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Fred Menino
Professor, Animal Sciences
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RESEARCH:
Our laboratory has been investigating mechanisms of regulation and the physiologic role of plasminogen activator (PA) during early embryo development. Researchers working with mouse embryos have demonstrated PA production during the peri-implantation period and believe PA facilitates penetration of the uterus in implantation. We have identified a temporal pattern of PA production by cultured rabbit, cattle, and sheep embryos. Rabbit embryos, which have an invasive implantation similar to the mouse, produc e PA at the time of uterine penetration. Sheep embryos begin to produce PA at the morula-blastocyst transition whereas cattle embryos initiate production after the blastocyst stage and throughout blastocoelic expansion. Sheep and cattle embryos, however, do not share the type of invasive implantation characteristic of mouse and rabbit embryos, yet produce PA during similar developmental periods. Zymographic analysis of Day 12-14 cattle embryonic tissues identified an urokinase-type PA (uPA, 49kD) produced by the trophoblast. A high molecular mass PA (85kD) was also detected in the trophoblast, suggesting either a novel tissue-type PA (tPA) or a uPA complexed with a PA inhibitor (PAI). Results of experiments with amiloride, a competitive inhibitor of uPA, indicated that all PA activity exhibited by Day 12-14 cattle embryos was amiloride-sensitive, suggesting the absence of tissue-type PA. A similar series of experiments was conducted with sheep embryos and both a uPA (50kD) and uPA-PAI complex (81kD) were identified. These observations suggest that in species like cattle and sheep, production of PAI by the embryo may regulate embryonic-induced proteolysis through the PA cascade and limit uterine penetration.
Oocyte-cumulus cell complexes from cattle and pig ovaries also produce PA and PAI during maturation in vitro. Cattle oocyte-cumulus cell complexes produce uPA, tPA and PAI-1 whereas pig oocyte-cumulus cell complexes produce tPA and PAI-1. Production of these proteins can be modulated by stimulators of the protein kinase A and C systems and reagents affecting cellular phosphorylation and dephosphorylation. Okadaic acid, a potent phosphatase inhibitor, drastically increased PAI-1 production in oocyte-cumulus cell complexes from cattle and induced the appearance of uPA in pig oocyte-cumulus cell complexes.
Experiments currently under way are aimed at identifying the developmental stages when transcriptional and translational events for embryonic PA production occur. Preliminary experiments with cattle and sheep embryos have suggested that uPA production is cell stage-specific and transcription and translation of the uPA zymogen may occur during the transition from maternal to embryonic genomic control of development. We are also conducting work to elucidate the physiological relevance of this enzyme to processes involved in cellular migration and differentiation during early embryogenesis.