J. Rozinek

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.

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.

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.

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.

Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187.

The pig ovary contains a large number of growing oocytes, which do not mature in vitro and cannot be readily used in various biotechnologies. This study was conducted to determine the possibility of inducing meiotic maturation in growing pig oocytes with an internal diameter of 110 microm, which had developed partial meiotic competence. Most of these oocytes spontaneously stopped maturation at the metaphase I stage (68%); a limited number proceeded to the metaphase II stage (26%). Treatment with calcium ionophore A23187 (50 microM for 5 or 10 min) after 24h in vitro culture overcame the block at the metaphase I stage, and treated growing pig oocytes matured to the metaphase II stage (66%). Oocytes in which maturation had been induced by calcium ionophore were again treated with calcium ionophore. Up to 58% of the treated oocytes were activated. Parthenogenetic development in oocytes treated with ionophore for meiosis induction and activation was very limited. The portion which reached morula stage did not exceed 8% and at most 3% developed to the blastocyst stage.

Effect of testosterone and dibutyryl c-AMP on the meiotic competence in pig oocytes of various size categories

Abstract Spontaneous meiosis resumption was investigated in small pig oocytes of various size categories. Two parameters were studied: 1) the possibility for inhibiting spontaneous meiosis resumption in small oocytes of various size categories and 2) the level of meiotic competence of small oocytes in which meiosis resumption had been temporarily blocked. It was observed that 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) combined with 0.5 μM testosterone were able to prevent spontaneous maturation in pig oocytes of various size categories cultured in vitro for up to 10 d. Moreover, a culture of these oocytes with dbcAMP and testosterone has a beneficial effect on the integrity of the oocyte-granulosa cell complex. When oocytes were allowed to mature after long-term inhibition of maturation, the percentage of oocytes able to resume maturation was significantly increased. However, the portion of oocytes which completed maturation, reaching the metaphase II (M II) stage, did not increase. It was found that dbcAMP, in combination with testosterone, also efficiently inhibited spontaneous maturation in fully grown pig oocytes. When the inhibitory effect of dbcAMP and testosterone was reversed in fully grown oocytes, maturation was significantly accelerated.

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.