96 EXPRESSION OF THE LONG FORM OF THE LEPTIN RECEPTOR IN BOVINE OVIDUCT EPITHELIAL, UTERINE EPITHELIAL, AND BLASTOCYST-STAGE EMBRYOS

Abstract

Critical reproductive events of animals are affected via metabolic cues that directly modulate the hypothalamic-pituitary-gonadal axis. The adipose-derived 146-amino-acid protein-hormone leptin has been associated with puberty and reproduction in many mammals and may be of critical importance in the regulation of reproduction. Leptin receptors are expressed in the brain, pituitary, and gonads, and there is growing evidence that leptin may be required for normal embryonic implantation and development in some animals. To date 6 isoforms of the leptin receptor have been identified; the long form (Ob-Rb) is the only isoform to be associated with signal transduction. The objective of this experiment was to test the hypothesis that the Ob-Rb isoform is expressed in cultured bovine oviduct and uterine epithelial cells, in vivo-derived mid-luteal uterine biopsies, and expanded bovine blastocyst-stage embryos. For cells, cell lines from mid-luteal abattoir-derived reproductive tracts (n = 5) were used; for uterine biopsies (n = 20), biopsies were collected from both uterine horns at the greater curvature from mature beef cows (n = 4) in mid-luteal phase; expanded IVP blastocyst-stage embryos (n = 40) were produced from abattoir-derived ovaries. For fluorescence microscopy samples were incubated in rabbit anti-human leptin receptor (#4781-L, Linco Research, St. Charles, MO) followed by Alexa Fluor 488 anti-rabbit IgG (#A-11008, Molecular Probes, Carlsbad, CA). We confirmed that the Ob-Rb isoform is expressed in both oviductal and uterine in vitro-cultured epithelial cells and uterine biopsies. All oviduct but not all uterine-cultured epithelial cells were immunopositive for the Ob-Rb isoform. All uterine biopsies were positive for the Ob-Rb isoform along the luminal side of the uterine endometrium with an apparent lack of staining occurring in myometrial tissue. Moreover, we demonstrated that all bovine blastocysts were immunopositive with staining occurring within the trophoblast cell type but not the inner cell mass. These results show for the first time that bovine oviduct and uterine epithelial cells express the long form of the leptin receptor and support earlier findings that blastocyst-stage embryos contain Ob-Rb isoforms. This suggests that the Ob-Rb isoform is a component of the bovine maternal-embryonic complex; however, its function in pre-implantation embryo development remains to be clarified.