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The fate of proteins formed during meiotic maturation was examined after fertilization. Rabbit ovarian oocytes were labelled in vitro with [3H]lysine and fertilized after transfer to recipients. A significant accumulatin of the label was detected autoradiographically only in fully grown male and female pronuclei. Pig oocytes at the germinal vesicle and metaphase I stages were labelled with [3H]lysine, [3H]methionine or [3H]tryptophan and fertilized. Pronuclei were labelled by all 3 precursors. During cleavage, eggs labelled with [3H]lysine lost the nuclear label by the 4-cell stage. However the [3H]methionine label was present in the cytoplasm and marked in the nuclei at the 4-cell stage, while the [3H]tryptophan label was still clear in 8-cell embryos.

One hundred and forty-two deep-frozen seven days old bovine embryos were, after thawing and stepwise dilution of cryoprotectant, morphologically evaluated under the stereomicroscope. According to the morphology the embryos were divided into four categories as follows: I--fully expanded blastocysts, II--slightly contracted embryos with a minute part of degenerated cells, III--contracted embryos with some degenerated cells, IV--degenerated embryos and those with heavy-damaged zona pellucida, or its complete loss. After surgical transfer of 37 embryos of class I, 16 (43.2%) recipients were pregnant. Transfer of 53 embryos of class II established 20 (37.7%) pregnancies. After transfer of 28 embryos of class III, 10 (35.7%) recipients were pregnant. Twenty four (17%) embryos included in class IV were excluded from transfer. These results indicate that simple morphological evaluation of embryo after freezing and thawing secures relatively objective selection in field conditions. At least this method enables to reveal morphological changes which are not compatible with further development.

Cumulus expansion and cumulus cell-oocyte coupling during in-vivo and in-vitro maturation of pig oocytes were studied by measuring [3H]uridine uptake. In vivo, cumulus expansion started before germinal vesicle breakdown (GVBD) (16 h versus 20 h after hCG) but no significant change occurred in the coupling index until 32 h after hCG. Intercellular coupling was decreasing at 32 h after hCG in oocytes at anaphase I and telophase I. Complete uncoupling was closely correlated with corona radiata expansion. In vitro, partial uncoupling was observed in oocyte-cumulus cell complexes from prepubertal and PMSG-stimulated gilts cultured for 16 and 32 h, respectively. The addition of FSH caused cumulus expansion, and the functional coupling between the cumulus cells and the oocyte was maintained up to at least 16 h of culture in complexes from prepubertal gilts. We conclude that, under our conditions, neither hormone-free nor FSH-supplemented medium ensured the same [3H]uridine uptake and uncoupling kinetics as during in-vivo maturation.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.

The superovulation of donors, heifers and cows, was induced by means of gonadotropin PMSG (Antex Leo) in combination with the synthetic prostaglandin analogue--PGF2 alpha (Estrumate, ICI) applied 48 hours later. The response of donors to this treatment was variable. The average number of ovulating follicles from one heifer was fourteen, and the number of embryos to be used was four. In cows the average number of ovulating follicles amounted to six, and on the average three embryos from one donor could be used. Seventy-four embryos were transferred to thirty-seven recipients, into each horn of uterus one embryo. Twenty-six (70%) cows were pregnant, out of this number seventeen (65%) recipients gave birth to twins. In some cases the isolated embryos were kept in the oviduct of female rabbit for 72 hours. It was proved that this deposition did not exert any negative influence on further development of embryos.

Growing mouse oocytes incompetent to resume meiosis were fused with fully grown immature mouse oocytes and cultured for 20-24 h. In giant cells that developed, two intact germinal vesicles remained well conserved in all cases. We propose that the cytoplasm of growing oocytes possesses a maturation-inhibiting activity which is able to arrest, after fusion, nuclear maturation in fully grown oocytes competent to mature spontaneously.