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.

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.

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.

[Cryopreservation of embryos in the IVF/ET program at the Maternal and Child Care Institute in Prague]. / Kryokonzervace embryí v programu IVF/ET v Ustavu pro péci o matku a díte v Praze.

Two methods for embryo cryopreservation using 1,2-propanediol were compared--ultra-rapid vitrification and controlled freezing. No significant differences were found in the pregnancy and implantation rates although better results were achieved by the controlled freezing method. Significantly better results both in pregnancy and implantation rates were obtained when the embryos were frozen in the pronuclear stage compared to cleaved stage embryos. In the group of pronuclear stage embryos 3 pregnancies were achieved as a result of 11 embryo transfers (27%, implantation rate 9%) vs 14 pregnancies from 193 embryo transfers (7%, implantation rate 2.5%) for the cleaved embryos.

Microtubule rearrangement during in vitro maturation of pig oocytes. Effect of cycloheximide.

In freshly isolated fully grown pig oocytes at the germinal vesicle (GV) stage, the cytoplasmic microtubules are arranged in a meshwork. This microtubule arrangement is also maintained during the initial phases of meiotic maturation in vitro. A perinuclear array of microtubules is formed immediately before germinal vesicle breakdown (GVBD). Short-term treatment of oocytes with taxol when oocytes are at metaphase I stage induced formation of cytoplasmic asters. The oocyte cytoplasm is unable to respond to the taxol treatment at the earlier stages of meiotic maturation. In oocytes cultured with the proteosynthesis inhibitor cycloheximide, meiotic maturation is blocked. Condensation of chromatin occurs but the nuclear envelope is preserved and the microtubule arrangement is unchanged. A perinuclear array of microtubules does not appear and oocyte cytoplasm does not respond to short-term taxol treatment by the formation of cytoplasmic asters. We can conclude that the microtubule rearrangement and the acquisition of competence for tubulin assembly are blocked by cycloheximide and are thus dependent on de novo proteosynthesis.

Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells.

Total inhibition of germinal vesicle breakdown (GVBD) was observed in growing pig oocytes (internal diameter 80, 90 and 100 microns) when they were cultured in a medium conditioned by cumulus oocyte complexes (COCs) of fully grown oocytes. In denuded growing oocytes, only partial inhibition was observed. The inhibitory effect was fully reversible. The addition of heparin (300 IU/ml) could overcome the effect of the conditioned medium. Transient exposure (6 h) of oocytes to dibutyryl cyclic adenosine monophosphate (dbcAMP) (1 mg/ml) could also partly reverse the effect of factor (s) produced by cumulus cells of fully grown oocytes. Follicle-stimulating hormone (5 micrograms/ml) was able to increase the percentage of maturing oocytes. The addition of luteinizing hormone (5 micrograms/ml) had no effect on GVBD inhibition by cumulus-conditioned medium.