Progress 09/01/01 to 08/31/06
Outputs
All mammalian uteri have endometrial glands that secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the completed studies were to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective utilized human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective was revised to
include a large-scale expressed tag sequencing project to identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high throughput cDNA microarray systems. The selected cDNAs were then be confirmed by real-time PCR and sequenced. This approach was feasible and successful. These studies identified novel expressed sequence tags (ESTs) in the endometrium and identified galectin-15, a secretory product of the endometrium. Current evidence indicates that galectin-15 plays an important role in conceptus growth and implantation. Further, a custom ovine endometrial cDNA array was used to identify genes different in the endometrium of pregnant and UGKO ewes as well as genes regulated by progesterone and interferon tau. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14
normal pregnant ewes. Culture media was separated by two-dimensional gel electrophoresis, and proteins present in pregnant, but absent in cyclic and UGKO ewes were sequenced by mass spectrometry. This proteomic analysis resulted in the identification of two proteins, carbonic anhydrase and isocitrate dehydrogenase. In summary, many biomarkers of progesterone and interferon tau action in the endometrium of the ovine uterus were identified by these studies. These biomarkers will be useful to predict endometrial function and may be useful to understand high rates of early pregnancy loss observed in beef cattle and dairy cattle.
Impacts
Specific information on the secretions of the uterus that regulate embryo growth and survival will help define genetic markers of fertility in ruminants and factors that might be used to increase the success of in vitro embryo culture and diagnose infertility. The expectation is that this research will increase reproductive efficiency of ruminants, thereby increasing profitability to the producer.
Publications
- Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that an absence of endometrial gland secretions in uterine gland knockout (UGKO) ewes compromises conceptus survival and elongation. Reproduction 2002; 124:289-300.
- Gray CA, Bazer FW, Spencer TE. Uterine glands: developmental biology and function during pregnancy. ARBS Ann Rev Biomed Sci 2002; 3:85-126.
- Gray CA, Adelson DL, Bazer FW, Burghardt RC, Meeusen EN, Spencer TE. Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in trophectoderm. Proc Natl Acad Sci USA 2004; 101:7982-7987.
- Gray CA, Dunlap KA, Burghardt RC, Spencer TE. Galectin-15 in ovine uteroplacental tissues. Reproduction 2005; 130:231-240.
- Spencer TE, Bazer FW. Uterine and Placental Factors Regulating Conceptus Growth in Domestic Animals. J Anim Sci 2004; 82 (E. Suppl.):E4-E13(http://www.asas.org/symposia/04esupp/E4.pdf)
- Spencer TE, Johnson GA, Burghardt RC, Bazer FW. Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biol Reprod 2004; 71:2-10.
- Spencer TE, Johnson GA, Bazer FW, Burghardt RC. Implantation mechanisms: insights from the sheep. Reproduction 2004; 128:657-668.
- Padua MB, Tekin S, Spencer TE, Hansen PJ. Actions of progesterone on uterine immunosuppression and endometrial gland development in the uterine gland knockout (UGKO) ewe. Mol Reprod Dev 2005; 71:347-257.
- Spencer TE, Gray CA. Sheep Uterine Gland Knockout (UGKO) Model. In: Methods in Molecular Medicine, Volume 121: Placenta and Trophoblast: Methods and Protocols, Volume 1 (Soares MJ and Hunt JS, Eds.). Humana Press, Inc., Totowa, NJ; 2005:85-94.
- Gray CA, Abbey CA, Beremand PD, Choi Y, Farmer JL, Adelson DL, Thomas TL, Bazer FW, Spencer TE. Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus. Biol Reprod 2006; 74:383-394.
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Progress 01/01/05 to 12/31/05
Outputs
All mammalian uteri have endometrial glands that secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the current studies are to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective was to utilize human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective was
revised to include a large-scale expressed tag sequencing project to identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high throughput cDNA microarray systems. The selected cDNAs will then be confirmed by real-time PCR and sequenced. This approach is feasible and has been successful. A directionally cloned cDNA library was generated from total endometrial RNA pooled from five Day 14 pregnant ewes. Currently, 3,388 clones have been sequenced, consisting of 294 clusters and 2,256 singletons for a total of 2,550 non-redundant sequences (75%). All sequences were deposited in GenBank. A number of the novel transcripts were identified that appear to regulate conceptus growth and survival, including galectin-15. Further, a custom ovine endometrial cDNA array was used to identify genes different in the endometrium of pregnant and UGKO ewes as well as genes regulated by
progesterone and interferon tau. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14 normal pregnant ewes. Culture media was separated by two-dimensional gel electrophoresis, and proteins present in pregnant, but absent in cyclic and UGKO ewes were sequenced by mass spectrometry. This proteomic analysis resulted in the identification of two proteins, carbonic anhydrase and isocitrate dehydrogenase.
Impacts
Specific information on the secretions of the uterus that regulate embryo growth and survival will help define genetic markers of fertility in ruminants and factors that might be used to increase the success of in vitro embryo culture and diagnose infertility. The expectation is that this research will increase reproductive efficiency of ruminants.
Publications
- Gray CA, Dunlap KA, Burghardt RC, Spencer TE. Galectin-15 in ovine uteroplacental tissues. Reproduction 2005; 130:231-240.
- Padua MB, Tekin S, Spencer TE, Hansen PJ. Actions of progesterone on uterine immunosuppression and endometrial gland development in the uterine gland knockout (UGKO) ewe. Mol Reprod Dev 2005; 71:347-257.
- Spencer TE, Gray CA. Sheep Uterine Gland Knockout (UGKO) Model. In: Methods in Molecular Medicine, Volume 121: Placenta and Trophoblast: Methods and Protocols, Volume 1 (Soares MJ and Hunt JS, Eds.). Humana Press, Inc., Totowa, NJ; 2005:85-94.
- Gray CA, Abbey CA, Beremand PD, Choi Y, Farmer JL, Adelson DL, Thomas TL, Bazer FW, Spencer TE. Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus. Biol Reprod 2006; 74:383-394.
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Progress 01/01/04 to 12/31/04
Outputs
All mammalian uteri have endometrial glands that secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the current studies are to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective was to utilize human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective was
revised to include a large-scale expressed tag sequencing project to identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high throughput cDNA microarray systems. The selected cDNAs will then be confirmed by real-time PCR and sequenced. This approach is feasible and has been successful. A directionally cloned cDNA library was generated from total endometrial RNA pooled from five Day 14 pregnant ewes. The library contained >2 million clones, with an average insert size of 1.2 kb, and less than 5% of the clones from genomic, mitochondrial or ribosomal origin. The cDNAs from 23,000 clones were isolated for sequencing. Currently, 3,388 clones have been sequenced, consisting of 294 clusters and 2,256 singletons for a total of 2,550 non-redundant sequences (75%). A number of the novel transcripts were identified that appear to regulate conceptus growth and survival, including
galectin 15. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14 normal pregnant ewes. To identify proteins important for conceptus survival, UGKO ewes (n=5) were synchronized to estrus (D0) and bred to fertile rams; normal ewes were bred at estrus to either vasectomized (n=5) or fertile rams (n=5). On D14, uteri were flushed with saline to determine conceptus presence, ewes were hysterectomized, and endometrium was isolated for explant culture. Culture media was separated by two-dimensional gel electrophoresis, and proteins present in pregnant, but absent in cyclic and UGKO ewes were sequenced by mass spectrometry. This proteomic analysis resulted in the identification of two proteins, carbonic anhydrase and isocitrate dehydrogenase.
Impacts
Specific information on the secretions of the uterus that regulate embryo growth and survival will help define genetic markers of fertility in ruminants and factors that might be used to increase the success of in vitro embryo culture. The expectation is that this research will increase reproductive efficiency of ruminants.
Publications
- Gray CA, Adelson DL, Bazer FW, Burghardt RC, Meeusen EN, Spencer TE. Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in trophectoderm. Proc Natl Acad Sci USA 2004; 101:7982-7987.
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Progress 01/01/03 to 12/31/03
Outputs
All mammalian uteri have endometrial glands that secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the current studies are to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective was to utilize human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective was
revised to include a large-scale expressed tag sequencing project to identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high throughput cDNA microarray systems. The selected cDNAs will then be confirmed by real-time PCR and sequenced. This approach is feasible and has been successful. A directionally cloned cDNA library was generated from total endometrial RNA pooled from five Day 14 pregnant ewes. The library contained >2 million clones, with an average insert size of 1.2 kb, and less than 5% of the clones from genomic, mitochondrial or ribosomal origin. The cDNAs from 23,000 clones were isolated for sequencing. Currently, 3,388 clones have been sequenced, consisting of 294 clusters and 2,256 singletons for a total of 2,550 non-redundant sequences (75%). A number of the novel transcripts were identified that appear to regulate conceptus growth and survival, including
galectin 11 and heparin sulfate proteoglycan. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14 normal pregnant ewes. To identify proteins important for conceptus survival, UGKO ewes (n=5) were synchronized to estrus (D0) and bred to fertile rams; normal ewes were bred at estrus to either vasectomized (n=5) or fertile rams (n=5). On D14, uteri were flushed with saline to determine conceptus presence, ewes were hysterectomized, and endometrium was isolated for explant culture. Culture media was separated by two-dimensional gel electrophoresis, and proteins present in pregnant, but absent in cyclic and UGKO ewes were sequenced by mass spectrometry. This proteomic analysis resulted in the identification of two proteins, carbonic anhydrase and isocitrate dehydrogenase.
Impacts
Specific information on the secretions of the uterus that regulate embryo growth and survival will help define genetic markers of fertility in ruminants and factors that might be used to increase the success of in vitro embryo culture. The expectation is that this research will increase reproductive efficiency of ruminants.
Publications
- Spencer TE, Bazer FW. Uterine and Placental Factors Regulating Conceptus Growth in Domestic Animals. J Animal Sci 2003; (in press).
- Spencer TE, Johnson GA, Burghardt RC, Bazer FW. Progesterone and placental hormone actions on the uterus: insights from domestic animals. Biol Reprod 2003; (in press).
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Progress 01/01/02 to 12/31/02
Outputs
All mammalian uteri have endometrial glands that secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the current studies are to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective was to utilize human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective has been
revised to include a large-scale expressed tag sequencing project that will identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high throughput cDNA microarray systems. The selected cDNAs will then be confirmed by real-time PCR and sequenced. This approach is feasible and should ensure success. A directionally cloned cDNA library was generated from total endometrial RNA pooled from five Day 14 pregnant ewes. The library contained >2 million clones, with an average insert size of 1.2 kb, and less than 5% of the clones from genomic, mitochondrial or ribosomal origin. The cDNAs from 23,000 clones were isolated for sequencing. Currently, 3,388 clones have been sequenced, consisting of 294 clusters and 2,256 singletons for a total of 2,550 non-redundant sequences (75%). A number of the transcripts have been previously identified in the early Px ovine endometrium (i.e.,
interferon regulatory factor two, beta-2-microglobulin, and isocitrate dehydrogenase). These cDNAs will be arrayed for transciptional profiling of endometrium from cyclic, pregnant and UGKO ewes, as well as isolated glandular epithelium. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14 normal pregnant ewes. To identify proteins important for conceptus survival, UGKO ewes (n=5) were synchronized to estrus (D0) and bred to fertile rams; normal ewes were bred at estrus to either vasectomized (n=5) or fertile rams (n=5). On D14, uteri were flushed with saline to determine conceptus presence, ewes were hysterectomized, and endometrium was isolated for explant culture. Culture media was separated by two-dimensional gel electrophoresis, and proteins present in pregnant, but absent in cyclic and UGKO ewes were sequenced by mass spectrometry. This proteomic analysis resulted in
the identification of two proteins, carbonic anhydrase and isocitrate dehydrogenase.
Impacts
The research generated by this project should identify genes and proteins that are important for the ability of the ruminant uterus to support early embryo survival and development before implantation. The genes that are identified will serve as critical markers of endometrial function and uterine capacity. In addition, it is expected that the identified genes can be used to select animals for superior uterine capacity, thereby enhancing the efficiency of reproduction and animal production agriculture.
Publications
- Gray CA, Burghardt RC, Johnson GA, Bazer FW, Spencer TE. Evidence that an absence of endometrial gland secretions in uterine gland knockout (UGKO) ewes compromises conceptus survival and elongation. Reproduction 2002; 124:289-300.
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Progress 01/01/01 to 12/31/01
Outputs
All mammalian uteri have endometrial glands which secrete a variety of substances hypothesized to support development of the conceptus (embryo and associated placental membranes) during pregnancy. This research program has utilized sheep that lack glands in the uterus or the uterine gland knockout (UGKO) model. UGKO ewes are unable to maintain peri-implantation conceptus survival and development, indicating that genes expressed by endometrial glands are required for successful maternal support of conceptus growth and development. Objectives of the current studies are to discover genes important for conceptus survival and development using UGKO ewes and modern genomics and proteomic techniques. The first objective was to utilize human cDNA microarrays to determine which genes are absent in the endometrium of UGKO ewes and present in that of normal ewes. A series of experiments determined that this approach did not yield good results. Therefore, this objective has been
revised to include a large-scale expressed tag sequencing project that will identify candidate genes by screening a Day 14 pregnant ovine endometrial cDNA library with probes from Day 14 normal pregnant, normal cyclic and bred UGKO ewes using high througput cDNA microarray systems. The selected cDNAs will then be confirmed by real-time PCR and sequenced. This approach is feasible and should ensure success. The second objective was to use a proteomics approach to identify proteins absent in the uterine flush of UGKO ewes that are present in the uterine flush of Day 14 normal pregnant ewes. The animal portion of this project has been completed. Preliminary proteomic analyses have identified several candidate proteins that are being submitted for mass spectrometry identification and sequencing.
Impacts
The research generated by this project should identify genes and proteins that are important for the ability of the ruminant uterus to support early embryo survival and development before implantation. The genes that are identified will serve as critical markers of endometrial function and uterine capacity. In addition, it is expected that the identified genes can be used to select animals for superior uterine capacity, thereby enhancing the efficiency of reproduction and animal production agriculture.
Publications
- No publications reported this period
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