Source: UNIVERSITY OF MISSOURI submitted to NRP
INTERLEUKIN-1B REGULATION OF CONCEPTUS DEVELOPMENT AND SURVIVAL IN THE PIG
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1002420
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 17, 2014
Project End Date
Jan 1, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Animal Sciences
Non Technical Summary
The increasing cost of production over the past several years dictates that the swine producer must maximize efficiency to maintain a profitable operation. Economic importance of increasing number of offspring born has been demonstrated in the production systems of numerous domestic farm animals [Bradford, 1985; Tess et al. 1983; Gregory et al. 1990]. Improving litter size would have a substantial impact on the efficiency of swine production. Tomes and Nielson [1982] demonstrated that overall profitability increased as the number of pigs/sow/year increased. Sows which produce small litters cause reduced financial gains, due to increased cost of feed, labor and under-utilized facilities per pound of pork produced. Although increasing litter size would increase the efficiency of pork production, large improvements have not been achieved. There is potential for average litter sizes of 14 or more pigs if even 75% all ovulated ova developed into live pigs at term. However, approximately 40-50% of the fertilized ova released from the ovary do not survive through gestation [Pope, 1994]. Fertilization of ova after ovulation in swine is generally greater than 95%, therefore the loss of potential piglets is the result of early embryonic and late fetal deaths. Although ovulation rate establishes the potential litter size, it by itself will not result in a marked improvement in litter size [see Pope, 1994]. Improvements in litter size will therefore come from understanding factors associated with conceptus development and survival. Establishment and maintenance of pregnancy involves closely orchestrated events in ovarian, uterine and conceptus development in all mammalian species [Bazer et al., 1982]. Alterations in the synchronous development of the uterine environment with the growing and differentiating conceptus during early pregnancy can result in a failure to establish pregnancy which results in a return to estrus during the 21 day estrous cycle [Polge, 1982]. The incidence of prenatal mortality in swine is quite high, ranging from 20 to 46 percent before term [Pope, 1994]. The majority of embryonic mortality usually occurs prior to Day 20 of gestation. Data for gilts indicate that, excluding females in which total fertilization failure occurs, little embryonic loss takes place before 7 days after the onset of estrus [Polge, 1982]. Estimates suggesting that the majority of embryonic mortality occurs between Days 10 and 20 of gestation is not surprising given the critical events which occur during this period. Deriving an in vivo approach to establish which uterine and conceptus factors may be involved with conceptus growth and survival would have definite advantages in enhancing our understanding of the interactions between the conceptus and its uterine environment which would lead to improving reproductive efficiency in swine. The objective of the present project is to to elucidate the function of conceptus synthesis of interleukin-1b (IL-1b) during conceptus elongation and uterine activation of nuclear factor-kB (NF-kB) with respect to early embryonic development and survival in the pig. Studies will utilize advanced molecular techniques and reproductive technologies to develop a specific conceptus IL-1b knockdown in the pig and evaluate the effects of a recombinant conceptus IL-1b protein on uterine function. Our central hypothesis is that conceptus IL-1b synthesis is involved with the rapid trophoblast elongation of the pig and uterine NF-kB activation which permits conceptus development and implantation.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013510105050%
3013510102050%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology; 1050 - Developmental biology;
Goals / Objectives
Understand early embryo development and establishment of pregnancy in the pig which may be utilized to improve overall embryo survival and reproductive efficiency. OBJECTIVES: (1) Determine the role of conceptus interleukin-1beta (IL-1b) synthesis in rapid trophoblastic elongation and establishment of pregnancy in the pig. (2) Determine the interaction between conceptus interleukin-1b and nuclear factor kappa B (NF-kB) activation within the uterine epithelium to initiate conceptus implantation.
Project Methods
Interleukin-1b knockouts will be created for both the conceptus and systemic form of IL-1b. We will select two specific loci on the conceptus IL1b gene and the same two loci on systemic IL1b gene for zinc finger nuclease (ZFNs) targeting. The two loci exist in the promoter region and in exon 1. These two regions differ between the two gene forms, while exons 2 to exon 7 are very similar, indicating that the two target sites are responsible for the functional differences between conceptus and systemic form of IL1b. The genetically modified pig fetal fibroblast cells containing the IL-1b knockout will be utilized to generate IL-1b-/- embryos. Two groups of swine nuclear transfer donor cells will be generated with ZFN-mediated inactivation of the conceptus IL1b and IL1 genes, respectively, by using established standard protocols in our laboratory. Cloned embryos (conceptus IL-1b-/-, IL-1b-/-, and controls) will be transferred to recipient females on day 4 of the recipients estrous cycle. Embryos will be flushed from the uterus on either Day 8, 12, or 18 of gestation when embryos will be evaluated for morphological stage of development. Embryos and corresponding endometrial tissue will be subjected to mRNA, protein, DNA analyses. Transcriptional profiling (RNA-seq) on conceptus and endometrial tissue will be conducted and a bioinformatic analysis of the data will determine any alteration in biological pathways specific to pregnancy recognition, elongation, and implantation. To test the hypotheses that conceptus IL-1b activation of NF-kB in endometrial LE and GE is required for establishment and maintenance of pregnancy. The approach is to inhibit NF-kB activation with the NF-kB inhibitor, sodium salicylate during the period of conceptus IL-1b expression between Days 11 and 13 of pregnancy in the pig. Bred gilts will be assigned randomly to receive either 1) no treatment (control) or 2) sodium salicylate on Days 9-13 of pregnancy. Gilts will be hysterectomized-ovariectomized on either Day 13 or 18 of gestation. Uterine flushing's and endometrial tissue will be collected processed immediately following removal of the uterus. Endometrial tissue will be evaluated for NF-kB activation and several genes (SAL1, PTGS2, and STC1) which are stimulated by IL-1b during the establishment of pregnancy in the pig. Uterine epithelial expression of progesterone receptor will be evaluated by immunohistochemistry. Inhibition of NF-kB activity will determine if 1) endometrial epithelium up regulates with loss of NF-kB inhibition and 2) the conceptus trophoblast can elongate and continue to establish pregnancy. Results from the research will establish the role of conceptus IL-1b in rapid of elongation of pig conceptuses during the establishment of pregnancy. The factor and pathway to initiate trophoblast remodeling in the pig is unknown and the establishment of IL-1b-/- embryos will provide a model to determine if IL-1b expression is involved with this amazing morphological change which essential to the establishment and maintenance of pregnancy. The study will also determine the regulation of endometrial NF-kB for establishment and maintenance of pregnancy in the pig. Determination of roles for conceptus IL-1b and endometrial NF-kB activation during pregnancy will help lead to possible therapies to improve reproduction in the pig. The increasing cost of animal production over the past several years dictates that the swine producer must maximize efficiency to maintain a profitable operation. Economic importance of increasing number of offspring born has been demonstrated in the production systems of numerous domestic farm animals. Improving litter size would have a substantial impact on the efficiency of swine production. Although increasing litter size would increase the efficiency of pork production, large improvements have not been achieved. There is potential for average litter sizes of 14 or more pigs if 75% all eggs ovulated developed into live pigs at term. However, approximately 40-50% of the fertilized eggs released from the ovary do not survive through gestation. The incidence of prenatal mortality in swine is quite high, ranging from 20 to 46 percent before term for which the majority of mortality occurs prior to Day 20 of gestation. Improvements in litter size will therefore come from understanding factors associated with conceptus development and survival. Conceptuses (embryo + membrane) undergo rapid differentiation and expansion of their trophoblast (elongation) between Days 11 to 12 of gestation as they develop from a 1-2 mm sphere to a 9-10 mm ovoid shape between Days 10 to 12 and then rapidly transition to tubular and filamentous forms by elongating at 30-40 mm/h to > 100 mm in length (elongation occurs in less than 2 hours). Conceptus elongation is associated with the highest rate of embryo loss in the pig and is followed by attachment to the uterus. Conceptuses produce high amounts of the cytokine interleukin 1b (IL-1b) during the period of rapid elongation. We hypothesize that cellular IL-1b signaling is critical to trophoblastic elongation and to initiation of events essential to implantation and placentation for establishment and maintenance of pregnancy. The study will generate embryos with a knockout of the conceptus form of the IL-1b gene. The factor and pathway to initiate trophoblast remodeling in the pig is unknown and the establishment of IL-1b knockout embryos will provide a model to determine if IL-1b expression is involved with this amazing morphological change which is essential to the establishment and maintenance of pregnancy in the pig. The study will also determine the IL-1b regulation of uterus through NF-kB activation for establishment and maintenance of pregnancy in the pig. NF-kB activates many genes involved with a proinflammatory tissue response. Determination of roles for conceptus IL-1b and endometrial gene activation during pregnancy will help lead to possible therapies to improve reproduction in the pig. There are natural plant dietary supplements that have inhibitory activity to NF-kB which can help reduce the inflammatory response during the establishment of pregnancy. These dietary supplements if effective might lead to improved reproductive function as well as increase animal health.

Progress 01/17/14 to 01/01/19

Outputs
Target Audience:Presentations have been made to scientific researchers at Regional and National Scientific Meetings in addition to publications available in research journals and books. Information from research is used for teaching, extension and increasing knowledge of reproductive biology of the pig. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The current project has trained two MS and one PhD graduate student with another graduate student currently completing a MS in 2020. How have the results been disseminated to communities of interest?Presentations at regional and national scientific meetings. Publication of manuscript research articles, reviews, book chapters. What do you plan to do during the next reporting period to accomplish the goals?The renewal of this Hatch project will investigate the role of pig conceptus interferons in early embryo development, and the establishment and maintenance of pregnancy. CRISPR/Cas9 will be utilized to edit conceptus interferons to establish a model for their role in pregnancy. We also develop of a double gene edit for aromatase (CYP19A1-/-) and prostaglandin-endoperoxide synthase 2 (PTGS2-/-) to directly address if both conceptus estrogen and PGE work together as maternal recognition of pregnancy signals in the pig.

Impacts
What was accomplished under these goals? We discovered that the gene encoding interleukin 1beta (IL1B) has duplicated in the pig, resulting in a novel gene (IL1B2) which is only expressed by pig conceptuses during the peri-implantation period. The unique conceptus expression of IL1B2 only occurs during the period of rapid conceptus elongation on Day 12 of pregnancy which activates endometrial nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts within the endometrium. We concluded that the peri-implantation pig conceptuses express a novel IL1B2 that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig. We established that both IL1B2 and aromatase (CYP19A1) are expressed at the same time by the pig conceptuses during the peri-implantation period. The unique conceptus expression and secretion of IL1B2 and estrogen (increase in aromatase enzyme expression) occurs during the period of rapid conceptus elongation on Day 12 of pregnancy. To understand the roles of IL1B2 and aromatase gene expression in during conceptus development and the establishment of pregnancy in the pig, we utilized the CRISPR/Cas9 genomic engineering to knockout IL1B2 and CYP19A1 (aromatase) gene expression. The rapid morphological transition of IL1B2-/- conceptuses was inhibited compared to normal elongation of IL1B2+/+ conceptuses. Thus, we have established that conceptus IL1B2 expression is essential to rapid elongation of the trophoblast for placental attachment to the uterine surface. Our current research has demonstrated that early pig embryo production of estrogen and prostaglandins (PG) are not essential for blastocyst or early conceptus development. CRISPR/Cas9 knockout of conceptus aromatase gene expression resulted in loss of conceptus estrogen production. However, loss of conceptus estrogen synthesis did not affect early placental development and establishment of pregnancy to day 25 of gestation. Conceptus estrogen production has been considered the maternal recognition of pregnancy signal in the pig for the last 40 years. Our results alter the accepted theory and suggest that other conceptus factors can maintain functional corpora lutea (CL) in the absence of estrogen. We suggest that conceptus production of prostaglandin E (PGE) prevents CL regression in the absence of estrogen. However, conceptus production of estrogen is essential for successful maintenance of pregnancy beyond day 30 as both conceptus and endometrial PGs are not successful in compensating for lost estrogen production at this time. Our results also establish that porcine conceptus prostaglandin-endoperoxide synthase 2 (PTGS2) derived prostaglandin production is not essential for early embryonic development, conceptus elongation, maternal recognition of pregnancy, attachment to the maternal epithelium, placentation, and maintenance of pregnancy to day 35. These results indicate that endometrial prostaglandin production induced by conceptus estrogen secretion can support pregnancy in the absence of conceptus PTGS2 expression. The interaction between PGE and estrogen in both the aromatase and PTGS2 knockout conceptus models cannot be ignored, as both estrogen and PGE appear to be able to compensate for one another during these essential events in early pregnancy in the pig. Development of a double gene edit (aromatase and PTGS2) in the future would be needed to directly address if both conceptus estrogen and PGs work together as maternal recognition of pregnancy signals in the pig. Pig conceptus express both Type I and II interferons. Interferon gamma (Type 1) is expressed from day 12 to 20 of gestation. There are 11 interferon delta's (IFND) expressed by pigs. However, conceptuses only express IFND 1 and 2 from day 14 to 35 of gestation while IFND 3,4 and 10 are expressed only on day 35 of gestation. The role of the conceptus IFN's will be evaluated through developing CRISPR/Cas 9 knockout of these specific conceptus genes. Utilizing CRISPR/Cas9 technology for selective gene editing of early embryos will continue to provide the model to gain fundamental knowledge of essential factors involved with development and survival of embryos which can lead to strategies for improving fertility and fecundity.

Publications


    Progress 10/01/18 to 01/01/19

    Outputs
    Target Audience:Presentations have been made to scientific researchers at Regional and National Scientific Meetings in addition to publications available in research journals and books. Information from research is used for teaching, extension and increasing knowledge of reproductive biology of the pig. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The current project has trained two MS graduate students with another graduate student currently completing a MS in 2020. How have the results been disseminated to communities of interest?Presentations at regional and national scientific meetings. Publication of manuscript research articles, reviews, book chapters. What do you plan to do during the next reporting period to accomplish the goals?The renewal of this Hatch project will investigate the role of pig conceptus interferons in early embryo development, and the establishment and maintenance of pregnancy. CRISPR/Cas9 will be utilized to edit conceptus interferons to establish a model for their role in pregnancy. We also develop of a double gene edit for aromatase (CYP19A1-/-) and prostaglandin-endoperoxide synthase 2 (PTGS2-/-) to directly address if both conceptus estrogen and PGE work together as maternal recognition of pregnancy signals in the pig.

    Impacts
    What was accomplished under these goals? Our current research has demonstrated that early pig embryo production of prostaglandin is not essential for blastocyst or early conceptus development. Knockout of conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) does not affect placental development, establishment of pregnancy or fetal survival. These results indicate that endometrial prostaglandin production induced by conceptus estrogen secretion can support pregnancy in the absence of conceptus PTGS2 expression. The pig conceptus expresses Type I and II interferons. Interferon gamma (Type 1) is expressed from day 12 to 20 of gestation. There are 11 interferon delta's (IFND) expressed by pigs. However, only IFND 1 and 2 are expressed from day 14 to 35 of gestation while IFND 3,4 and 10 are expressed only on day 35 of gestation.

    Publications

    • Type: Books Status: Awaiting Publication Year Published: 2020 Citation: Placentation in Mammals: Tribute to EC Amorosos contributions to Viviparity. 2020. Eds. R.D. Geisert and T. Spencer. Advances in Anatomy, Embryology and Cell Biology, Springer.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Meyer A.E., C.A. Pfeiffer, K.E. Brooks, L.D. Spate, J.A. Benne, R.Cecil, M.S. Samuel, C.N. Murphy, S. Behura, M.K. McLean, L.A. Ciernia, M.F. Smith, K.M. Whitworth, K.D. Wells, T.E. Spencer, R.S. Prather and R.D. Geisert. 2019. New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig. Biol Reprod 101: 148161. https://doi.org/10.1093/biolre/ioz058
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Pfeiffer C.A., A.E. Meyer, K.E. Brooks, P.R. Chen, J. Milano-Foster, L.D. Spate, J.A. Benne, R. Cecil, M.S. Samuel, L.A. Ciernia, M.F. Smith, K.D. Wells, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Ablation of conceptus PTGS2 provides a new understanding of early pregnancy events in the pig. Biol Reprod doi.org/10.1093/biolre/ioz192.
    • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Adur M.K., J.W. Ross, F.J. White, E.M. Harder, D.R. Stein, R.D. Geisert, M.D. Ashworth. 2020. Estrogen regulates the uterine inflammatory response induced by interleukin-1 beta during the establishment of pregnancy in the pig. Reprod Sci
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Pfeiffer C.A., A.E. Meyer., L.D. Spate, J.A. Benne, R.F. Cecil, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Conceptus Prostaglandin Synthase 2 is Not Essential for Early Development and the Establishment of Pregnancy in the Pig. 52nd Annual Meeting of SSR, P432.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johns D.N., C.G. Lucas, C.A. Pfeiffer, L.D. Spate, J.A. Benne, R.F. Cecil, K.M. Wells, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2019. Pig Conceptus Expression of Type I Interferon Delta and Type II Interferon Gamma During Early Pregnancy. The 16th Annual Gilbert S. Greenwald Symposium on Reproduction and Regenerative Medicine, P25.
    • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Pfeiffer C.A. Ablation of conceptus PTGS2 provides a new understanding of early pregnancy events in the pig. MS Thesis, University of Missouri.


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:Presentations of the current research objective for the research on estrogen knockout in pig conceptuses were made at an international and national meeting. The meetings included scientists, graduate students and major swine industry producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently there are two graduate students working on the project. One studentfinished her Master's thesisin summer of 2018 another will finish her Master's thesis this summer.Graduate students have gained a great deal of experience with utilizing CRISPR technology for selective gene editing of early embryos and have presented their researchat national meeting. Ashley Meyer's poster presentation at the annual Society for the Study of Reproduction meeting received 2nd place. How have the results been disseminated to communities of interest?The results of our research has presented at professional scientific meetings, published inBiology of Reproduction,and presented to Veterinarian's and swine producer's at a regional meeting. The meetings included scientists, graduate students and major swine industry producers. What do you plan to do during the next reporting period to accomplish the goals?We willinvestigate the role of conceptus interferon production in establishment of pregnancy utilizing CRISPR technology for selective gene editing of early embryos . We are currently completing the study investigating the role of conceputsprostaglandin-endoperoxidesynthase 2in early conceptus development and establishment of pregnancy.

    Impacts
    What was accomplished under these goals? Interleukin 1beta2 (IL1B2), aromatase (CYP19A1) and prostaglandin-endoperoxidesynthase 2 (PTGS2) are expressed by the pig conceptuses during the peri-implantation period. The unique conceptus expression and secretion of IL1B2, estrogen (increase in aromatase enzyme expression) and prostaglandins occurs during the period of rapid conceptus elongation on Day 12 of pregnancy and thought to be essential to establishment of pregnancy. To understand the roles of IL1B2 and aromatase gene expression in during conceptus development and the establishment of pregnancy in the pig, we utilized the CRISPR/Cas9 genomic engineering to knockout conceptus IL1B2, CYP19A1 (aromatase) and PTGS2 gene expression. Trophoblast elongation and attachment occurred on Day 14 in wild type (IL1B2+/+) conceptuses but did not occur in IL1B2-/- null conceptuses which lacked IL1B2 secretion. Although the morphological transition of IL1B2-/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. Trophoblast elongation and attachment occurred normally in both Day 14 wild type (CYP19A1+/+) and null (CYP19A1-/-) conceptuses. Although estrogen production by CYP19A1-/- conceptuses was inhibited, pregnancy was established and embryo development continued to Day 25 of pregnancy. However, gilts carrying CYP19A1-/- embryos aborted after Day 30 of pregnancy. The pregnancy of CYP19A1-/- embryos was rescued when embryos were co-transferred with wild type in vitro produced embryos which produce estrogen. Thus, estrogen is essential for the maintenance of pregnancy beyond 25 days of gestation. These results indicate that conceptus estrogen production from Day 12 to 18 of pregnancy is not essential for the establishment of pregnancy but is clearly needed to maintain pregnancy beyond the first one third of gestation. To investigate the possible role of conceptus prostaglandins (PGE production) in the establishment and maintenance of pregnancy, we utilized CRISPR/Cas9 gene editing to generate (PTGS2-/-) embryos. The PTGS2-/- blastocysts did not express PTGS2 or produce prostaglandins in culture. Conceptus elongation and embryo development were normal and the pregnancy was maintained beyond day 30 of gestation. Since both PTGS2-/- and CYP19A1-/- (aromatase) conceptuses can establish and maintain early pregnancy, these results suggest both endometrial PGE production stimulated by conceptus estrogen or conceptus PGE provide alternative pathways by which maternal recognition of pregnancy can occur in the pig. Utilizing CRISPR technology for selective gene editing of early embryos will continue to provide the model to gain fundamental knowledge of essential factors involved with development and survival of embryos which can lead to strategies for improving fertility and fecundity.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2018 Citation: Geisert R.D. and M.C. Lucy. 2018. Comparative Mammalian Female Reproduction-Pig. In: The Encyclopedia of Reproduction Vol 2., 641-649 Academic Press: Elsevier.
    • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Meyer A.E., C.A. Pfeiffer, K.E. Brooks, L.D. Spate, J.A. Benne, R.Cecil, M.S. Samuel, C.N. Murphy, S. Behura, M.K. McLean, L.A. Ciernia, M.F. Smith, K.M. Whitworth, K.D. Wells, T.E. Spencer, R.S. Prather and R.D. Geisert. 2019. New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig. Biol Reprod (In Press)
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pfeiffer C.A., A.E. Meyer, L.D. Spate, J.A. Benne, R.F. Cecil, T.E. Spencer, R.S. Prather and R.D. Geisert. 2018. Biological Role of Prostaglandin Synthase 2 (PTGS2) on Early Conceptus Development in Pigs. 51st Annual Meeting of SSR, P399
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Meyer A.E., C.A. Pfeiffer, K.E. Brooks, L. Spate, J. Benne, C. Murphy, R. Cecil, T.E. Spencer, R.S. Prather and R.D. Geisert. 2018. New Perspecitive on Conceptus Estrogens and Pregnancy Establishment in Pigs. 51st Annual Meeting of SSR, P410.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Pfeiffer C.A., A.E. Meyer., L.D. Spate, J.A. Benne, R.F. Cecil, T.E. Spencer, R.S. Prather, and R.D. Geisert. 2018. Biological Role of Prostaglandin Synthase 2 (PTGS2) on Early Conceptus Development in Pigs. The 15th Annual Gilbert S. Greenwald Symposium on Reproduction and Regenerative Medicine, P40.
    • Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Geisert R.D., P. Sutvosky, M.C. Lucy, F.F. Bartol and A.E. Meyer. 2019. Chapter 15: Reproductive Physiology of Swine. In: Animal Agriculture: Sustainability, Challenges and Innovations. Eds. F.W. Bazer, G.C. Lamb and G. Wu. Academic Press, Elsevier Inc. (In Press)
    • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Meyer, A.E. 2018. New Perspective on the Role of Conceptus Estrogens in Conceptus Development, Maternal Recognition and the Establishment of Pregnancy in Pigs. Master of Science Thesis.


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:We have made presentations of our research findings to research scientists, graduate students, and pig producersat international (10th International Conference of Pig Reproduction) and national meetings (Society for the Study of Reproduction and the 14th Annual Greenwald Symposium on Reproduction and Regenerative Medicine). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently there are two graduate students working on the project. One student will finish her Master's thesis this summer, 2018. How have the results been disseminated to communities of interest?The results of our research has presented at professional scientific meetings, published in the Proceedings of the National Academy of Science and contributed to a conference journal review published in Molecular Reproduction andDevelopment. What do you plan to do during the next reporting period to accomplish the goals?We will continue to investigate the role of conceptus estrogen production in establishment of pregnancy in the pig with our aromatase null embryos. We are currently investigating whether the pregnancy of aromatase null embryos can be rescuedthrough treatment of pigs with estrogen orco-transferred with wild type embryos.

    Impacts
    What was accomplished under these goals? We have established that interleukin 1beta2 (IL1B2) and aromatase (CYP19A1) are expressed by the pig conceptuses during the peri-implantation period. The unique conceptus expression and secretion of IL1B2 and estrogen (increase in aromatase enzyme expression) occurs during the period of rapid conceptus elongation on Day 12 of pregnancy. To understand the roles of IL1B2 and aromatase gene expression in during conceptus development and the establishment of pregnancy in the pig, we utilized the CRISPR/Cas9 genomic engineering to knockout IL1B2 and CYP19A1 (aromatase) gene expression. Trophoblast elongation and attachment occurred on Day 14 in wild type (IL1B2+/+) conceptuses but did not occur in IL1B2-/- null conceptuses which lacked IL1B2 secretion. Although the morphological transition of IL1B2-/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion was decreased, indicating that IL1B2 may be involved with spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the pro-inflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface. Trophoblast elongation and attachment occurred normally in both Day 14 wild type (CYP19A1+/+) and null (CYP19A1-/-) conceptuses. Although estrogen production by CYP19A1-/- conceptuses was inhibited, pregnancy was established and embryo development continued to Day 25 of pregnancy. However, gilts carrying CYP19A1-/- embryos aborted after Day 30 of pregnancy. These results indicate that conceptus estrogen production from Day 12 to 18 of pregnancy is not essential for the establishment of pregnancy but is clearly needed to maintain pregnancy beyond the first one third of gestation. Utilizing CRISPR technology for selective gene editing of early embryos will continue to provide the model to gain fundamental knowledge of essential factors involved with development and survival of embryos which can lead to strategies for improving fertility and fecundity.

    Publications

    • Type: Book Chapters Status: Published Year Published: 2017 Citation: Geisert R.D., J.J. Whyte, A.E. Meyer, D.J. Mathew, M.C. Lucy, R.S. Prather and T.E. Spencer 2017. Rapid conceptus elongation in the pig: Interleukin 1 beta 2 and estrogen regulated phenomena. Mol Reprod Dev 84:760-774. doi: 10.1002/mrd.22813.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Whyte W.W., A.E. Meyer., L.D. Spate, J.A. Benne, R. Cecil, M.S. Samuel, C.N. Murphy, R.S. Prather and R.D. Geisert. 2018. Inactivation of porcine interleukin-1? (IL1B2) results in failure of rapid conceptus elongation. PNAS 115 (2) 307-312. doi:10.1073/pnas.1718004115.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Rapid Conceptus Elongation in the Pig: Interleukin 1 beta 2 and Estrogen Regulated Phenomena. 2017. 10th International Conference on Pig Reproduction, Columbia, Missouri.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Meyer A.E., R.D. Geisert, T.E. Spencer, R.S. Prather, K.E. Brooks, L. Spate, J. Benne amd S. Murphy, R. Cecil. 2017. Generation of aromatase (CYP19A1) null embyros for evaluation of estrogens role in conceptus development and establishment of pregnancy. 10th International Conference on Reproduction, June 11-14.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Meyer A.E., Geisert R.D., Spencer T.E., Prather R.S., Brooks K.E., Murphy C., Spate L., Benne J., Murphy S., Cecil R. 2017. Effect of Aromatase (CYP19A1) Deletion on Pre-Implantation Embryo Development and Conceptus Elongation in Pigs. 50th Annual Meeting of SSR, P118.


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have an undergraduate Discovery Fellow helping conduct research on the project. Dr. Whyte developed the conceptus IL1B2 knockouts and was the first to demonstrate the importance of IL1B2 in during early conceptus development and survival. My graduate student, Ashley Meyer is currently completing experiments with aromatase and prostaglandin endoperoxide synthase 2 knockouts to continue to evaluate key genes involved with early conceptus development in the pig. Ashley will complete her Master of Science degree in the fall of 2017. How have the results been disseminated to communities of interest?We have published one journal review of our research in Reproduction and will provide an internationally invited presentation during the 10th International Conference on the Control of Pig Reproduction in June. The manuscript for the ICAR presentation will be published in Molecular Reproduction and Development. The discoveries from our research is included in our undergraduate and graduate courses in reproductive biology at the University of Missouri. What do you plan to do during the next reporting period to accomplish the goals?We will complete the experiment and analyses of the IL1B2 knockout pigconceptuses. Currently, a manuscript is being prepared for the submission of our results for publication in PNAS. Research experiments on evaluating the role of aromatase and prostaglandin endoperoxide synthase 2 through CRISPR knockout conceptuses will be completed. The experiments will provide cutting edge knowledge for the role of genes regulating conceptusdevelopment and survival.

    Impacts
    What was accomplished under these goals? Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin-1 beta (IL1B), a master proinflammatory cytokine, is released by pig conceptus during the peri-implantation period and is believed to be essential for establishment of pregnancy. We discovered that the gene encoding IL1B has duplicated in the pig, resulting in a novel gene (IL1B2). Our research has indicated that the novel IL1B2 is uniquely expressed by pig conceptuses during the peri-implantation period. We recently utilized the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nuclease technology to generate IL1B2 null embryos. Null mutation of the IL1B2 did not affect development of the embryos to the blastocyst stage. Although the IL1B2 null blastocysts develop to spherical conceptuses (Day 10), the conceptuses fail to rapidly elongate or survive in utero as compared with wild type derived conceptuses. These results demonstrate that the unique and specific expression of pig conceptus IL1B2 is essential to the rapid remodeling and elongation within the uterine horn. Therefore, the unique expression of IL1B2 in the pig conceptus is the critical factor involved with the rapid trophoblast elongation to establish and maintain pregnancy.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mathew DJ, M.C. Lucy and R.D. Geisert. 2016. Proinflammatory cytokines and establishment of pregnancy: The porcine model. Reproduction pii: REP-16-0047.
    • Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Geisert RD, Whyte J.J., Meyer A.E., Mathew D.J., Lucy M.C., Prather R.S., and Spencer T.E. 2017. Rapid Conceptus Elongation in the Pig: Interleukin 1 beta 2 and Estrogen Regulated Phenomena. Molecular Reproduction & Development (in press)
    • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Meyer A.E., Geisert R.D., Spencer T.E., Prather R.S., Brooks K.E., Spate L. , Benne J., Murphy S., Cecil R. 2017. Generation of aromatase (CYP19A1) null embryos for evaluation of estrogens role in conceptus development and establishment of pregnancy.Molecular Reproduction & Development (In Press) A.E. Meyer, R. D. Geisert*, T.E. Spencer, R.S. Prather, K.E. Brooks, L. Spate, J. Benne, and S. Murphy, R. Cecil
    • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Geisert R.D. Rapid Conceptus Elongation in the Pig: Interleukin 1 beta 2 and Estrogen Regulated Phenomena. 10th International Conference on Control of Pig Reproduction. Columbia, MO (June 11-16, 2017)


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My graduate MS studentAshleyHobbs will conductresearch on using the CRISPR technology to knockout aromatase and estrogen receptor in the pig conceptuses. Dr. Whyte (Research Associate)is developing conceptus IL1B2 knockouts to the functional role during early conceptus development.These studies willhelp determine biological pathways involved with inducing conceptus elongation for subsequent placental attachment and development and the conceptus-uterine interactions essential for embryo survival. How have the results been disseminated to communities of interest?We have published journal articles, review papers and recurrently a book chapter that will provide the newest information to students, graduate students and researchers. What do you plan to do during the next reporting period to accomplish the goals?We will utilize the CRISPR technology to knockout conceptus estrogen synthesis andits receptor to evaluate the interaction with the conceptusIL1B2that is being developed in our laboratory. These studies willhelp determine biological pathways involved with inducing conceptus elongation for subsequent placental attachment and development and the conceptus-uterine interactions essential for embryo survival.

    Impacts
    What was accomplished under these goals? We discovered that the gene encoding interleukin 1beta (IL1B) has duplicated in the pig, resulting in a novel gene (IL1B2) which is onlyexpressed by pig conceptuses during the peri-implantation period. The unique conceptus expression of IL1B2 only occurs during the period of rapid conceptus elongation on Day 12 of pregnancy which activates endometrial nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts within the endometrium. We concluded that the peri-implantation pig conceptuses express a novel IL1B2 that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig. Conceptus IL1B2 has a slightly different response on the endometrium compared withsystemic IL1B1which we believe is due to slight differences in the structure of IL1B2 compared to ILB1which can alter its activity when bindingwith its receptor. The only method to truly understand the role of conceptus IL1B2 expression during early pregnancy is to either use a receptor inhibitoror knockout conceptus expression of IL1B2. Although receptor inhibitors can be effective, there are always questions as to other effects of the inhibitor on the system. The innovative and novel CRISPR/Cas9 genomic engineering to knockout specific genes provides the ideal method to evaluate the role of conceptus gene expression during early development in the pig. Using the CRISPR technology will allow us to knockout conceptus IL1B2 synthesis and help determine biological pathways involved with inducing conceptus elongation for subsequent placental attachment and development and the conceptus-uterine interactions essential for embryo survival. Utilizing CRISPR technology for selective gene editing of early embryos will provide the model to gain fundamental knowledge of essential factors involved with development and survival of embryos which can lead to strategies for improving fertility and fecundity.

    Publications

    • Type: Books Status: Published Year Published: 2015 Citation: Regulation of Implantation and Establishment of Pregnancy in Mammals: Tribute to 45 Year Anniversary of Roger V. Shorts Maternal Recognition of Pregnancy. 2015. Ed. R.D. Geisert. Advances in Anatomy, Embryology and Cell Biology, Springer.
    • Type: Book Chapters Status: Published Year Published: 2015 Citation: Geisert R.D., G.A. Johnson and R.C. Burghardt. 2015. Implantation and Establishment of Pregnancy in the Pig. In: Regulation of Implantation and Establishment of Pregnancy in Mammals: Tribute to 45 Year Anniversary of Roger V. Shorts Maternal Recognition of Pregnancy. Ed. R.D. Geisert. Advances in Anatomy, Embryology and Cell Biology, Springer, pp. 137-164.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mathew D.J, Newsom E.M., Guyton J.M., Tuggle C.M., Geisert R.D. and Lucy M.C. 2015. Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin-1 beta 2: a novel interleukin-1 expressed by the elongating pig conceptus. Biol. Reprod. 92:1-13.


    Progress 01/17/14 to 09/30/14

    Outputs
    Target Audience: Nothing Reported Changes/Problems: We have used the new CISPR technology for knocking out IL1B2 in early embryos. These are less costly then other methods we previously proposed and are very efficient in Dr. Prather's laboratory. This method will provide a proof of principle for investigating conceptus genesesstenial of early development and survival in the future. What opportunities for training and professional development has the project provided? We have an undergraduate helping conduct researchon the project. My graduate student Daniel Mathew conducted the research and completed his PhD in the fall of 2014. Dr. Whyte is developing conceptus IL1B2 knockouts to the functional role during early conceptus development and has an undergraduate assisting him in the lab. How have the results been disseminated to communities of interest? We have published a journal manuscript in Biology of Reproduction concerning the discovery of the pig conceptues IL1B2. Two review papers have been published to demonstrate the role of IL1B2 in establishment of pregnancy. Dr. Whyte will present his data concerning the IL1B2 knockout at an international meeting. What do you plan to do during the next reporting period to accomplish the goals? We are currently completing the study to knockout IL1B2 in conceptus. This study will determine the need or not for conceptus IL1B2 in development and establishment of pregnancy in the pig. The experiment will provide cutting edge knowledge for the role of IL1B2 in embryo survival.

    Impacts
    What was accomplished under these goals? Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin-1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. We discovered that the gene encoding IL1B has duplicated in the pig, resulting in a novel gene (IL1B2). Preliminary observations indicate that the novel IL1B2 is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B2 was cloned from mRNA isolated from d 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBL). The pig conceptuses but not PBL expressed a novel IL1B2. Porcine endometrium was treated with recombinant porcine IL1B and IL1B2 proteins. Immunohistochemistryand real-time reverse transcriptase-polymerase chain reactionwere used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts within the endometrium, respectively. Both IL1B and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lesser in endometrium treated with IL1B2 suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. We conclude thatthe peri-implantation pig conceptus expresses a novel IL1B2 that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig. To investigate differences in protein structure between IL1B and IL1B2, we predicted and aligned the atomic structures of pro and mature systemic IL1B1 and conceptus IL1B2. Alignment of predicted pro and mature proteins resulted in an RMSD of (3.47 Å) and (0.70 Å), respectively, indicating that IL1Band IL1B2 are highly similar in structure in absence of the pro-domains. Viewing the solvent accessible surface area of the pro-proteins suggested that there is a possible steric hindrance of the first CASP1 site in IL1B2 compared with that of IL1B. For the mature proteins, Novafold predicted that IL1B and IL1B2 bind the IL-1RI with 35 and 32 binding sites, respectively. Thirty-two binding sites were shared between the two proteins. Of these binding sites, IL1B2 had three non-conserved amino acid substitutions that resulted in a complete change of charge and solvent accessible surface areas compared with IL1B. Overall, differences in protein structure, the number of IL-1RI binding sites and amino acid side chain charges could affect the availability and activity of IL1B2 compared with IL1B.

    Publications

    • Type: Other Status: Published Year Published: 2014 Citation: Geisert RD, Lucy MC, Whyte JJ, Ross JW, Mathew DJ (2014) Cytokines from the pig conceptus: roles in conceptus development in pigs. J Anim Sci Biotechnol 7:51 doi: 10.1186/2049-1891-5-51
    • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Mathew DJ, Newsom EM, Guyton JM, Tuggle CK, Geisert RD, Lucy MC (2015) Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin-1 beta 2: a novel interleukin-1 expressed by the elongating pig conceptus. Biol Reprod. 2015 Mar 11. pii: biolreprod.114.126128
    • Type: Other Status: Submitted Year Published: 2015 Citation: Mathew, D.J., Lucy M.C. and Geisert R.D. 2014. Pro-imfammatory cytokines and the establishment of pregnancy: The porcie model. Cell & Developmental Biology (Review)
    • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Whyte J.J., M.E.Hennessy, R.D. Geisert, R.S. Prather. 2014. 115 Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-directed inactivation of porcine interleukin-1B as a model to to study endometrial receptivity and conceptus attachment. Reprod Fertil Dev. 27:149-50
    • Type: Book Chapters Status: Accepted Year Published: 2015 Citation: Geisert R.D., G.A. Johnson and R.C. Burghardt. 2015. Implantation and Establishment of Pregnancy in the Pig. In: Regulation of Implantation and Establishment of Pregnancy in Mammals: Tribute to 45 Year Anniversary of Roger V. Shorts Maternal Recognition of Pregnancy. Ed. R.D. Geisert. Advances in Anatomy, Embryology and Cell Biology, Springer