Progress 09/01/02 to 08/31/06
Outputs
The overall goal of the present research project is to improve embryonic survival in the pig during early pregnancy. Majority of loss for a potential litter occurs during the peri-implantation period when pig conceptuses undergo rapid differentiation and expansion of their trophoblastic membrane between Days 11 to 12 of gestation, which is also the period when conceptuses secrete estrogen to establish pregnancy. Release of estrogen and cytokines by developing conceptus during the peri-implantation period of embryonic development serve critical roles in cellular growth, differentiation and remodeling of the embryo and its extraembryonic membrane. Previous research in our laboratory established that treatment of pregnant gilts with estrogen on Days 9 and 10 of pregnancy causes total embryonic loss in the pig. Specifically this project determined: 1) Alteration of conceptus gene expression during the period of period of rapid trophoblast elongation and 2) Differential
expression of endometrial genes following endocrine disruption of pregnancy with estrogen treatment on Days 9 and 10 of pregnancy. Through the utilization of suppressive subtraction hybridization we made a novel discovery that conceptus production of interleukin 1beta may induce trophoblast elongation in pig conceptus and stimulate proinflammatory response within the uterus to establish pregnancy in the pig. Conceptus secretion of interleukin 1beta could serve an essential function in initiation of nuclear factor kappa B activation to induce secretion of cytokines and adhesion factors for establishment of pregnancy. A number of genes are up-regulated during the period of trophoblast elongation and attachment to the uterine surface providing information for future studies concerning pathways for normal conceptus development in the pig. Uterine expression of inter-alpha-trypsin inhibitor heavy chains and bikunin play an important function in the formation of the uterine surface
glycocalyx for placental attachment and inhibition of invasive implantation. We hypothesized that early exposure of pregnant gilts to estrogen disrupted expression of inter-alpha inhibitor heavy chains and bikunin but our studies failed to demonstrate any alteration in gene expression. However, our studies did indicate that early estrogen exposure alters the insulin-like growth factor system within the uterine lumen. Early exposure of pregnant gilts to exogenous estrogen stimulates premature cleavage of the insulin-like growth factor binding proteins within the uterine lumen resulting in an earlier disappearance of insulin-like growth factor 1. Removal of the binding proteins causes a mistiming for the presence of a high concentration of insulin-like growth factor 1 in the uterine lumen during the critical period of conceptus elongation and development. Early exposure of pregnant gilts to estrogen advances expression of a number of endometrial genes suggesting that the endocrine
disruptive effect of estrogen during pregnancy in the pig occurs through alteration of normal synchrony between maternal uterine environment and conceptus development.
Impacts
Research from the present grant has helped unravel the mystery of how the length of estrous cycle is controlled through down-regulation of uterine progesterone receptor in the pig. Future emphasis on research determining the uterine factors involved with triggering nuclear kappa B activation during the estrous cycle will provide possible methods to synchronize gilts for breeding, greatly improving reproductive management for swine producers. The discovery that conceptus secretion of interleukin 1beta is involved with establishment of pregnancy through activation of uterine nuclear factor kappa B gives new direction for research to improve reproductive efficiency in swine. Understanding the basic pathway for regulating the uterine inflammatory response during early pregnancy in the pig has provided valuable information on the factors regulating embryo survival and growth during gestation. Through determination of the uterine factors involved with embryo survival
and growth, we will be able establish treatments to influence growth and survival piglets in utero to increase litter size and improve profitability of the swine producer in the future.
Publications
- Fernando, S. C., Najar, F. Z., Guo, X., Zhou, L., Fu, Y., Geisert, R. D., Roe, B. A., and DeSilva, U*. 2006 Isolation and Characterization of the Porcine Kallikrein Gene Family: Genomic Structure, Radiation-Hybrid Mapping and Differential Expression Analysis. Genomics (in press)
- Ashworth, M. D., White, F. J., Ross, J. W., Hu, J., DeSilva, U., Johnson, G. A., and Geisert, R. D., 2006 Characterization of porcine endometrial cyclooxygenase expression during estrous cycle, early pregnancy, and following endocrine disruption of pregnancy. Biology of Reproduction 74, 1007-1015
- Fernando, S. C., Ashworth, M.S., Ross, J. W., Geisert, R. D., and DeSilva, U*. 2006 Porcine endometrial and conceptus tissue kallikrein 1,4,11 and 14 gene expression. Reproduction 132,1-10
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Progress 10/01/04 to 09/30/05
Outputs
The long-term goal of our research is to identify and understand the factors involved with uterine regulation of early porcine conceptus development and survival to develop effective methods for improving litter size in the pig. Our laboratory has identified and characterized 13 tissue kallikrein (KLK) genes present in the porcine genome. The porcine kallikrein gene family is clustered on chromosome 6 and lacks orthologues to human KLK2 and KLK3 as occurs in rodents. Tissue kallikreins are known to regulate the activation of many growth factors and extracellular proteases in a variety of tissues. The pig endometrium expresses tissue KLK1, KLK4, KLK6, KLK9, KLK10, KLK11, KLK14 and KLK15 during the estrous cycle and early pregnancy. Pattern of endometrial gene expressed is different for each of the tissue kallikreins. Endometrial KLK14 expression appears to be regulated by the presence of progesterone receptor in the uterine epithelium as its expression is not
detectable until progesterone receptor is down-regulated in the uterine epithelium after Day 10 of the estrous cycle or pregnancy. A pregnancy specific induction of endometrial KLK9 expression occurs between Days 12 to 17 of gestation when placental attachment to the uterine surface occurs in the pig. Tissue KLK9, also known as epidermal growth factor binding protein, functions in the activation of epidermal growth factor which is a major growth factor in embryonic development. Endometrial expression of KLK9 may serve an important role in placental attachment and embryo survival in the pig. Exposure of pregnant gilts to estrogen prior to Day 10 of gestation results in total embryonic loss in the pig. Changes in endometrial gene expression following estrogen treatment were investigated utilizing a porcine specific microarray. Results indicated that expression for a number of endometrial genes is altered by early estrogen exposure. The vast majority of genes are altered on Day 10 to 13
of gestation. Estrogen treatment advances or alters expression of several endometrial genes involved with immunoregulation and cell adhesion. Pregnant gilts treated with estrogen have a premature loss of insulin-like binding proteins which results in a 24 to 48 hour earlier decline of insulin-like growth factor-1 within the uterine lumen. Results of our studies indicate that early embryonic mortality following estrogen treatment may result from asynchrony of endometrial secretion with conceptus development.
Impacts
There is a great deal of excitement concerning the utilization of the kallikrein family as markers of cancer in various tissues in human medicine. Isolation of the porcine tissue kallikreins allows the pig to be used as a model for expression of kallikreins in a disease state. However, the specific biological function of the various kallikreins is largely unknown. We have established the expression of multiple tissue kallikreins in the porcine endometrium and conceptus during the oestrous cycle and early pregnancy. Each of the tissue kallikreins investigated displayed a different expression pattern suggesting that the various kallikreins initiate many biological processes during early conceptus attachment and survival. We have also determined the role of estrogen as an endocrine disruptor in the establishment of pregnancy. Alteration in endometrial secretion of insulin-like growth factor-1 as well as other growth factors, immunoregulatory agents and cell adhesion
factors by environmental estrogens, indicates that later embryonic loss can be programmed by exposure to estrogen very early in gestation.
Publications
- Geisert R.D., Ross, J.W., Ashworth, M.D., White, F.J., Johnson, G.A., Hu, J. and DeSilva, U. 2005. Maternal recognition of pregnancy signal or endocrine disruptor: The two faces of oestrogen during establishment of pregnancy in the pig. In: Control of Pig Reproduction VII. R. Kraeling and C. Ashworth, Eds, Nottingham University Press, pp 131-145.
- Ashworth, M.D., White, F.J., Ross, J.W., Hu, J., DeSilva, U., Johnson, G.A. and Geisert, R.D. 2005. Characterization of Porcine Endometrial Cyclooxygenase Expression During the Estrous cycle, Early Pregnancy and Following Endocrine Disruption of Pregnancy. Biol. Reprod. (In revision for acceptance).
- White F.J., Ross, J.W., Joyce, M.M., Geisert, R.D., Burghardt, R.C. and Johnson, G.A. 2005. Steroid regulation of cell specific osteopontin expression in the pregnant porcine uterus. Biol. Reprod. (In Press) Ashworth, M.D., Ross, J.W., Allen, D.T., Stein, D.R., Spicer, L.J., DeSilva, U. and Geisert, R.D. 2005. Endocrine disruption of uterine insulin-like growth factor (IGF) expression in the pregnant gilt. Reprod. 130:545-551.
- Geisert R.D., Ross, J.W., Ashworth, M.D. and Malayer, J.R. 2004. Estrogen regulator and/or endocrine disruptor in establishment of pregnancy. Current Topics in Steroid Research 4:69-84.
- Vonnahme K.A., Ferando, S.C., Ross, J.W., Ashworth, M.D., DeSilva, U., Malayer, J.R. and Geisert, R.D. 2004. Porcine endometrial expression of kininogen, factor XII and plasma kallikrein in cyclic and pregnant gilts. Biol. Reprod. 70:132-138.
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Progress 10/01/03 to 09/30/04
Outputs
The noninvasive form of implantation in swine involves many uterine and conceptus factors. Early pregnancy in the pig is sensitive to environmental conditions and endocrine disruptors which can cause embryonic death if the pig is inappropriately exposed to estrogenic compounds prior to placental attachment to the uterine surface. The long-term goal of our research is to identify and understand factors involved with uterine regulation of early porcine conceptus development to establish effective methods for improving reproductive efficiency in swine. Our research has established that conceptus secretion of interleukin 1 beta activates endometrial acute phase proteins and kallikrein-kininogen-kinin system prior to placental attachment. Tissue kallikreins belong to a gene family of 10 to 15 serine proteases in the human and mouse which have different substrate specificities. Tissue kallikrein 1 (KLK1) releases bradykinin from low molecular weight kininogen to stimulate
uterine vascular hyperemia. Gene sequences for the porcine tissue kallikrein family are not available. Therefore, objective of our research was to isolate and sequence the porcine kallikrein genes for future evaluation of uterine and conceptus expression. Our laboratory isolated and sequenced 13 porcine kallikrein genes (KLK1, KLK4-KLK15) which have significant homology to the human and mouse tissue kallikreins. The porcine kallikrein genes are clustered within pig chromosome 6 (6q12-q21). Evaluation of endometrial and conceptus KLK1, KLK4 and KLK14 gene expression indicate that day (endometrium) and development (conceptus) changes occur during the estrous cycle and early pregnancy. Endometrial KLK14 expression was up-regulated on Day 0 and 12-18 of the estrous cycle and early pregnancy but was not detectable on Days 5 and 10. KLK14 is reported to be expressed in prostate secretory epithelial cells and is regulated by androgens and estrogens. Our results suggest that expression of
endometrial KLK14 is inhibited through progesterone binding of progesterone receptor in the uterine surface and glandular epithelium. Expression of KLK14 is temporally associated with loss of progesterone receptor from the uterine surface and glandular epithelium which occurs after Day 10 of the estrous cycle and pregnancy. Endometrial expression of KLK14 may play an important role in porcine conceptus development and implantation. Our research has also indicated that early exposure of pregnant gilts to estrogen alters the normal pattern of endometrial insulin-like growth factor 1 (IGF-1) secretion. There is a 3 to 4 fold decrease in uterine lumenal content of IGF-1 on Day 12 and 13 of gestation in estrogen treated gilts. The decreased availability of IGF-1 during a critical period in conceptus development could cause loss of embryos after Day 14 of gestation. Identification of the porcine tissue kallikreins will provide the necessary information to investigate the role of the various
tissue kallikreins in establishment and maintenance of pregnancy in the pig.
Impacts
Investigation has demonstrated the presence of the kallikrein-kininogen-kinin system during establishment of early pregnancy in the pig. Therapies to enhance bradykinin production during pregnancy may improve litter size in the pig. Exposure of the early pregnancy to estrogen causes breakdown of the uterine surface glycocalyx. Understanding regulation of components involved with extracellular matrix formation will help us determine how environmental estrogens can negatively effect embryonic survival in a number of species.
Publications
- Geisert R. D., J.W. Ross, M.D. Ashworth and J.R. Malayer. 2005. Estrogen regulator and/or endocrine disruptor in establishment of pregnancy. Current Topics in Steroid Research (In Press)
- Vonnahme K.A., S.C. Ferando, J.W. Ross, M.D. Ashworth, U. DeSilva, J.R. Malayer and R.D. Geisert. 2004. Porcine endometrial expression of kininogen, factor XII and plasma kallikrein in cyclic and pregnant gilts. Biol. Reprod. 70:132-138.
- Fernado, SC. 2004. Isolation, characterization, mapping and expression analysis of porcine tissue kallikreins. M.S. Thesis
- Ashworth M.D., J.W. Ross, J.L. Roberts, J.R. Malayer, A.J. Day and R.D. Geisert. 2004. Porcine endometrial expression of extracellular matrix components following estrogen disruption of pregnancy. Biol. Reprod. Special Issue p159.
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Progress 10/01/02 to 09/30/03
Outputs
Swine can experience a high rate of embryonic mortality during the second to third week of gestation. Long-term goal of our research is to identify and understand the factors involved with uterine regulation of early porcine conceptus development to establish effective methods for improving litter size. Implantation in the pig elicits a generalized acute inflammatory response in the uterus. We have established that the elongating pig conceptuses release the pro-inflammatory cytokine, interleukin 1beta during the period of placental attachment to the uterine surface. IL-1beta would stimulate the release of acute phase proteins such as the inter-alpha-trypsin inhibitor family and activate kallikrein-kininogen-kinin system present in the uterus of the pig. The inter-alpha-trypsin inhibitor family is involved with stabilization of the uterine surface glycocalyx, which can be disrupted by estrogen if administered prior to the period of conceptus trophoblastic elongation.
Release of bradykinin from kininogen by tissue kallikrein stimulates a uterine vascular response. Objective of our research is to determine the role of estrogen in the regulation of uterine gene expression for the inter-alpha-trypsin inhibitor family, plasma and tissue kallikrein, Factor XII, HMW- and LMW-kininogen and the release of kallikrein, bradykinin and insulin-like growth factors into the uterine lumen. Gene expression for plasma kallikrein, Factor XII and HMW-kininogen was detected in endometrium but not early conceptuses. Although endometrial plasma kallikrein and Factor XII gene expression is unique as the liver is the tissue of normal expression for these particular genes, endometrial expression is low. Tissue kallikrein and LMW-kininogen were detected in early conceptuses and endometrium. Combined with our previous studies that have demonstrated the porcine uterus secretes tissue kallikrein and bradykinin increases during pregnancy indicates the kallikrein-kininogen
system is activatived during pregnancy. Tissue kallikrein family is composed of 10 to 15 homologous genes in the rat and human. Currently, we have isolated several BAC clones from the region of the porcine genome that contains the kallikrein genes and are in the process of sequencing a BAC clone. Isolation of all members of the tissue kallikrein gene family will allow us to investigate the variants expressed in conceptuses and endometrium during early development. Our laboratory has established that attachment of the placenta to the uterine surface involves inter-alpha-trypsin inhibitor family which forms the apical extracellular matrix on the uterine surface. We have evaluated gene expression for the inter-alpha-trypsin inhibitor family during conceptus degeneration following treatment of pregnant gilts with estrogen. Treatment of pregnant gilts with estrogen on Days 9 and 10 of pregnancy caused conceptus degeneration on Day 17 of gestation. Endometrial gene expression for the
inter-alpha-trypsin inhibitor heavy chains or bikunin was not altered by early estrogen exposure. Research will continue to investigate the effects estrogen on components of the apical glycocalyx on the uterine luminal epithelium.
Impacts
Investigation has demonstrated the presence of the kallikrein-kininogen-kinin system during establishment of early pregnancy in the pig. Therapies to enhance bradykinin production during pregnancy may improve litter size in the pig. Exposure of the early pregnancy to estrogen causes breakdown of the uterine surface glycocalyx. Understanding regulation of components involved with extracellular matrix formation will help us determine how environmental estrogens can negatively effect embryonic survival in a number of species.
Publications
- Vonnahme, K.A., Ferando, S.C., Ross, J.W., Ashworth, M.D., DeSilva, U., Malayer, J.R. and Geisert, R.D. 2003. Porcine endometrial expression of kininogen, factor XII and plasma kallikrein in cyclic and pregnant gilts. Biol. Reprod. (In Press).
- Geisert, R.D., Ashworth, M.D. and Malayer, J.R. 2003. Endometrial expression of inter-alpha-trypsin inhibitor heavy chains in cyclic and pregnant gilts. Reproduction 126:621-626.
- Ross J.W., Malayer, J.R., Ritchey, J.W. and Geisert, R.D. 2003. Involvement of the interleukin-1beta system in porcine trophoblastic elongation and at the fetal-maternal interface during peri-implantation development. Biol. Reprod. 69:1251-1259.
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Progress 10/01/01 to 09/30/02
Outputs
During early conceptus development of pig, there is a high loss of embryos during the second to third week of pregnancy. For the conceptuses to survive, the placenta must form a loose attachment to the uterine surface. To fully understand how embryos are selected to survive in the uterus, it is necessary to identify and understand the factors involved with uterine regulation of early porcine conceptus and placental development. We have previously established that components of the inter-alpha-trypsin inhibitor family and kallikrein-kininogen-kinin system are present in the uterus of the pig. The inter-alpha-trypsin inhibitor family is involved with stabilization of the uterine surface glycocalyx, which can be disrupted by estrogen if administered prior to the period of conceptus trophoblastic elongation. Objective of our research is to determine the role of estrogen in the regulation of uterine gene expression for the inter-alpha-trypsin inhibitor family, plasma and
tissue kallikrein, Factor XII, HMW- and LMW-kininogen and the release of kallikrein, bradykinin and insulin-like growth factors into the uterine lumen. We have demonstrated gene expression for plasma and tissue kallikrein, Factor XII and HMW- and LMW-kininogen by porcine conceptuses and endometrium. Alteration in endometrial gene expression for these specific genes during the estrous cycle and early pregnancy are currently being evaluated. Gene expression of endometrial plasma kallikrein and HMW-kininogen is unique as the liver is the tissue of normal expression for these particular genes. We have evaluated gene expression for the inter-alpha-trypsin inhibitor family during conceptus degeneration following treatment of pregnant gilts with estrogen. Treatment of pregnant gilts with estrogen on Days 9 and 10 of pregnancy caused conceptus degeneration on Day 17 of gestation. Total RNA was extracted from the endometrial tissue and Realtime PCR was utilized to determine gene expression for
the four inter-alpha-trypsin inhibitor heavy chains and bikunin. No change in endometrial gene expression for the inter-alpha-trypsin inhibitor heavy chains or bikunin was detected. Endometrial tissue from the study will be utilized to perform suppressive subtraction hybridization to detect possible alterations in gene expression following estrogen treatment. Studies to determine the uterine change in tumor necrosis factor and interleukin-1beta gene and protein production following estrogen treatment are also currently underway.
Impacts
Discovery of plasma kallikrein and HMW-Kininogen gene expression by the endometrium, indicates that kinins can play a major role in the development and survival of porcine embryos. Development of possible methods to enhance kinin production during pregnancy may improve litter size in the pig. Understanding the role of estrogen in pregnancy loss in the pig will help establish therapies to inhibit the effects of environmental estrogens on embryonic mortality in the pig.
Publications
- Ashworth, M.D., Ross J.W., Malayer J.R. and Geisert R.D. 2002. Effect of estrogen administration on endometrial acute phase protein gene expression in pregnant gilts. Biol. Reprod. 66, Suppl. 1, 315
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