Brief Summary of Minutes of Annual Meeting

W3171 Meeting LSU Ag Center February 11, 2015 In attendance: Milan Shipka, Alaska, Administrator Clay Isom, Utah State University, Chair Quinton Winger, Colorado State University, Secretary (Ad Hoc) Brett White, Nebraska Edward Schmidt, Montana Rick Rorie, Arkansas Ken Bondioli, Louisiana State University Guest: Carlos Pinto, Louisiana State University LSU Graduate Students: Brittany Foster, LSU Fabian Diaz, LSU 8:20 Meeting called to order by Clay Isom Minutes from annual meeting W2171 Reno, NV were read by Clay Isom and approved. 8:30 Video Conference Call with Mark Mirando, USDA President’s Budget FY 2016 AFRI discretionary funding increased from 325 million to 450 million, foundational grants will use 40% of the budget. 2016 budget included an increase of 5.1% for Hatch fund dollars. A 12.5 million dollar increase put in specifically for a competitive grant program within the Hatch program. This is at the proposal level. Dr. Mirando highlighted 2 areas in the Food Security area that have animal sections. One contains Animal Breeding in title. May be an opportunity for Animal Reproduction? 10:00 Earle Pope, ACRES Audubon Center for Research of Endangered Species presentation, “Assisted Reproductive Technologies for Conservation of Endangered Cats” 1:00 Station Reports Arkansas Colorado Louisiana 3:00 Station Reports Montana Nebraska Utah Business Meeting: Discussed the successful re-write and award of the W3171. Discussed the importance of using the meetings as well as improving open communication within the group between meetings to foster collaborations. Money is needed to foster collaborations, therefore a strategy to improve the ability of members of the group to be co-investigators on grants with agricultural reproduction focus. Discussed strategies to improve competitiveness of grant applications using collaborative approaches. Clay Isom brought up the possibility of writing a peer-reviewed publication together by members of the group that would highlight the possible similarities of the groups research focus. Discussed the use of a web based system to make resources available more easily visible to members of the group. All agreed that we should use the email address lists of the group to improve communication about research collaborations. Collaborations: Isom and Schmidt talked about using the “embryo cradle” for nuclear transfer. Isom and Bondioli talked about in vitro gene expression similarities. Several talks focused on diet issues. Also, placental and fetal development due to androgen. Selection of the date for the 2016 meeting January 22, 2016 12:00-6:00pm in Louisville, Kentucky to coincide with the IETS meeting. Nomination for incoming secretary Rick Rorrie nominated Brett White, seconded by Clay Isom. Unanimously passed vote. Jerry Bouma will take over the chair position 60 days after the meeting. Discussed reaching out to new W3171 members that did not make it to the meeting. Milan mentioned we could also reach out to other Western region universities for any researchers that might be a good fit for the W3171 to fill empty slots. 6:40pm Meeting adjourned

Objective 1. Understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis Novel findings suggest a significant role for cytoplasmic polyadenylation during bovine oocyte maturation considering changes in polyadenylated transcript levels occur without changes in total RNA abundance. Also, changes in transcript abundance are found to corresponded to differences in poly(A) tail length at specific maturation stages. Comprehensive transcriptome analyses also were conducted on single matured bovine oocytes and pre-implantation embryos and novel stage-specific modules of co-expressed genes were identified that are potential master regulators of pre-implantation development. In separate studies RNA sequencing experiments on MII oocytes matured in vitro and in vivo reveal transcripts that are impacted by the in vitro maturation process. Finally, microfluidics-based qPCR was conducted on single oocytes and their associated cumulus cells and identified candidate genes whose expression was affected by age in the mare. Similarly, relative patterns of gene expression were compared based on follicular size and oocyte competence in goat oocytes. DPPA3 is identified as a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, and suggests that the role of DPPA3 during early development is conserved between species. In separate studies, histone methyltransferases were found to play a role in heterochromatin stabilization and potentially embryo developmental potential. Studies focused on improving in vitro maturation of oocytes and embryo culture reveal that polydimethylsiloxane well-insert systems successfully matures oocytes and culture embryos in an individually-identifiable manner, while possibly enhancing developmental potential. The endometrial transcriptome was characterized in pregnant and non-pregnant sows, and maternal pathways that could be key to embryo implantation and survival are uncovered. In addition, the cervical transcriptome was uncovered in cattle during different stages of the reproductive cycle that provide new insight into the mechanisms/factors that play a role in cervical permeability and antimicrobial activity. Ovarian responses to dietary conjugated linoleic acid uptake was studied and the observed highly regulated mechanisms involved in fatty acid uptake by ovarian components could explain the lack of ovarian responses in different cattle breeds. Metabolic studies in cultured bovine embryos were developed and data reveal that substrate balance in media greatly affects embryo metabolic pathways. Apical blebbing by epithelial cells in the epididymis was identified as a process by which proteins are transferred to sperm as they travel through the epididymis, and this process is regulated by testicular luminal factors. Objective 2. Refine methods for production of genetically modified animals to improve livestock production efficiency Laser-assisted intracytoplasmic injection was discovered to be an efficient technique to inject CRISPR/Cas9 RNA in bovine zygotes for genome editing purposes, which are a first step towards generation of genetically modified livestock. In addition, transgenic goats were generated using CRISPR/Cas9 technology to knockout myostatin, as well as using homologous recombination to knockin human K-ras oncogene. Placenta-specific gene targeting approaches have been developed by incubating sheep hatched blastocysts with lentiviral-based shRNA constructs to study stem cell factor LIN28 and transcription factor AR in placental development and function. In addition, data thus far suggest a role for LIN28 in trophoblast cell proliferation, and AR in placental angiogenesis. Separate studies were conducted to characterize trophoblast-derived stem-like cells from porcine blastocysts, and experiments are underway to assess their utility in donor nuclear transfer exeriments. A new system was developed and tested involving transduced mammary epithelial cells to evaluate vector construction that target recombinant protein expression in milk. Novel findings are reported on improved derivation efficiency and pluripotency of embryonic stem cells by supplementation of small molecules to culture media. In addition, new data reveals the importance of Akt signaling in somatic cell reprogramming. Separate experiments reveal that electromagnetic biostimulation of fibroblast cells can be used as an alternative to reprogram cells. In vitro transcribed mRNA was successfully used to transfect bovine fetal fibroblasts and induce expression of pluripotency factor OCT4, as an alternative, novel approach to generate stem cells without genetic modifications. Potential improvements were tested involving HAGM hydrogels and micromass culture systems for in vitro chondrogenic differentiation of porcine adipose derived stem cells, as well as addition of platelet-rich plasma derived growth factors to support proliferation and migration of adipose derived stem cells. Continued improvements are made to the Dracula micro-manipulator that will allow for its broad application in embryo research and cryopreservation.

Publications Rorie, R.W., A. J. Davis, T. D. Lester, and J. G. Powell. 2014. Comparison of two estrous synchronization protocols for use with X sorted semen in lactating beef cows. Prof. Anim. Sci. 30:620-624. Rorie, R.W., P.A. Delgado and T.D. Lester. 2014. Variation among beef bulls in the ratio of X- to Y-chromosome bearing spermatozoa. Adv. Reprod. Sci. 2:69-75. Rowe, M.P., J.G. Powell, E.B. Kegley, T.D. Lester and R.W. Rorie. 2014. Effect of supplemental trace mineral source on bull semen quality. Prof. Anim. Sci. 30:68-73. Bakhtari A, Ross PJ. DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos. Epigenetics. 2014 Sep;9(9):1271-9. doi: 10.4161/epi.32087. Epub 2014 Aug 1. Berger, T., and A. Conley. 2014. Reduced endogenous estrogen and hemicastration interact synergistically to increase porcine sertoli cell proliferation. Biology of reproduction 90: 114. Reyes JM, Chitwood JL, Ross PJ. RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation. Mol Reprod Dev. 2015 Jan 5. doi: 10.1002/mrd.22445. Campos-Chillon F, Farmerie TA, Bouma GJ, Clay CM, Carnevale EM. The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle. Reprod Fertil & Develop, 2015 Mar 19. doi: 10.1071/RD14472. da Silveira JC, Winger QA, Bouma GJ, Carnevale EM. Age effects on follicular fluid exosomal miRNAs and TGF? signaling during follicle development in the mare. Reprod Fertil & Develop, Accepted. Cleys ER, Halleran JL, Enriquez VA, da Silveira JC, West RC, Winger QA, Anthony RV, Bruemmer JE, Clay CM, Bouma GJ. Androgen Receptor and Histone Lysine Demethylases in Ovine Placenta. PLoS ONE 2015 Feb 12;10(2):e0117472.. Hatzel JN, Bouma GJ, Cleys ER, Bemis LT, Ehrhart EJ, McCue PM. Identification of heat shock protein 10 within the equine embryo, endometrium, and maternal peripheral blood mononuclear cells. Theriogenology 2015 Mar 15;83(5):832-9.. Nemere I, Garbi N, Winger Q. The 1,25D3 -MARRS Receptor/PDIA3/ERp57 and Lifespan. J Cell Biochem. 2014 Oct 6. doi: 10.1002/jcb.24986. [Epub ahead of print] Cleys ER, Halleran JL, McWhorter E, Hergenreder J, Enriquez VA, da Silveira JC, Bruemmer JE, Winger QA, Bouma GJ. Identification of microRNAs in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep. Mol Reprod Dev. 2014 Nov;81(11):983-93. da Silveira JC, Carnevale EM, Winger QA, Bouma GJ. Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare. Reprod Biol Endocrinol. 2014 May 26;12:44. Szerlong H, Herman JA, Krause CM, Deluca JG, Skoultchi A, Winger QA, Prenni JE, Hansen JC. Proteomic characterization of the nucleolar linker histone H1 interaction network. Journal of Molecular Biology, J Mol Biol. 2015 Jan 10. pii: S0022-2836(15)00006-6. Jeckel KM, Bouma GJ, Hess AM, Petrilli EB, Frye MA. Dietary fatty acids alter left ventricular myocardial gene expression in Wistar rats. Nutr Res. 2014 Aug;34(8):694-706. Le CH, Mulligan CM, Routh MA, Bouma GJ, Frye MA, Jeckel KM, Sparagna GC, Lynch JM, Moore RL, McCune SA, Bristow M, Zarini S, Murphy RC, Chicco AJ. Delta-6-desaturase links polyunsaturated fatty acid metabolism with phospholipid remodeling and disease progression in heart failure. Circ Heart Fail. 2014 Jan;7(1):172-83. Lin, CJ; Amano, T; Tang Y; TianXC. 2014. Improved derivation efficiency and pluripotency of stem cells from the refractory inbred C57BL/6 mouse strain by small molecules. PLoSONE 9(9): e106916 published online Sept 11, 2014. Jiang Z, Sun J, Dong H, Luo O, Zheng X, Obergfell C, Tang Y, Bi J, O’Neill R, Ruan Y, Chen J, Tian XC. (2014) Complete Transcriptional Profiles of Bovine In Vivo Pre-implantation Development. BMC Genomics 15:756; published online Sept 4, 2014. Tang Y, Jiang Z, Luo Y, Zhao X, Wang L, Tian XC. (2014). Differential Effects of Akt Isoforms on Somatic Cell Reprogramming J Cell Sci 127:3998-4008. Zopf D.A., Flanagan, C.L., Wheeler, M.B., Hollister, S.J., Green, G.E. 2013. Treatment of severe porcine tracheomalacia with a 3-dimensionally printed, bioresorbable, external airway splint. JAMA Otolaryngol Head Neck Surg. 2014 Jan;140(1):66-71. Yuan, Y., Paczkowski, M., Wheeler, M.B., Krisher, R.L. 2014. Use of a novel polydimethylsiloxane well insert to successfully mature, culture and identify single porcine oocytes and embryos. Reproduction, Fertility and Development 26:375-384. Monzani, P.S., Guemra, S., Adona, P.A., Ohashi, O.M., Meirelles, F.V., Wheeler, M.B. 2014. MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk. Animal Biotechnology, 26:2, 136-142. Zopf D.A., Mitsak, A.G., Flanagan, C.L., Wheeler, M.B., Green, G.E., Hollister, S.J. 2014. Computer-Aided Designed, 3-Dimensionally Printed Porous Tissue Bioscaffolds For Craniofacial Soft Tissue Reconstruction. J Otolaryngol Head Neck Surg. Online: DOI: 10.1177/0194599814552065 Bondioli, K.R. Cryopreservation of Bovine Embryos. In: Hopper, R. (ed) Bovine Reproduction. John Wiley and Sons, 2015 pp. 718-722. Chen, H., Fu, J., Chen, H., Hu, Y., Soroka, D. N., Prigge, J. R., Schmidt, E. E., Yan, F., Major, M. B., Chen, X. and Sang, S. (2014) Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo. Chemical research in toxicology. 27, 1575-1585. Sonsteng, K. M., Prigge, J. R., Talago, E. A., June, R. K. and Schmidt, E. E. (2014) Hydrodynamic delivery of Cre protein to lineage-mark or time-stamp mouse hepatocytes in situ. PLoS ONE. 9, e91219 Gorrini, C., Gang, B. P., Bassi, C., Wakeham, A., Baniasadi, S. P., Hao, Z., Li, W. Y., Cescon, D. W., Li, Y. T., Molyneux, S., Penrod, N., Lupien, M., Schmidt, E. E., Stambolic, V., Gauthier, M. L. and Mak, T. W. (2014) Estrogen controls the survival of BRCA1-deficient cells via a PI3K-NRF2-regulated pathway. Proc Natl Acad Sci USA. 111, 4472-4477 Saunders G, Stevens JR, and Isom SC. A shortcut for multiple testing on the directed acyclic graph of Gene Ontology. BMC Bioinformatics 2014, 15:349 DOI: 10.1186/s12859-014-0349-3 Li Q, Suasnavas E, Xiao L, Heywood S, Qi X, Zhou A, Isom SC. Label-free and non-invasive monitoring of porcine trophoblast derived cells: differentiation in serum and serum-free media. J Biophotonics. 2014 Sep 22;9999(9999). doi:10.1002/jbio.201400062. Suasnavas E, Heywood S, Ward A, Cox L, O’Grady M, Zhao Y, and Isom SC. Characterization of trophoblast-derived stem-like cells from peri-implantation porcine embryos. Animal Reproduction Science. In Press. Ni W., Qiao J., Hu S., Zhao X., Regouski M., Yang M., Polejaeva I.A.*, Chen C.* Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One 2014, 9 (9), e106718. Gong J., Chen G., Wang Z., Polejaeva I., Silisga R., Chen C-T., Kao C-M., and Chen L. Activating the expression of human K-rasg12d stimulates oncogenic transformation in transgenic goat fetal fibroblast cells. PLoS ONE. 2014, 9(3): e90059. Zhou W.*, Gosch G., Guerra T., Broek D., Wu G., Walker S., Polejaeva I*. Amino acid profiles in first trimester amniotic fluids of healthy cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies. Theriogenology. 2014, 81 (2), 225-229