The effects of hormones and inducers of intracellular messengers on bovine embryo development in vitro : plasminogen activator production and changes in embryonic size Public Deposited

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  • The effects of several hormones and inducers of intracellular messengers on plasminogen activator (PA) production and changes in embryonic size by cultured bovine embryos were evaluated. Day 8 embryos were cultured in Ham's F-12 with 1.5 mg/ml bovine serum albumin (BSA) containing different levels of progesterone (P), estradiol -17fl (E₂), dexamethasone (Dex), retinoic acid (RA), dibutyryl cyclic AMP (dbcAMP), or phorbol myristate acetate (PMA) for 5 days under paraffin oil in a humidified atmosphere of 5% CO₂ in air at 37°C. The concentrations of PA in the conditioned media were determined by a caseinolytic assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography were used to determine the molecular weight of PA in the medium and in the embryo homogenate. Changes in embryonic size were determined by measuring overall embryo diameter (OD) at 24-h intervals. None of the hormones and agents tested herein had a significant effect on PA production. Dimethyl sulfoxide (DMSO) which was used to dissolve PMA significantly inhibited PA production during the first 72 h of culture. Time of culture, however, exerted a significant effect on PA production by cultured embryos. The production of this protease was low during the first 48 h, increased during 72 and 96 h, and either remained high or slightly decreased toward the end of the culture period. Furthermore, the peak production of PA was attained 48 h after hatching. The molecular weight of PA in the conditioned medium and embryo tissues suggested that the bovine embryo at this developmental stage produced an urokinase-type PA. With the exception of dbcAMP and PMA, the hormones tested in this study did not affect embryonic size. While dbcAMP decreased OD later in culture, PMA enhanced OD throughout culture. The mechanism by which dbcAMP and PMA modulated embryonic size is not clear. These results suggest that cultured bovine embryos produce urokinasetype PA in a time dependent manner and the production of this enzyme is independent of exogenous hormonal regulation.
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