TISSUE INHIBITOR OF METALLOPROTEINASES (TIMP-1) PRODUCED BY GRANULOSA AND OVIDUCT CELLS ENHANCES IN-VITRO DEVELOPMENT OF BOVINE EMBRYO

Embryogenesis-stimulating activity (ESA) was found in serum-free conditioned media (CM) of bovine cumulus/granulosa cells (BGC) and bovine oviductal epithelial cells (BOEC). The CM of BGC (BGC-CM) contained two molecular species of ESA, one with a low molecular weight (M(r) 30 000) and another with a high molecular weight (M(r) 80 000); but only the activity with low molecular weight was detected in CM of BOEC by gel-permeation chromatography- The smaller ESA (embryogenin-1) in BGC-CM was purified to homogeneity, as a common activity in both CM by a combination of gel-permeation chromatography ion-exchange chromatography, and reverse-phase HPLC. Embryogenin-1 has a molecular weight of 31 100 (reduced) and has been identified as a bovine tissue inhibitor of metalloproteinase-1 by NH2-terminal amino acid sequence analysis, Western blot analysis, anti-proteinase activities against metalloproteinases, and the nucleotide sequence of cDNA isolated from a lambdagt11 cDNA library of the bovine ovary by a polyclonal antibody against embryogenin-1. These data suggest that the tissue inhibitor of metalloproteinase-1 produced by BGC and BOEC is a major ESA for in vitro development of bovine embryos.