Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: Three aims were funded to investigate: (1) extracellular galectin-15 (LGALS15) regulation of conceptus trophectoderm cell attachment, migration, proliferation and apoptosis; (2) intracellular galectin-15 regulation of apoptosis in the conceptus trophectoderm; and (3) functional role of endometrial galectin-15 in peri-implantation conceptus survival and growth. All of the goals and objectives outlined in Aims 1 and 2 were completed and published. Those experiments and others completed with the funds contributed to the training of three Ph.D. students and one M.S. student. In addition, data generated from the funds was used to prepare a new grant application that was recently funded. The results were disseminated at national and international scientific meetings (Society for the Study of Reproduction and Gordon Research Conference on Reproductive Tract Biology) and in form of scientific publications. PARTICIPANTS: The project was initiated and managed by the principal investigator (Thomas E. Spencer) and a co-investigator (Robert C. Burghardt). Those investigators managed the project in order to complete the goals and objectives. Graduate students who were supported by the project and completed the goals and objectives included Jennifer L. Farmer, Shaye K. Lewis, M. Carey Satterfield and Rebecca M. Simmons. Collaborators on the project within Texas A&M University included David Adelson, Fuller W. Bazer, Sarah G. Black, Chris G. Elsik, David Erikson, Kanako Hayashi, Jinyoung Kim, Kelli Kochan, Greg A. Johnson, Penny K. Riggs, Gwonhwa Song, Thomas H. Welsh, Jr. and Huaijun Zhou. Collaborators outside of the recipient's organization included F. Dean Jousan and Peter J. Hansen (University of Florida) and Gary Newton (Prairie View A&M University). The project supported the training of 3 doctoral students (Jennifer L. Farmer, Shaye K. Lewis, M. Carey Satterfield) and one M.S. student (Rebecca M. Simmons). TARGET AUDIENCES: The target audience is scientific professionals engaged in agricultural research. Research from the project was presented at the annual meeting of the Society for the Study of Reproduction and at the Gordon Research Conference on Reproductive Tract Biology. In addition, the principal investigator presented the project at several international meetings including the International Conference on Ruminant Reproduction. PROJECT MODIFICATIONS: Technical difficulties prevented the completion of Aim 3. Although the morpholinos are delivered very well to the conceptus trophectoderm, we found that delivery to the endometrial luminal and superficial glands is exceedingly poor. Indeed, the endometrial luminal epithelium does not take up enough of the morpholino antisense oligonucleotides to effectively knockdown LGALS15 mRNA translation. We suspect that the differences in morpholino delivery into the conceptus as compared to the endometrial epithelia are due to the lack of cell proliferation.
Impacts
Collectively, the experiments found that the galectin-15 (LGALS15) gene is induced by progesterone and stimulated by interferon tau (IFNT) specifically in the endometrial luminal and superficial glands in sheep. Recombinant ovine and caprine LGALS15 stimulated trophectoderm cell migration and attachment via a RGD-integrin binding domain present in the C-terminus. Interestingly, the carbohydrate recognition domain of LGALS15 did not play a role in those biological effects. Further, experiments found that LGALS15 does not have effects on trophectoderm cell proliferation or elicit apoptosis, but rather partially protects cells against apoptosis induced by agents such as staurosporine. Integrin activation, cell migration and cell attachment are critical processes in conceptus elongation and implantation. One interesting finding was that the LGALS15 gene is present in cattle, sheep and goats, but is only expressed in the uterus of sheep and goats. Thus, significant differences in gene expression exist between the endometria of the sheep and bovine uterus. Using a microarray and candidate gene analysis approach, we found that insulin-like growth factor binding protein one (IGFBP1) and hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) is a progesterone-induced and IFNT-stimulated gene that is expressed specifically in the endometrial LE/sGE of both the ovine and bovine uterus in association with conceptus elongation. In addition to IFNT, available data supports the idea that both IGFB1 and HSD11B1 are also regulated by prostaglandins from either the conceptus and/or endometrium itself. IGFBP1 contains a RGD-integrin binding domain in the C-terminus and also stimulates trophectoderm cell migration and attachment. Thus, common genes and pathways are present in uterus of both sheep and cattle during early pregnancy. Technical difficulties prevented the completion of Aim 3. Although the morpholinos are delivered very well to the conceptus trophectoderm, we found that delivery to the endometrial luminal and superficial glands is exceedingly poor. Indeed, the endometrial luminal epithelium does not take up enough of the morpholino antisense oligonucleotides to effectively knockdown LGALS15 mRNA translation. We suspect that the differences in morpholino delivery into the conceptus as compared to the endometrial epithelia are due to the lack of cell proliferation.
Publications
- Simmons RM, Satterfield MC, Welsh TH Jr, Bazer FW, Spencer TE. 2009. HSD11B1, HSD11B2, PTGS2 and NR3C1 expression in the peri-implantation ovine uterus: effects of pregnancy, progesterone and interferon tau. Biol Reprod; Aug 19. [Epub ahead of print].
- Simmons RM, Erikson D, Kim J, Burghardt RC, Bazer FW, Johnson GA, Spencer TE. 2009. IGFBP1 in the ruminant uterus: potential marker of conceptus elongation and functions in trophectoderm migration and attachment. Endocrinology; Jun 4. [Epub ahead of print].
- Satterfield, MC, Song G, Kochan KJ, Riggs PK, Simmons RM, Elsik CG, Adelson DL, Bazer FW, Zhou H, Spencer TE. 2009. Discovery of candidate genes in the endometrium regulating ovine blastocyst growth and conceptus elongation. Physiological Genomics; Aug 18. [Epub ahead of print].
- Song G, Satterfield MC, Kim J, Bazer FW, Spencer TE. 2008. Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy. Reproduction; 137:1-14.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Three aims were funded to investigate: (1) extracellular galectin-15 regulation of conceptus trophectoderm cell attachment, migration, proliferation and apoptosis; (2) intracellular galectin-15 regulation of apoptosis in the conceptus trophectoderm; and (3) functional role of endometrial galectin-15 in peri-implantation conceptus survival and growth. All of the experiments outlined in Aims 1 and 2 were completed and published. Those experiments and others completed with the funds contributed to the training of three Ph.D. students and one M.S. student. Collectively, the experiments found found that LGALS15 is induced by progesterone and stimulated by IFNT specifically in the endometrial LE/sGE in sheep. Recombinant ovine and caprine LGALS15 stimulated trophectoderm cell migration and attachment via a RGD-integrin binding domain present in the C-terminus. Interestingly, the carbohydrate recognition domain of LGALS15 did not play a role in those biological effects. Further, experiments found that LGALS15 does not have effects on trophectoderm cell proliferation or elicit apoptosis, but rather partially protects cells against apoptosis induced by agents such as staurosporine. Integrin activation, cell migration and cell attachment are critical processes in conceptus elongation and implantation. One interesting finding was that the LGALS15 gene is present in cattle, sheep and goats, but is only expressed in the uterus of sheep and goats. Thus, differences in gene expression exist between the sheep and bovine uterus. Using a microarray and candidate gene analysis approach, we found that IGFBP1 is a progesterone-induced and IFNT-stimulated gene that is expressed specifically in the endometrial LE/sGE of both the ovine and bovine uterus in association with conceptus elongation. IGFBP1 contains a RGD-integrin binding domain in the C-terminus and also stimulates trophectoderm cell migration and attachment. Thus, common genes and pathways are present in uterus of both sheep and cattle during early pregnancy. Technical difficulties prevented the completion of Aim 3. Although the morpholinos are delivered very well to the conceptus trophectoderm, we found that delivery to the endometrial LE/sGE is exceedingly poor. Indeed, the endometrial LE does not take up enough of the morpholino antisense oligonucleotides to effectively knockdown LGALS15 mRNA translation. We suspect that the differences in morpholino delivery into the conceptus as compared to the endometrial LE/sGE are due to the lack of cell proliferation in the LE/sGE. As a possible solution to this problem, we are currently investigating the feasibility of lentiviral transgenesis as a method to modify gene expression patterns in the endometrial epithelia. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The target audience is scientific professionals engaged in agricultural research. This research was presented by the two graduate students at the annual meeting of the Society for the Study of Reproduction in 2008. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Infertility and pregnancy loss represent major constraints to reproductive efficiency in domestic livestock that decrease profitability of agricultural enterprises. Studies on the genes and proteins produced by the uterus may lead to the identification of factors that can enhance the efficiency of in vitro embryo production and the post-transfer survival of embryos as well as identify infertile animals. Currently, there are few markers to predict the fertility of female ruminant animals. Knowledge gained from these studies will be useful to design management, biotechnology and genetic applications aimed at enhancing production efficiency in animal agriculture.
Publications
- Satterfield MC, Song G, Hayashi K, Bazer FW, Spencer TE. 2008. Progesterone regulation of the endometrial WNT system in the ovine uterus. Reprod Fertil Dev; 20:935-946.
- Satterfield MC, Hayashi K, Song G, Black SG, Bazer FW, Spencer TE. 2008. Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus. Biol Reprod; 79:1226-1236.
- Song G, Satterfield MC, Kim J, Bazer FW, Spencer TE. 2008. Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone. Biol Reprod; 79:376-386.
- Song G, Satterfield MC, Kim J, Bazer FW, Spencer TE. 2008. Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy. Reproduction; 137:1-14.
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Long-term research goals are to: (a) identify and determine the physiological function(s) of genes expressed in uterine epithelia; and (b) determine the biological function(s) of endometrial secretions in conceptus (embryo/fetus and associated extraembryonic membranes) survival and growth. In order to test the central hypotheses, we will pursue three specific aims to investigate: (1) extracellular galectin-15 regulation of conceptus trophectoderm cell attachment, migration, proliferation and apoptosis; (2) intracellular galectin-15 regulation of apoptosis in the conceptus trophectoderm; and (3) functional role of endometrial galectin-15 in peri-implantation conceptus survival and growth. During the second year, we completed most of the studies outlined in specific aims 1 and 2. Studies tested the hypothesis that LGALS15 is a secreted regulator of blastocyst development, as well as growth, migration, adhesion, and apoptosis of trophoblast. We found that LGALS15 in both sheep
and goats contains a conserved RGD recognition sequence in the C-terminus for integrin binding. Bovine embryos were produced in vitro by standard conditions, and putative zygotes were cultured in the presence of recombinant ovine LGALS15. Rates of embryo cleavage and blastocyst formation were not affected by LGALS15. LGALS15 moderately increased proliferation of ovine trophectoderm (oTr) cells. Staurosporine elicited apoptosis of oTr cells, which could be partially inhibited by LGALS15. Migration of oTr cells was stimulated by LGALS15 that was dependent on Jun N-terminal kinase (JNK). 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 was abolished by mutation of
the LDVRGD sequence. Although the bovine genome contains an LGALS15-like gene, expressed sequence tags encoding LGALS15 mRNA were found only for sheep, and full-length LGALS15 cDNAs were cloned only from endometrial total RNA isolated from pregnant sheep and goats, but not pregnant cattle or pigs. Ovine and caprine LGALS15 were highly homologous at the mRNA (95%) and protein (91%) levels. In situ hybridization detected abundant LGALS15 mRNA in endometrial LE and sGE of early pregnant goats, but not in cattle or pigs. Recombinant ovine and caprine LGALS15 proteins elicited a dose-dependent increase in ovine trophectoderm cell attachment in vitro that was comparable to bovine fibronectin. These results support the hypotheses that LGALS15 is uniquely expressed in Caprinae endometria and that it stimulates trophectoderm cell migration and attachment via integrin binding and activation which are critical to blastocyst elongation and implantation.
PARTICIPANTS: Drs. Thomas E. Spencer and Robert C. Burghardt are co-principal investigators and worked together to design experiments and supervise two graduate students working on this research. Ms. Jennifer L. Farmer and Mr. Shaye Lewis are two graduate students who are pursuing their doctoral studies. The two scientific papers generated from this research are part of their dissertation studies.
TARGET AUDIENCES: The target audience is scientific professionals engaged in agricultural research. This research was presented by the two graduate students at the annual meeting of the Society for the Study of Reproduction in 2007.
Impacts
Infertility and pregnancy loss represent major constraints to reproductive efficiency in domestic livestock that decrease profitability of agricultural enterprises. Studies on the genes and proteins produced by the uterus may lead to the identification of factors that can enhance the efficiency of in vitro embryo production and the post-transfer survival of embryos as well as identify infertile animals. Currently, there are few markers to predict the fertility of female ruminant animals. Knowledge gained from these studies will be useful to design management, biotechnology and genetic applications aimed at enhancing production efficiency in animal agriculture.
Publications
- Farmer JL, Burghardt RC, Jousan FD, Hansen PJ, Bazer FW, Spencer TE. 2007. Galectin 15 (LGALS15) functions in trophectoderm migration and attachment. FASEB J 2007 Sep 21; [Epub ahead of print].
- Lewis SK, Farmer JL, Burghardt RC, Newton GR, Johnson GA, Adelson DL, Bazer FW, Spencer TE. 2007. Galectin-15 (LGALS15): a gene uniquely expressed in the uteri of sheep and goats that functions in trophoblast attachment. Biol Reprod 2007; 77:1027-1036
- Spencer TE, Johnson GA, Bazer FW, Burghardt RC, Palmarini M. 2007. Pregnancy recognition and conceptus implantation in domestic ruminants: roles of progesterone, interferons and endogenous retroviruses. Reprod Fertil Dev 2007; 19:65-78.
- Spencer TE, Johnson GA, Bazer FW, Burghardt RC. 2007. Fetal-maternal interactions during the establishment of pregnancy in ruminants. Soc Reprod Fertil Suppl 2007; 64:379-396.
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Progress 09/01/05 to 08/31/06
Outputs
Long-term research goals are to: (a) identify and determine the physiological function(s) of genes expressed in uterine epithelia; and (b) determine the biological function(s) of endometrial secretions in conceptus (embryo/fetus and associated extraembryonic membranes) survival and growth. In order to test the central hypotheses, we will pursue three specific aims to investigate: (1) extracellular galectin-15 regulation of conceptus trophectoderm cell attachment, migration, proliferation and apoptosis; (2) intracellular galectin-15 regulation of apoptosis in the conceptus trophectoderm; and (3) functional role of endometrial galectin-15 in peri-implantation conceptus survival and growth. During the first year, uterine flushes were collected for analysis of galectin-15 protein abundance. Large amounts of pure recombinant ovine galectin-15 and mutated galectin-15 were produced in bacteria and purified using affinity chromatography. A high affinity polyclonal antibody to
recombinant ovine galectin-15 was produced in rabbits. The carbohydrate binding specificity of galectin-15 was determined by glycan array analysis, which found specific binding to alpha-fucose and mannose carbohydrate structures. Two ovine mononuclear trophectoderm cell lines were established from Day 15 conceptuses. Attachment assays using the trophectoderm cells revealed that galectin-15 promoted attachment that was dependent on a LDVRGD sequence in the C-terminus. Further, galectin-15 increased the proliferation of one of two trophectoderm cell lines. However, galectin-15 did not affect trophectoderm cell migration or inhibit apoptosis. Further, galectin-15 did not promote maturation of in vitro produced bovine blastocysts or inhibit lymphocyte proliferation in response to an activation agent. Recent results indicate that galectin-15 is also present in the goat uterus. Available results support the hypothesis that galectin-15 is a progesterone induced and interferon tau stimulated
secreted protein from the endometrium that mediates conceptus attachment and elongation during the peri-implantation period of pregnancy in ruminants.
Impacts
Infertility and pregnancy loss represent major constraints to reproductive efficiency in domestic livestock that decrease profitability of agricultural enterprises. Studies on the genes and proteins produced by the uterus may lead to the identification of factors that can enhance the efficiency of in vitro embryo production and the post-transfer survival of embryos as well as identify infertile animals. Currently, there are few markers to predict the fertility of female ruminant animals. Knowledge gained from these studies will be useful to design management, biotechnology and genetic applications aimed at enhancing production efficiency in animal agriculture.
Publications
- Satterfield MC, Bazer FW, Spencer TE. Progesterone regulation of pre-implantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus. Biol Reprod 2006; 75:289-296.
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