Summary. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and zymography were used to determine the tissue source and to characterize the types of plasminogen activator (PA) produced by bovine blastocysts. Day 12–14 blastocysts were collected at slaughter from oestrus-synchronized, superovulated and artificially inseminated Holstein cows. In Expt 1, blastocysts were cultured for 24 h in Ham's F-12 in a humidified atmosphere of 5% CO₂ in air at 37°C. After culture, blastocysts and medium were recovered and stored separately at −20°C. In Expt 2, embryonic discs were separated from trophoblast by microdissection. Intact blastocysts, embryonic discs and trophoblast were then cultured for 24 h and recovered as in Expt 1. In both experiments, embryonic tissues and media were electrophoresed with PA and molecular mass standards. Polyacrylamide gels were laid onto casein–agar gel plates (zymograms) and incubated at room temperature for 24–48 h. Caseinolytic zones in zymograms containing plasminogen were evidence of PA. In Expt 1, bovine blastocysts contained and secreted light and heavy forms of PA (47·0 ± 1·0 and 86·1 ± 0·7 kDa, respectively). In Expt 2, intact blastocysts and trophoblast produced both forms of PA (41·5 ± 1·5 and 92·2 ± 2·7 kDa) but PAs were not detected in embryonic discs. The results suggest that Day 12–14 bovine blastocysts produce urokinase-type PA (41·5–47·0 kDa) and a form of high molecular mass (86·1–92·2 kDa) that is either a novel tissue-type PA or a PA inhibitor which complexes with the lighter form.

Keywords: cow; blastocyst; plasminogen activator; zymography; electrophoresis

Effects of the metabolic inhibitors cycloheximide and ouabain on development in vitro and plasminogen activator production by sheep embryos were investigated. Embryos (n = 152) from the eight-cell to the morula stage were surgically collected from naturally mated, oestrus-synchronized and superovulated Polypay ewes. In Expt 1, embryos (n = 104) were grouped by cell stage, cultured in Whitten's medium with 1.5% BSA containing 0, 0.1 or 1.0 μg cycloheximide ml⁻¹ for 24 h, washed and cultured in this medium for 168 h. In Expt 2, morulae (n = 48) were cultured for 48 h in Whitten's medium with 1.5% BSA transferred to the same medium containing 0 or 1.0 mmol ouabain l⁻¹ and cultured for 24 h, and then washed and cultured in this medium for 120 h. At 24 h intervals in both experiments, the medium was recovered and analysed for plasminogen activator. In Expt 1, eight-cell embryos underwent limited development; little difference in the production of plasminogen activator due to cycloheximide treatment was therefore observed. Compared with medium without cycloheximide, treatment with 1.0 μg cycloheximide ml⁻¹ reduced the number of 16-cell embryos (P < 0.05) and morulae (P < 0.05) (60% versus 10% and 77% versus 8%, respectively) that began to hatch. The mean production of plasminogen activator was greatest in embryos cultured initially as morulae compared with that of 16-cell and eight-cell embryos (P < 0.05). Cycloheximide treatment suppressed the mean production of plasminogen activator in a dose-dependent manner (P < 0.05). In Expt 2, fewer embryos (P < 0.05) developed to the blastocyst and expanded blastocyst stages following ouabain treatment (83% and 4%, respectively) compared with embryos not exposed to ouabain (100% and 100%, respectively). Embryos treated with ouabain produced less plasminogen activator than did untreated embryos (P < 0.05). These results suggest that developmental changes caused by treating sheep embryos with cycloheximide or ouabain are reflected by changes in the production of plasminogen activator.

Summary. Mice were induced to superovulate and 2-cell embryos were cultured in Whitten's medium with 10 mg bovine serum albumin/ml (WM) as control, Medium WM with 2·3, 4·6, 23·1 or 46·2 μg plasmin/ml, Medium WM with 14·6, 29·1 or 145·7 μg plasminogen/ml, Medium WM with 0·1, 0·2, 1·1 or 2·2 μg trypsin/ml, Medium WM with 0·2, 0·3, 1·6 or 3·3 μg pronase/ml and Medium WM with 10% heat-treated bovine serum (HTBS). Proteolytic activities in the culture media were evaluated at the start of the culture period and 10 days later. Blastocyst formation was significantly reduced in cultures supplemented with pronase and in the two higher levels of trypsin when compared to that in Medium WM. More embryos developed to the blastocyst stage in Medium WM + 2·3 or 23·1 μg plasmin/ml and Medium WM + 14·6 μg plasminogen/ml than in Medium WM (P < 0·05). The incidence of hatching was significantly greater in Medium WM than in all plasminogen- and plasmin-supplemented media except for Medium WM + 29·1 μg plasminogen/ml. Although not significantly different, hatching was lower in Medium WM and Medium WM + 0·1 μg trypsin/ml when compared to Medium WM + HTBS. Similar numbers of embryos completed the hatching process in Media WM, WM + 0·1 or 0·2 μg trypsin/ml and WM + 0·3 μg pronase/ml. Since dissolution of the zona pellucida occurred within 96 h for embryos cultured in Media WM + 1·6 or 3·3 μg pronase/ml and WM + 1·1 or 2·2 μg trypsin/ml, hatching could not be evaluated. The incidence of attachment and subsequent trophoblastic outgrowth was significantly greater in media with plasmin, plasminogen and HTBS than in pronase- and trypsin-supplemented media or in Medium WM alone. The results suggest that the enhancement in embryo development observed in plasmin-and plasminogen-supplemented media is due not only to the provision of protease but to an additional trophic effect as well. Simple medium supplemented with plasmin or plasminogen is as able to support in-vitro development as is medium supplemented with serum.

The effects of varying the potassium concentration in Whitten's medium on the development of pig embryos in vitro was investigated. Osmolality was maintained by adjusting the NaCl concentration. In Expt 1, 209 one-cell to early blastocyst stage embryos were individually cultured in 50 μl microdrops of glucose-free Whitten's medium containing 0.4% (w/v) BSA and 1.5, 3.0, 6.0 or 12.0 mmol potassium l⁻¹. The percentages of embryos undergoing blastocoel formation and expansion for each cell stage evaluated did not differ (P >0.10) among the various concentrations of potassium. However, more four-cell embryos commenced hatching in 3.0 mmol potassium l⁻¹ (53%) than in 1.5 mmol potassium l⁻¹ (16%) (P <0.05) and 12.0 mmol potassium l⁻¹ (24%) (P <0.10). The time required for embryos to develop blastocoels, expand and initiate hatching was not affected (P > 0.10) by the concentration of potassium in the medium. However, blastocoel formation by one- to two-cell embryos was delayed (P < 0.05) in medium with 1.5 mmol potassium l⁻¹ In Expt 2, 89 four-cell embryos were cultured in medium containing 6.0 or 24.0 mmol potassium l⁻¹. No differences (P >0.10) were observed in the percentages of embryos undergoing blastocoel formation, expansion and hatching or in the time required to develop to these stages. The number of cells in embryos recovered at 96 h was greater (P < 0.05) in medium containing 6.0 compared with 24.0 mmol potassium l⁻¹ (32.9±1.7 versus 26.9 ± 2.1, respectively). These results demonstrate that pig embryos are tolerant to a wide range of potassium concentrations when the osmolality of the medium is maintained by adjusting the NaCl concentration. These results also suggest that limitations in development due to culture conditions are not due to inappropriate concentrations of this ion.