Source: UNIVERSITY OF GEORGIA submitted to NRP
MEIOSIS IN PORCINE IPSCS IN A TESTIS NICHE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1005834
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Feb 28, 2015
Project End Date
Feb 27, 2019
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Animal & Dairy Science
Non Technical Summary
Developmental errors in germ cell (sperm and oocyte) meiosis is major cause of infertility in pigs and small litter sized directly affecting the profitability of swine farming operations. In this proposal, porcine induced pluripotent stem cells in a testicular microenvironment are used to study meiotic cell signaling that may one day lead to treatments, cures and preventative measures for many of these germ cell related conditions.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30135101030100%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
Reproductive challenges in livestock populations are of significant concern and are often a direct result of abnormal meiosis. Aberrant meiosis significantly affects fertility [1-3], is the leading cause of early pregnancy loss [4] and results in birth defects [5, 6]. Failed gametogenesis in livestock, including errors in meiosis, have significantly increased because of intense selection for commercial traits and decreased emphasis on reproductive success [7]. Porcine sub-fertility and sterility, spontaneous fetal abortion, decrease litter size and abnormal offspring have all been linked to errors in meiosis [8-11]. Advances in studying meiosis in livestock have been hindered by a lack of a biologically relevant system to explore the mechanisms and causes of meiotic failure, some of which are unique to each species and cannot be studied in rodent models.We have recently developed two novel technologies enabling systematic study of meiosis in livestock: 1) A robust in vitro pluripotent stem cell to germ cell differentiation culture system capable of generating large numbers of germ like-cells [12, 13], 2) the development of the only known chimera competent porcine induced pluripotent stem cells [14] . We propose to overcome significant challenges in the paucity of material obtained from current meiotic models by combining these technologies; providing a system with a robust cell source that can be easily expanded, differentiated and genetically manipulated. Our system overcomes species specific differences between rodent models and pigs and utilizes a meiotic competent microenvironment that is absent in current pluripotent stem cell to germ cell differentiation cultures that unreliably produce < 15% haploid cells [15-18]. This innovative stem cell to germ cell differentiation culture system will help us address our long term goal to identify and understand meiotic regulatory signaling in pigs that could provide invaluable insight into the mechanistic causes of abnormal meiosis. The use of porcine testicular aggregates is a logical first step being the best understood testicular aggregate system and the availability of porcine induced pluripotent stem cells for side along studies.The objective of this proposal is to interrogate the role of the essential meiotic gene STRA8 and gene families DAZ (BOULE, DAZL and DAZ genes) and Argonaute (PIWIL1, 2 and 4 genes) in meiosis through inducible overexpression in porcine induced pluripotent stem cells in a testicular aggregate microenvironment. Failure of these genes to be expressed in mouse and/or humans results in abnormal meiosis indicating their critical roles, yet their exact functions remain enigmatic [19-30]. We hypothesize that overexpression of STRA8 and individual DAZ and Argonaute family genes in porcine induced pluripotent stem cells will result in 1) improved meiotic phenotypic fidelity in cohesion, synapsis, genetic recombination, chromosome segregation and increased numbers of germ cells that enter and progress through meiosis and 2) an increase in expression of germ cell specific genes and pathway expression at the pre-meiotic, meiotic and post-meiotic stages of development. We will test this hypothesis with the following aims:Aim 1. Determine the effect of the testicular aggregate microenvironment on porcine induced pluripotent stem cells and derived germ-like cells to differentiation into meiotic competent gametes. We anticipate that the complex porcine testicular aggregate microenvironment will provide important factors for porcine iPSCs to develop and that transplant of iPSC derived germ cells at different stages of differentiation will potentially provide important insight into the biological capabilities and differentiation signals occurring within the aggregate.Aim 2. Define the functional role of STRA8, DAZ (BOULE, DAZL and DAZ genes) and Argonaute (PIWIL1, 2 and 4 genes) family genes in meiotic development in porcine iPSC derived cells using a tetracycline inducible overexpression system. Phenotypic data derived from the overexpression of these important meiotic genes will enable us to determine the specific meiotic stages (e.g. Prophase I, Metaphase II), sub-stages (Prophase I- Leptotente, Zygotene) and processes (e.g. synapsis) where these genes may play key roles. The use of a tet inducible system allows for more in-depth analysis than previously done in rodent knockouts.Aim 3. Determine changes in global gene expression in isolated pre-meiotic, meiotic and haploid cells derived from porcine induced pluripotent stem cells overexpressing STRA8, DAZ and Argonaute family genes. Comparative gene expression analysis of cells that do and do not overexpress meiotic genes at specific germ cell stages will aid in determining direct and indirect gene targets, effected pathways and further define the stage and role of overexpressed genes in meiosis. We are uniquely positioned to isolate highly enriched populations of pre-meiotic, meiotic and post-meiotic cells utilizing a validated and published STRA8 GFP meiotic reporter system.
Project Methods
Aim 1. Generation of Porcine Pre-Meiotic and Meiotic Germ-Like Cells.Germ cells will be differentiated for 10 days to generate initial populations of pre-meiotic (DDX4+/SYCP3-) cells. To produce an enriched (>90% DDX4+) population of pre-meiotic GLCs, cells will be clonally isolated by single cell FACS sorting of individual cells into wells of a 96 well plate. Clonal populations will be expanded and characterized by immunocytochemistry and flow cytometry to identify DDX4+ populations for use in aggregate studies. To derive enriched (>70%) early meiotic (DDX4/SYCP3+) GLCS, clonal lines of pre-meiotic GLCs will undergo an additional 10 days of differentiation and will be analyzed as before for co-expression of DDX4 and SYCP3. These protocols have been developed using human cells, however initial germ cell differentiation of piPSCs suggest that these time points are reasonable and additional time points can be added if needed.Testicular Aggregate Formation and Transplantation.Testicular aggregates will be generated by isolating testis from 5 day old pigs. Testis will be subjected to enzymatic digestion and GFP+ porcine iPSCs, pre-meiotic or early meiotic GLCs will be incorporated so that approximately 5% of the cells are iPSCs, the average amount of germ cells previously shown to be in successful aggregates. A total of 112 aggregates will be formed to assess meiotic differentiation for each developmental stage (iPSC, pre-meiotic and meiotic GLCs) in each species. Four aggregates will be transplanted under the dorsal skin into each of 96 castrated SCID mice. Four mice with aggregates from each of the three iPSC derived cell types will be randomly assigned to 7 analysis time points: 7, 14, 28, 70, 175, 210 and 280 days. These time points were selected based on data from over 100 independent trials across species as being likely points where key differences in development can be observed. In past experience, seven time points have been sufficient to observe all possible stages of development, despite delays likely due to the reorganization of the aggregate tissue, and has provided robust coverage of meiotic cells at various stages. The final time point of 280 days was chosen as full spermatogenesis in porcine aggregates typically occurs by this point. As positive control, aggregates will be generated containing only endogenous germ cells and transplanted into 4 mice for each time point.Testicular Aggregate Analysis.Transplants will be recovered at each time point with cells being analyzed for localization, morphology, protein and DNA changes indicative of developmental stage. Cells will be categorized as type A spermatogonia, type B spermatogonia, meiotic (including sub categories of meiotic cells), round and elongated spermatids by analysis of sectioned aggregates and single cells. Aggregates selected for section analysis will be fixed, sectioned and hematoxylin stained for each time point. Differentiated iPSCs in sectioned tissue will be identified by GFP expression. The spatial localization, cellular morphology and organization of each aggregate will be assessed to determine developmental progression. Additionally, immunohistohemistry will be performed for proteins indicative of each developmental stage to further support morphological findings. Single cell analysis will be conducted by flow cytometry, immunocytochemistry and chromosome spreads. Single cells will be isolated from aggregates by multiple rounds of trypsinization followed by flow cytometry and immunocytochemistry analysis of germ cell stage specific markers. Flow cytometry of stained cells will be utilized to determine the percentage of iPSCs at specific germ cell stages, while immunocytochemistry will be used to assess the proper subcellular distribution and formation of key meiotic landmarks including cohesin (cohesin proteins REC8, SMC1, SMC1β & SMC3) and synaptomal (synaptomal axial/ lateral element proteins SYCP3, SYCP1) complexes, double stranded breaks (DSB formation protein SPO11 and marker γH2AX) and DNA repair foci (DNA repair proteins MLH1, DMC1). Changes in DNA content will be initially determined by Hoechst DNA staining of single cells and flow cytometry analysis for changes in fluorescence indicating differentiation of cells into haploid gametes and confirmed by chromosome spreads.Aim 2. Generation of Meiotic Gene Overexpressing iPSC Derived Cells.TZV family vectors will be generated containing a tetracycline responsive element (TRE-t) driving STRA8, BOULE, DAZL, DAZ , PIWIL1, 2 and 4 genes. Additionally, the vectors will also contain ubiquitin (Ubi) driven reverse tetracycline transactivator 3 (rTTA3), which binds and activates expression of the TRE-t promoter in the presence of Dox and results in the activation of target genes and puromycin resistance genes utilizing an internal ribosome entry site (IRES). Porcine cells will be transduced and tested for Doxycycline (Dox) induced gene expression by quantitative RT-PCR (qRT-PCR) and flow cytometry with transduced cells being selected for based on puromycin resistance. Cells will then be incorporated into the testicular aggregate bioassay.Analysis of Tet Inducible Aggregates.Aggregates containing one of 8 constructs (7 meiotic genes and empty vector control) will be transplanted into mice and activated by Dox presented in feed. Gene overexpression will be continuous or inactivated at pre-meiosis or post-prophase I and transplants will be harvested at 4 time points spanning pre-meiotic to post-meiotic developmental stages. Four mice with 4 individual transplants will be used per treatment for each gene. Transplants will be recovered and analyzed as described above for localization, morphology, protein and DNA changes in whole sections and at the single cell level.Aim 3. Isolation of Meiotic Stage Specific Cells and mRNA. Porcine iPSCs that contain one of 7 tet inducible meiotic genes plus the empty vector control line will be transfected with the STRA8 GFP reporter vector that also contains genes that confer constitutively active CMV promoter driven puromycin resistance. Additionally, these cells will be transduced to express red florescent protein (RFP) as a means of tracking and isolating them in the aggregate. iPSCs containing the STRA8 GFP vector will be selected based on puromycine resistance and cells will be FACs sorted to ensure a pure population of RFP+ cells.Similar to Aim 2, aggregates containing one of 8 and now the STRA8 GFP reporter vector will be transplanted into mice and activated by feeding of Dox. Gene overexpression will be continuous or inactivated at pre-meiosis or post-prophase I and transplants will be harvested at 4 time points spanning pre-meiotic to post-meiotic developmental stages. Four mice with 4 individual transplants will be used per treatment for each gene. Transplants will be recovered and disassociated to a single cell state. RFP+ pre-meiotic cells will be isolated by FACs at a time point previously determined to contain no meiotic cells, while meiotic will be isolated on RFP plus STRA8 GFP expression and post-meiotic cells on Hoechst DNA staining showing haploid DNA content plus RFP expression. RNA will be isolated.Gene Expression Analysis of Tet Inducible Aggregates.Analysis of gene expression will be performed on starting iPSC derived cell populations and isolated cells from each treatment using the Porcine Gene Expression Microarrays. The porcine microarray uses 43,803 probes to interrogate well over 20,000 genes including alternative splice variants. Probes are based on the most up to date sequences in NCBI Unigene, Reference Sequence and other major public gene databases.

Progress 02/28/15 to 02/27/19

Outputs
Target Audience:The main audience was academic, industry and government scientist. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided an equivalent of 2 student EFTs training in cell culture, induced pluripotent stem cell reprogramming, cell differentiation, immunocytochemistry, semi-quantitative and quantitative RT-PCR, and other critical laboratory techniques. Students also were trained to write abstracts and manuscripts and to present posters and give scientific oral presentations. Students were also provided a number of opportunities to present their research at regional, national and international conferences. How have the results been disseminated to communities of interest?Yes. In the form of abstracts and publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Under these goals, several major accomplishments were achieved: 1. Multiple new male and female pig induced pluripotent stem cells were generated for the production of germ cells. 2. Under unique culture conditions, piPSCs were demonstrated to show increased gene and protein expression consistent with an early germ cell-like state. However, it was difficult to generate consistentcultures showing this phenotype.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Controlled Cortical Impact Severity Results in Graded Cellular, Tissue and Functional Responses in a Piglet Traumatic Brain Injury Model. Emily W. Baker*, Holly A. Kinder*, Jessica M. Hutcheson, Kylee Jo J. Duberstein, Simon R. Platt, Elizabeth W. Howerth, Franklin D. West. Journal of Neurotrauma. [Epub ahead of print 2018 Jun]. 2019 Jan 1;36(1):61-73.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: LiF@SiO2 nanoparticles for controlled lithium release and osteoarthritis therapy. Trever Todd, Zhenhui Lu, Benjamin Cline, Jinmin Zhao, Weizhong Zhang, Hongmin Chen, Anil Kumar, Wen Jiang, Franklin West, Samuel Franklin, Li Zheng, and Jin Xie. Nano Research. [Epub ahead of print 2018 Apr]. 2018. Oct;11(10): 5751-5760.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Human neural stem cell extracellular vesicles improve recovery in a porcine model of ischemic stroke. Robin L. Webb*, Erin E. Kaiser*, Brian J. Jurgielewicz, Samantha E. Spellicy, Shelley L. Scoville, Tyler A. Thompson, Raymond L. Swetenburg, David C. Hess, Franklin D. West and Steve L. Stice. Stroke. 2018 May;49(5):1248-1256.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Human iNPC therapy leads to improvement in functional neurologic outcomes in a pig ischemic stroke model. Vivian W. Lau, Simon R. Platt, Harrison E. Grace, Emily W. Baker, and Franklin D. West. Brain and Behavior. [Epub ahead of print 2018 Apr]. 2018 Apr 18;8(5).
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Willard KA, Elling CL, Stice SL, Brindley MA. The Oxysterol 7-Ketocholesterol Reduces Zika Virus Titers in Vero Cells and Human Neurons. Viruses 11(1) 30 Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Goodfellow FT, Willard KA, Wu X, Scoville S, Stice SL, Brindley MA. Strain-Dependent Consequences of Zika Virus Infection and Differential Impact on Neural Development. Viruses 10(10) 09 Oct 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Latchoumane C-FV, Jackson L, Sendi MSE, Tehrani KF, Mortensen LJ, Stice SL, Ghovanloo M, Karumbaiah L. Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro Scientific Reports 8(1) Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Saxena T, Lyon JG, Pai SB, Pare D, Amero J, Karumbaiah L, Carroll SL, Gaupp E, Bellamkonda RV. Engineering Controlled Peritumoral Inflammation to Constrain Brain Tumor Growth. Adv Healthc Mater e1801076 11 Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Logun M, Zhao W, Mao L, Karumbaiah L. Microfluidics in Malignant Glioma Research and Precision Medicine Advanced Biosystems 2(5):1700221-1700221 May 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Forouhesh Tehrani K, Pendleton E, Leitmann B, Mortensen L. Fast axial scanning using liquid lens technology for 2-photon in vivo microscopy Biomedical Optics Express 17 Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Forouhesh Tehrani K, Pendleton E, Southern W, Call J, Mortensen L. Spatial frequency metrics for analysis of musculoskeletal tissues using multi-photon microscopy Tissue Engineering - Part C: Methods 16 Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Southern W, Nichenko A, Forouhesh Tehrani K, McGranahan M, Krishnan L, Qualls A, Jenkins N, Mortensen L, Yin H, Yin A. PGC-1? overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury Scientific Reports 12 Oct 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Selma JM, Das A, Awojoodu AO, Wang T, Kaushik AP, Cui Q, Song H, Ogle ME, Olingy CE, Pendleton EG. Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization During Bone Repair Cellular and Molecular Bioengineering 11(4):241-253 Aug 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Mortensen L, Pendleton E, Forouhesh Tehrani K, Barrow R. Second Harmonic Generation Polarimetry of Collagen Organization in Whole Bone Scientific Reports 22 Jun 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Shao L, Chang J, Feng W, Wang X, Williamson EA, Li Y, Schajnovitz A, Scadden D, Mortensen LJ, Lin CP. The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow Nature Communications 9(1) Dec 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Tehrani KF, Pendleton EG, Southern WM, Call JA, Mortensen LJ. Two-photon deep-tissue spatially resolved mitochondrial imaging using membrane potential fluorescence fluctuations Biomedical Optics Express 9(1):254-254 01 Jan 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang Z, Yoshida Y, Kramer NE, Kawabata F, Tabata S, Kim WK, Liu H-X. Abundant proliferating cells within early chicken taste buds indicate a potentially "built-in" progenitor system for taste bud growth during maturation in hatchlings. Histol Histopathol 18055 31 Oct 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang Z, Yoshida Y, Kramer N, Kawabata F, Tabata S, Kim W, Liu H. Abundant proliferating cells within early chicken taste buds indicate a potentially "built-in" progenitor system for taste bud growth during maturation in hatchlings. Histology and Histopathology Article number 18055 31 Oct 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu H-X, Rajapaksha P, Wang Z, E Kramer N, J Marshall B. An Update on the Sense of Taste in Chickens: A Better Developed System than Previously Appreciated Journal of Nutrition & Food Sciences 08(02) 2018


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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? See Final Report

Publications


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

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? See Final Report

    Publications


      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? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

      Impacts
      What was accomplished under these goals? See Final Report

      Publications


        Progress 02/28/15 to 09/30/15

        Outputs
        Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

        Impacts
        What was accomplished under these goals? See final Report

        Publications