Performing Department
Animal Dairy & Veterinary Scie
Non Technical Summary
Discovery of the role of interferon-tau (IFNT) in pregnancy recognition is considered a seminal finding in ruminant reproduction. All type I interferons, including IFNT, activate the type I interferon receptor, IFNAR, which is a heterodimer made up of two subunits: IFNAR1 and IFNAR2. We have produced genetically modified sheep with IFNAR2 inactivated on either one or both chromosomes. Experiments conducted over the past year have established that biallelic IFNAR2 knockout ewes that do not have a functional type I interferon receptor can become pregnant and have a completely normal pregnancy. This unexpected and novel discovery implies that the accepted paradigm for pregnancy recognition in ruminants needs to be revised. This project is based on the general hypothesis that since IFNT signaling via IFNAR is not required for pregnancy in sheep, there must be a redundant or alternative mechanism of pregnancy recognition in ruminants. The specific aims for the project are to: (1) determine differences in trophoblast, endometrial epithelial cell, and corpus luteum gene expression in wildtype versus biallelic IFNAR2 knockout pregnancies where neither the dam nor conceptus expresses IFNAR; and (2) identify candidate antiluteolytic or luteotropic agents present in uterine luminal fluid and secreted by cultured trophoblast cells. For aim one, gene expression will be characterized by single-cell RNA-seq. For aim two, LC-MS/MS will be used to characterize uterine luminal fluid and trophoblast cell supernatants. This proposal directly addresses two Animal Reproduction Program priority areas, embryonic and fetal development,and gonadal function.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
This project has two specific aims:1) Determine differences in trophoblast, endometrial epithelial cell, and corpus luteum (CL) gene expression in wildtype versus biallelic IFNAR2 knockout pregnancies where neither the dam nor conceptus expresses IFNAR.2) Identify candidate antiluteolytic or luteotropic agents present in uterine luminal fluid and secreted by cultured trophoblast cells.
Project Methods
Specific aim #1:Breeding of WT and IFNAR2-/- ewes for collection of reproductive tissues. Starting in year one (fall of 2024), four IFNAR2-/- will be bred to an IFNAR2-/- ram of proven fertility wearing a marking harness. Likewise, four age-matched WT ewes will be bred to a WT ram of proven fertility wearing a marking harness. The ewes will be examined twice a day to determine if they have been bred (i.e., marked by the ram). Blood samples will be collected for P4 ELISA assays prior to introduction of the ram, and then every other day until the ewes are sacrificed for tissue collection on day 14-16 of pregnancy or, in the case of non-pregnant ewes, the estrus cycle. Ewes will be sacrificed 14-16 days after they are marked by the ram. From pregnant ewes, the conceptus, endometrium and the dominant CL will be collected, whereas endometrium and the dominant CL will be collected from ewes found to be non-pregnant. Once tissues have been collected from two day 14-16 pregnant ewes in each group (WT and IFNAR2-/-) the remaining pregnancies will be allowed to continue until day 30 of pregnancy. Pregnancies that continue beyond day 16 of pregnancy will continue to have serum samples collected every other day for measurement of P4 levels and pregnancy confirmation with the bioPRYN Pregnancy-Specific Protein B (PSPB) ELISA (BioTracking Inc). PBSB assays will be run on samples collect between days 22 and 30 of pregnancy. For animals sacrificed on day 30 of pregnancy, trophoblast, endometrium (including both placentomal and interplacentomal areas) and the dominant CL will be collected. Once the samples from day 14-16 and day-30 pregnancies have been collected, if we have not already obtained tissues from two non-pregnant ewes in each group, additional ewes will be sacrificed on day 14-16 of the estrus cycle (diestrus). These ewes will be closely monitored and P4 will be measured every other day to determine when they are in estrus. As with the bred, non-pregnant ewes, endometrium and the dominant CL will be collected. While we may be able to collect all these samples in year one, we anticipate that it will probably take two breeding seasons to obtain all the samples.Single-cell library preparation and sequencing. Immediately after tissues are collected, single-cell suspensions will be prepared using a combination of enzymatic digestion and mechanical dissociation. Single-cell RNA-seq libraries will be prepared using the 10x Genomics Chromium Single Cell 3' Reagent Kit v2 according to the recommended protocol. Single-cell libraries will be sequenced on an Illumina NextSeq 2000 sequencer using a P2 200 Cycle Kit with four libraries multiplexed per chip. This should provide sufficient sequencing depth for sequencing 2,000 cells per sample. Sequence data will be analyzed using the 10x Genomics Loupe Browser.Specific aim #2:Production of WT and IFNAR2-/- blastocysts by somatic cell nuclear transfer (SCNT). Sheep embryos will be produced by SCNT as previously described. Ovine oocytes will be matured for 21 hours at 38.5°C in Bo-IVM commercial medium, after which nuclear maturation will be evaluated based on polar body extrusion. Matured MII oocytes will undergo our previously described standard ovine SCNT procedure using sheep fetal or neonatal fibroblasts. As nuclear donors for derivation of WT and IFNAR2-/- embryos, we will use the SFF5 (male) or SFF3 (female) WT, Romney fetal fibroblast cell line, and a fetal fibroblast cell line derived from a colony 57 IFNAR2-/- fetus (male) or fibroblasts isolated from an IFNAR2-/- ewe, probably IFN2211 or IFN2262. After activation, embryos will be cultured under mineral oil in 40 ml droplets of Bo-IVC at 38.5°C in an atmosphere of 5% CO2, 5% O2 for 7 days. Medium will be changed every 48 h.Establishment of long-term 3D cultures from SCNT blastocysts. For establishment of long-term 3D trophoblast cultures, hatched 7-8 day old ovine SCNT blastocysts will be transferred to wells of a 24-well plate containing a feeder cell layer of 10,000 mitomycin C treated mouse embryonic fibroblast cells in 0.5 ml of media. The feeder cells will be incubated overnight at 38.5°C in an atmosphere of 5% CO2 before adding day 7 or 8 blastocysts. Trophoblast stem cell (TSC) cultures will be derived following the method described by Wang et al. 2023.Blastocysts will be cultured in TSC medium comprised of DMEM:F12 and Neurobasal medium (1:1) plus supplements. Both the blastocysts and isolated trophoblast cells will be cultured at 38.5°C in an atmosphere of 5% CO2. After 48 hours unattached embryos will be pressed against the feeder cells with needles to get them to adhere. Culture medium will be changed daily. At day 7 or 8, outgrowths will be dissociated by Dispase for 5-10 mins at 38.5°C, followed by two washes with DMEM/F12. Isolated TSCs will be cultured in 12-well plates. The TSCs will be cultured in TSC medium supplemented with 10 mM Rho-associated protein kinase (ROCK) inhibitor Y-27632 for the first 24 hours and then switched to TSC media without ROCK inhibitor. Once TSC cultures are established, TSCs will be passaged every 6 days at a 1:6 split ratio using Accutase. Trophoblast cell lines will be cryopreserved in ProFreeze freezing medium and long-term 3D cultures will be established.Long-term 3D trophoblast cultures will be grown in a slightly modified version of the trophoblast organoid medium (TOM) described by Sheridan et al. (2020). Tissues will be cultured at 38.5°C in 5% CO2. After 15, 30 and 60 days of culture in TOM supernatants will be collected for LC-MS/MS analysis and cells will be snap frozen for isolation of RNA and next-generation sequencing (RNA-seq).Establishment of long-term 3D cultures from flushed day 14 or 15 conceptuses. Day 14 or 15 conceptuses will be flushed from the uteri of anesthetized ewes. A midline laparotomy will be performed, and the uterus will be exposed. Briefly, individual uterine horns will be flushed by placing a glass cannula into the tip of the uterine horn and infusing 10 ml of phosphate buffered saline (pH 7.4) into the base of the horn. Uterine luminal contents will be massaged toward the tip of the uterine horn and out through the glass cannula into a petri dish or test tube. The flush medium will be kept for analysis by LC-MS/MS to identify potential mediators of pregnancy recognition and a piece of chorion will be snap frozen for isolation of RNA and RNA-seq.To establish long-term 3D cultures, chorionic tissue will be digested for 5 minutes with warm 0.25% trypsin-EDTA solution and cell aggregates will be seeded at approximately 200,000 cells/well in 6-well culture plates. The trophoblast cells will be grown in TOM as described above. After 15, 30 and 60 days of culture, supernatants will be collected for LC-MS/MS analysis and cells will be snap frozen for isolation of RNA and RNA-seq.Analysis of trophoblast supernatants by LC-MS/MS. Mass spectrometry will be performed with a Sciex Triple Quad 7500 LC-MS/MS. In these studies, we will target both lipids and proteins secreted by trophoblast cells. Samples for analysis will include both uterine luminal fluid collected during embryo flushes and trophoblast culture supernatants from long-term 3D trophoblast cultures.Characterization of gene expression in cultured trophoblast cells by RNA-seq. RNA will be isolated with the RNeasy Mini Kit and cDNA will be made by reverse transcription with the SuperScript VILO IV cDNA Synthesis Kit. Sequencing libraries will be prepared following standard protocols and sequenced on an Illumina NextSeq 2000 sequencer using a P2 100 Cycle Kit with up to 20 samples multiplexed per run. This depth of sequencing will yield approximately 20 million reads/sample.