ABSTRACT. This study was conducted to purify a tissue inhibitor of metalloproteinase (TIMP)-1 in a serum-free medium conditioned with bovine oviduct epithelial cells (BOEC) and to evaluate its effect on development of "HanWoo" (Bos taurus coreanae) embryos to the blastocyst stage. In the first study using SDS-PAGE electrophoresis, the presence of 32 kDa proteins, which contains TIMP-1, was detected in the medium conditioned with BOEC, and TIMP-1 was then purified from the medium by gel filtration and HPLC techniques. When examined TIMP-1 secretion, fluorescent foci indicating the secretion of TIMP-1 were found after stained BOEC with fluorescein isothiocyanate. In the next experiment, two-cell embryos derived from in vitro-fertilization were cultured in a serum-free medium, to which 0, 1.25, 2.5 or 5 µg/ml of purified TIMP-1 was supplemented. More (P<0.05) embryos developed to the morula and blastocyst stages after the addition of 2.5 µg/ml to culture medium than after no addition. In conclusion, our data indicate that BOEC secrete TIMP-1 and this glycoprotein promotes the prehatched development of "HanWoo" embryos derived from in vitro-fertilization.-KEY WORDS: bovine embryo, bovine oviduct epithelial cell (BOEC), in vitro-fertilization (IVF), prehatched development, tissue inhibitor of metalloproteinase-1 (TIMP-1).J Recent findings that embryo development is enhanced by culture in either BOEC-conditioned medium [2,3,11,12] or a perifusion culture chamber [18] suggest that BOEC have a positive paracrine action on development of embryos to the blastocyst stage. Attempts to purify the embryotropic factors secreted by BOEC have been made in several studies [15,22]. In our preliminary study conducted to identify embryotropic substances produced by BOEC [14], we found a strong band at the molecular weight of 32 kDa in BOECconditioned medium. We further acknowledged that this protein fraction contains bovine tissue inhibitor of metalloproteinase-1 (TIMP-1), which might have an embryotropic action [22]. From these studies, it was hypothesized that TIMP-1 possibly secreted from BOEC might stimulate "HanWoo" embryo development. Consequently, we firstly attempted to purify TIMP-1 in the conditioned medium with BOEC collected from "HanWoo" species. A fluorescein isothiocyanate-conjugated (FITC) staining method using a rabbit polyclonal antibody was employed for examining the secretion of TIMP-1 by BOEC. Secondly, embryotropic activity of TIMP-1 was evaluated by use of 2-cell embryos derived from in vitrofertilized "HanWoo" oocytes. A serum-free medium was used for evaluating TIMP-1 action on embryo development."HanWoo" (Bos taurus coreanae) inhabits only in the region of Korea peninsula and becomes endangered animal in the bovine species. When considering the meat productivity and a strong disease resistance of this species, development of a reproduction system for this species has an important value. Due to their relatively small size, however, newly-developed technologies for enhancing reproductive efficacy have been li...