a significant increase (P < 0.05) in the activation rate when compared to the respective pulsatile SNAP-treatment without 6-DMAP or CHX (63 vs. 78% of activated oocytes for 6-DMAP; 63 vs. 83% of activated oocytes for CHX). However, the development of parthenogenetic embryos was not enhanced when the pulsatile SNAP-treatment was combined with 6-DMAP or with CHX.

“…The ratio of aged oocytes spontaneously activated in vitro is generally low (with maxima up to 45%) and is lower than the activation rate a�er the artificial parthenogenesis with various artificial stimuli in pig oocytes matured in vitro under our culture conditions (Jílek et al , 2001Petr et al, 1995Petr et al, , 2000Petr et al, , 2002. Both diploid parthenogenetic embryos (i.e.…”

Section: Discussionmentioning

confidence: 99%

In vitro aging of porcine oocytes

Petrova¹,

Sedmı́ková²,

Eva³

et al. 2004

Porcine oocytes matured in vitro develop in various ways if they are further cultivated. In our studies these oocytes were cultivated for 1 to 5 days (in vitro aging). During the 1st day of aging, most of them remained at the stage of metaphase II (98%). Then many oocytes underwent the spontaneous parthenogenetic activation. The portion of activated oocytes reached its peak a�er 2 or 3 days of aging in vitro (39 or 45%). The portion of fragmented oocytes peaked at the same time (28%). During subsequent aging in vitro (i.e. day 4 or 5 of aging), the portion of lysed oocytes significantly increased (30 or 37%). The highest portion of spontaneously activated parthenogenetic embryos at a pronuclear stage (35%) was observed during the 2nd day of aging in vitro. These pronuclear embryos had mainly one polar body with two pronuclei (47% of all pronuclear embryos) or two polar bodies with one pronucleus (38% of all pronuclear embryos). During the 3rd and 5th day of in vitro aging, there was a significant increase in the portion of parthenogenetic embryos cleaved to the 2-cell or 3-cell stage. When considering the prolonged in vitro culture of porcine oocyte, only the first day of aging should be taken into account, since beyond this time significant changes, i.e. parthenogenesis, fragmentation or lysis, occurred in oocytes under in vitro conditions.