Purification and Embryotropic Roles of Tissue Inhibitor of Metalloproteinase-1 in Development of "HanWoo"(Bos taurus coreanae) Oocytes Co-cultured with Bovine Oviduct Epithelial Cells.

Abstract

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...

Ovulation rate and early embryonic survival rate in female rabbits of a synthetic line and a local Algerian population

“…The oviduct synthesizes and secretes many proteins in many species including rabbit (García et al, 2010), swine (Buhi and Alvarez, 2003), sheep and cattle (Nancarrow and Hill, 1995) which influence the gene expression of the developing embryos. Several proteins play an important role in embryo development and embryogenesis regulation (Insulin-like growth factor 1, Herrler et al, 1998;oviductin, Buhi and Alvarez, 2003; TIMP metallopeptidase inhibitor 1, Hwang et al, 2000;uteroglobin, Riffo et al, 2007;leptin, Zerani et al, 2005). The asynchrony associated with the advanced uterine environment could cause the death of less developed embryos (Wilde et al, 1988;Xie et al, 1990).…”

Candidate gene analysis for reproductive traits in two lines of rabbits divergently selected for uterine capacity1

“…These differences in early embryo development seem to be associated with greater ES in the AA genotype than in the GG and AG genotypes at this stage of gestation, representing 32 and 25% of the SD for EES, and 80 and 65% of the SD for % CM, respectively. A disruption of the TIMP1 gene product is associated with a significant increase in uterine matrix metalloproteinase activity, leading to a negative effect on the development of embryos (Hwang et al, 2000;Nothnick, 2000Nothnick, , 2001a. Estellé et al (2006) reported greater expression of the TIMP1 gene in the oviduct at 62 h of gestation in the High line.…”

“…Some proteins have an important role in embryo development. Secretoglobin family 1A member 1 (SCGB1A1, also known as uteroglobin; Riffo et al, 2007), IGF1 (Lin et al, 2003), and tissue inhibitor of metalloproteinases 1 (TIMP1; Hwang et al, 2000) are involved in regulating the embryogenesis, angiogenesis, morphogenesis, and endometrial tissue remodeling processes that take place during gestation. Therefore, SCGB1A1, IGF1, and TIMP1 could explain part of the embryonic losses before implantation in rabbits.…”

Methanobrevibacter boviskoreani sp. nov., isolated from the rumen of Korean native cattle

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