Browsing by Subject "ovine"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Biological functions of galectin 15 (lgals15) in the ovine uterus(2009-05-15) Farmer, Jennifer LynnGalectins are proteins with 15 known members found in nearly all living organisms. They share a conserved CRD that binds beta-galactoside sugars, and functions to cross-link glycoproteins as well as glycolipid receptors on the surface of cells to initiate biological responses. Functional studies on the extracellular and intracellular roles of galectins implicate them in cell adhesion, chemoattraction and migration as well as growth, differentiation and apoptosis. Therefore, studies were conducted to identify functional roles of galectin 15 (LGALS15) during the periimplantation period of pregnancy in the sheep. The first study was designed to develop and characterize primary ovine trophectoderm cell lines for the study of the biological functions of LGALS15. Once characterized, these cell lines were used to investigate the role of LGALS15 in trophectoderm gene expression, development, growth, and survival. Two primary trophectoderm cell lines (oTr1 and oTrF) were developed, and they had characteristics similar to in vivo conceptus trophectoderm relative to gene expression, morphology, and migration and proved suitable as an in vitro model to investigate functional roles of LGALS15. The second study investigated LGALS15 function in trophectoderm cell adhesion. A dose-dependent increase in oTr cell attachment to LGALS15 was found that could be inhibited by cyclic GRGDS, but not GRADS, peptides. Mutation of the LDVRGD integrin binding sequence of LGALS15 to LADRAD decreased its ability to promote oTr cell attachment, whereas mutation of the CRD had little effect. LGALS15 induced formation of robust focal adhesions in oTr cells that were abolished by mutation of the LDVRGD sequence. The third study tested the hypothesis that LGALS15 is a secreted regulator of trophectoderm development and gene expression, as well as growth, migration, and apoptosis of trophoblast. LGALS15 moderately increased cellular proliferation, partially inhibited staurosporine elicited apoptosis, stimulated migration that was dependent on Jun N-terminal kinase (JNK), and initiated differential gene expression of oTr cells. Collectively, these results support the hypothesis that LGALS15 has a biological role in the peri-implantation stage of early pregnancy in the ovine uterus and stimulates trophectoderm cell gene expression, migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation.Item Identification of endometrial genes important for conceptus survival and development in sheep(Texas A&M University, 2005-08-29) Gray, Catherine AllisonRecurrent early pregnancy loss in the ovine uterine gland knockout (UGKO) ewe model manifests on Day 14 of pregnancy, indicating that endometrial secretions are critical for peri-implantation conceptus development. Therefore, the following studies were conducted with fertile ewes and infertile UGKO ewes to identify candidate endometrial factors essential for normal conceptus survival, utilizing both genomics and proteomics approaches. The first study used transcriptional profiling of endometrium from Day 14 cyclic, pregnant, and bred UGKO ewes, as well as ewes treated with interferon tau (IFN??) and progesterone, to identify genes important for conceptus development. A number of novel and previously known IFN??-stimulated genes, as well as progesterone-stimulated genes were identified that are higher in fertile ewes, such as galectin-15. Interactive effects of progesterone and IFN?? regulate endometrial gene expression in a temporal and cell-type specific manner. The second study characterized the endometrial expression and hormonal regulation of galectin-15, a member of the galectin family of secreted ??-galactoside lectins. Galectin-15 was secreted into the uterine lumen by the lumenal (LE) and superficial glandular epithelium (sGE), where it may promote adhesion during implantation, as well as was phagocytosed by the trophectoderm and formed intracellular crystals. The third study determined the endometrial expression of galectin-15 throughout gestation. Galectin-15 was secreted into the uterine lumen, where it was phagocytosed by the trophectoderm/chorion, transferred through placental vasculature to the fetus, and cleared through the fetal kidney to be stored in allantoic fluid. The fourth study utilized proteomic analysis of uterine flushes and endometrial explant cultures from Day 14 cyclic, pregnant and UGKO ewes to identify differences in uterine secretions. Analyses identified several genes that were expressed by the LE and sGE and may be involved in prostaglandin production and/or pH regulation. Collectively, results of these studies suggest that transcriptional profiling and analysis of uterine secretions are effective tools to determine genes important for early pregnancy. Further, identified genes are expected to reveal novel endometrial factors and metabolic pathways for support of conceptus survival and implantation, as well as provide improvements for embryo culture methods and diagnose endometrial dysfunctions leading to infertility.Item Recovery and evaluation of somatic cells from ovine and bovine semen for use in nuclear transfer(2009-05-15) Liu, JieSomatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.