Performing Department
Animal Sciences
Non Technical Summary
Improvements to developmental competence of bovine embryos is a high priority area to increase reproductive efficiencies. Targeted approaches to modulate gene-environment interactions that influence blastocyst development through conceptus elongation and maternal-embryo communication are attractive strategies to increase pregnancy success. Peroxisome-proliferator activated receptor gamma (PPARG) is a ligand-activated nuclear transcription factor expressed in the bovine blastocyst that responds to fatty acid (FA) stimulation and is essential for placental function and pregnancy maintenance in other species. PPARG requirements for bovine embryo development are unknown.The overall goal of this proposal is to determine regulatory roles of PPARG for embryo development. Objective 1 will determine the requirement of PPARG for blastocyst formation and cell lineage specification while Objective 2 will define developmental and transcriptomic roles of PPARG in response to embryo FA availability. Objective 3 will determine roles of embryonic PPARG in response to maternal FA availability for conceptus elongation. An experimental model consisting of PPARG knockout embryos and supplementation of FA to embryo culture and cow diets for embryo transfer will determine developmental, transcriptomic, and morphological characteristics regulated by PPARG. Functions in maternal-embryo communication will be quantified by targeted transcriptome analyses of the endometrium.Results from these studies will positively impact the cattle industry by providing guidance for embryo requirements that improve early development success. This proposal specifically addresses Program Priority Area A1211 - "cellular, molecular, genomic and whole aspects relevant to improving reproductive efficiency" and the sub-priority area "embryonic and fetal development (including interaction between the conceptus and its uterine environment)."
Animal Health Component
0%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Long-term GoalThe long-term goal is to improve reproductive success by targeting gene-environment interactions that influence early embryo development. Results from these projects will help to close the gap between blastocyst formation and conceptus elongation as a powerful tool to determine environmental factors that condition the preimplantation embryo for developmental success. Information gained from these objectives are directly applicable to optimizing embryo environment and maternal uterine dietary conditions for early pregnancy. The overall goal is to improve embryo developmental competence and reproductive efficiency in US cattle.Specific ObjectivesDetermine the requirement of PPARG for bovine blastocyst formation and cell lineage specification.Determine developmental and transcriptomic roles of PPARG in response to FA availability for bovine embryos.Determine roles of embryonic PPARG in response to maternal FA availability for conceptus elongation.
Project Methods
Objective 1. Determine the requirement of PPARG for bovine blastocyst formation and cell lineage specification. CRISPR design and embryo microinjectionBovine oocytes sourced from abattoir-derived ovaries that are routinely used for IVF will be matured for zygotic microinjection of CRISPR/Cas9 with two synthetic guide RNAs (gRNA) targeting exon 2 (E2) of bovine PPARG in accordance with the PD's previously established protocols. Parthenogenetic activation of oocytes for fertilization will initially be employed to increase biallelic KO efficiency by tightening the window of CRISPR delivery prior to initial DNA replication (S-phase). Synthetic gRNAs (Synthego) and Cas9 protein (PNA Bio) will be delivered via intracytoplasmic injection.Assessment of editing efficiencyDNA from single embryos at the 8-16 cell stage of development will be extracted in DNA lysis buffer and amplified by PCR. Reactions, PCR purification, and Sanger sequencing will be carried out in accordance with the PD's published protocol. Sequences will be analyzed using TIDE Analysis Software for assessment of gene editing by sequence trace decomposition. SnapGene software will be used in complement with TIDE software to align sequences for characterization of mutation frequency and allele editing.Blastocysts derived from PPARG targeted deletion (Injected), IC and UC groups will be subjected to immunocytochemistry for co-staining of PPARG, CDX2 (trophectoderm) and SOX2 (inner-cell mass). Following data collection, in vitro fertilization using frozen-thawed bull sperm from n = 3 different pooled bulls routinely used for IVF will be performed to increase rigor of findings achieved with parthenogenetically activated oocytes.Objective 2: Determine developmental and transcriptomic roles of PPARG in response to FA availability for bovine embryos.Bovine embryos will be cultured as previously described with the addition of eicosapentaenoic acid (EPA) to culture medium on D5. Fatty acid challenges will be applied to WT and KO embryos. Response to FA availability ( + ) and presence of a functional PPARG gene (KO vs WT) will be determined by developmental rates to include blastocyst formation, total cell number, and ratio of trophectoderm (CDX2) to total cell number (DAPI). At the termination of culture on D7.5, single embryos will be snap frozen followed by RNA isolation, cDNA amplification and RT-PCR to determine differences in expression of genes within the PPARG pathway in response to 1) PPARG genotype (WT vs KO), and 2) FA supplementation (+ or - EPA). Quantitative RT-PCR of transcripts previously optimized and validate in the PD's laboratory will be amplified and subjected to expression analyses using H2.A as a standard housekeeping gene. Differences in transcript abundance will be determined using the ΔΔCT method. A Student's t-test will be employed to determine significance (p <0.05) based on differences in mean values between samples.Objective 3: Determine roles of embryonic PPARG in responding to maternal FA availability for conceptus elongation.Lactating recipient Holstein cows will be used for this experiment. The experimental approach is a randomized block design with a 2 x 2 factorial arrangement of treatments. Cows will be blocked by parity group (lactation 1 vs. lactation > 1) and genomic breeding value for cow conception rate and, within block, assigned randomly to receive a diet without supplemental unsaturated FA (CON) or a diet supplemented with 20 g/d of a combination of EPA and docosahexaenoic (DAH, C22:6n3) FA to increase the supply of n3 FA (n3FA) known to affect reproductive response in cows. Cows will be housed in the same barn and fed individually using Calan gates at the University of Florida Dairy Research Unit. Within each dietary treatment, cows will be assigned to receive a PPARG KO or WT embryo, thus creating 4 treatments, CON-KO, CON-WT, n3FA-KO, and n3FA-WT. Fatty acids in n3FA treatment will be supplemented as calcium salts and the CON diet will be adjusted to equal amounts of nutrients, including FA, but from saturated FA sources (palmitic and stearic). Cows will be assigned to treatments at 14 ± 3 d postpartum when dietary treatments start. Cows will be fed the respective diets for 40 days until embryos are transferred (54 d postpartum) and 49 days until conceptus are recovered. The objectives are to have 10 conceptuses collected per treatment (n = 40 total), which requires 96 cows enrolled in the experiment.Endometrial sampling for transcriptomic analysesEndometrial samples from control and FA supplemented cows will be collected via Cytobrush following protocols established by Co-PD Binelli prior to embryo transfer and on D16 immediately before flushing PPARG KO and WT conceptuses from control or FA supplemented cows. Endometrial tissue collected for biopsy will be stored in RNA later solution and then frozen at -80°C until nucleic acid extraction.RNA Isolation and Real-Time PCR from endometrial tissueEndometrial RNA will be extracted using the PureLink RNA Mini Kit (Invitrogen, Carlsbad, CA) according to manufacturer instructions. A targeted approach will be used to identify key transcripts with proven response to PPARG regulation in the embryo.Estrous synchronization for embryo transferLactating dairy cows will have the estrous cycle synchronized to receive timed embryo transfer on day 7 of the cycle (day 0 is the expected day of estrus). The estrous synchronization protocol will start at 28 ± 3 d postpartum, an IVP embryo will be transferred on d 54 ± 3 postpartum, and conceptus will be recovered on d 63 ± 3 postpartum (D16 of embryo development).Conceptus recovery for morphology characterization, transcriptomic & genomic analysesUpon collection of the endometrial fluid sample, the uterine horn ipsilateral to the CL will be flushed by transcervical catheterization as previously describedIFN-τ will be quantified by ELISA. The recovered conceptus will be washed, the morphology and length recorded, and then the embryonic disc dissected to separate from the trophoblast for separate storage in RNA later solution and then frozen at -80°C until nucleic acid extraction.