In vitro culture of ovine embryos up to early gastrulating stages

ABSTRACT Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes – trophoblast and hypoblast – and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated ‘The people behind the papers’ interview.

Blastocysts developed at days 6 and 7 after IVF were transferred to agarose-coated four-well dishes in groups of 10-15.Agarose-coated dishes were prepared 3 days before use.Gel was prepared by solving 2.4 % ultrapure low melting point agarose (Thermo Fisher Scientific) in PBS.
In subsequent experiments, blastocysts were randomly cultured in hIVC alone or supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor (Y-27632, Stem Cell Technologies); or in N2B27 alone or supplemented with 10 μM ROCK inhibitor, 20 ng/ml activin A (Stem Cell Technologies), 100 ng/ml Insulin Growth Factor 1 (IGF1, Thermo Fisher Scientific) or 20 ng/ml basic Fibroblast Growth Factor (bFGF, Thermo Fisher Scientific), or a combination of 10 μM ROCK inhibitor and 20 ng/ml activin A.
All post-hatching development conditions were tested at 38.5 ºC in a water saturated atmosphere of 5 % CO2, 5 % O2, and 90 % N2 and half of the culture medium was replaced every other day.At the end of the culture at D14, pictures from the embryos were taken on a stereo microscope (Zeiss Stemi 305) and embryo area was measured using Fiji software (4).Embryo survival was analyzed following similar criteria than under conventional culture up to expanded blastocysts: alive embryos were able to maintain the blastocoel, whereas dead embryos collapsed (Fig. S10A).Surviving embryos were collected for further analyses.

Immunofluorescence and lineage development analysis
Embryos were fixed in 4 % paraformaldehyde (PFA) for 15 minutes at room temperature (RT), washed in PBS -1 % BSA, permeabilized in 1 % Triton X-100 in PBS for 15 min at RT and blocked in 10 % Donkey Serum-0.02 % Tween 20 in PBS for 1 h at RT.Then, embryos were incubated overnight at 4 ºC with primary antibodies to detect epiblast (SOX2), hypoblast (SOX17), mesoderm

Post-hatching development system
Development: doi:10.1242/dev.199743:Supplementary information Development • Supplementary information (BRACHYURY), trophectoderm (GATA3), the principal kinase of the apical Par polarity complex (aPKC) or basal membrane (LAMININ).After 4 washes in PBS-1% BSA, embryos were incubated in the appropriate secondary Alexa-conjugated antibodies or in Alexa 488 Phalloidin to detect Factin (Table S3) and counterstained with DAPI for 1 h at RT, followed by 4 washes in PBS-1 % BSA.Finally, embryos were mounted and imaged at a structured illumination equipment composed by a Zeiss Axio Observer microscope coupled to ApoTome.2 or at a fluorescence stereomicroscope (Zeiss V20).For tridimensional images, embryos were placed on PBS -1 % BSA microdrops made by drawing circles with a PAP pen (Kisker Biotech GmbH) on a coverslide as previously described (5).Microdrops were covered by an incubation chamber (Sigma Z37,9467) to prevent embryo crushing.Cells were counted using the ZEN software (Zeiss).Following immunofluorescence analysis, lineage development was analyzed.Epiblast survival was identified by the presence of SOX2+ cells in the embryo (Fig. S10b, d-f), whereas ED formation was identified by the presence of a compact structure of at least 30 SOX2+ cells (Fig. S10d-f).Hypoblast migration was considered complete when all the inner surface of the trophectoderm was covered by SOX17+ cells (Fig. S10b-c).

Apoptotic cells detection
The TdT-mediated dUTP-biotin Nick end-labeling (TUNEL) assay was employed for apoptotic cell detection using the In Situ Cell Death Detection Kit, TMR Red (Roche) according to the manufacturer's instructions with minor modifications.Briefly, after fixation, embryos were permeabilized in 0.5% Triton X-100 and 0.1% Sodium Citrate in PBS for 20 min at RT.Then, embryos were incubated in 30 μl drops of TUNEL reaction mixture for 1 h at 37 ºC in a humidified chamber and then washed in PBS -1 % BSA.Finally, embryos were mounted in Fluoroshield with DAPI in slides with 8 mm diameter rings and round coverslips (Thermo Fisher).Embryos were imaged at the structured illumination equipment previously described.Z-stack images were taken to detect all apoptotic cells along the Z axis.Cells were counted using the Fiji software (4) and apoptotic rate was determined by calculating the ratio of the total number of TUNEL positive cells/number total cells in the embryo.

RNA isolation, cDNA synthesis and qPCR
Poly (A) RNA was extracted from 4 individual whole D14 embryos of each group and 4 pools of 10 D7 blastocysts using the Dynabeads mRNA Purification Kit (Life Technologies, Oslo, Norway) following the manufacturer's instructions with minor modifications (6).Briefly, 50 µl of lysis buffer were added to the sample and incubated at RT for 10 min with gently shaking.Then, 10 µl of beads were added and samples were incubated at RT for 5 min with gentle shaking, allowing beads/mRNA complexes formation.Finally, beads/mRNA complexes were washed twice in washing buffer A and twice in washing buffer B, and resuspended in 10 mM Tris-HCl pH 7.5.The Development: doi:10.1242/dev.199743:Supplementary information Development • Supplementary information amount of mRNA/sample was roughly similar, being around 4 ng.Immediately after extraction, samples were treated with DNAse (Promega, Madison, WI, USA) at 37 ºC for 5 min followed by enzyme denaturalization at 90 ºC for 5 minutes, and then the reverse transcription reaction was carried out with qScript cDNA Supermix (Quantabiosciences, Gaithersburg, MS, USA) in a total volume of 20 µl.Tubes were first incubated at 25°C for 5 min and then at 42°C for 60 min to allow the reverse transcription of RNA, followed by 85°C for 5 min to denature the reverse transcriptase.mRNA transcripts were quantified by real-time quantitative PCR (qPCR).Two replicate PCR experiments were conducted for all genes of interest and qPCR efficiency was tested beforehand, all primers used showing efficiencies above 0.9.PCR was performed by adding a 2-µl aliquot of each sample to the PCR mix (GoTaq qPCR Master Mix, Promega, Madison, WI, USA) containing the specific primers.Primer sequences are provided in Table S4.The comparative cycle threshold (CT) method was used to quantify expression levels.Fluorescence was acquired in each cycle to determine the threshold cycle.According to the comparative CT method, the CT value was determined by subtracting the endogenous control H2AFZ CT value (7) for each sample from the CT value of each gene in the sample.CT was calculated using the highest sample CT value (i.e., the sample with the lowest target expression) as an arbitrary constant to be subtracted from all other CT sample values.Fold changes in the relative gene expression of the target were determined using the formula 2 -ΔΔCT (8).

RNA sequencing
Total RNA was extracted from 3 D14 in vitro, 3 E11 and 3 E12.5 in vivo embryos using MagMAX TM mirVana TM Total RNA Isolation kit according to the manufacturer´s protocol.cDNA was synthetized with SMART-Seq™ v4 Ultra™ Low Input RNA Kit (Clontech) and amplified ds-cDNA was purified with AMPure XP beads (Beckman Coulter) and quantified with Qubit (Life Technologies).Libraries were prepared using Covaris shearing system and a size selection of 200 bp was performed.
Library concentration was first quantified using Qubit and then diluted to 2 ng/µl before checking insert size on an Agilent 2100 and quantifying by qPCR.Libraries were pooled and sequenced on a HiSeq 2500 Sequencing System (Illumina).
The number of raw reads (150-bp paired-end reads) ranged from 40 to 63 million per sample.
Resulting files were pseudoaligned and quantified using kallisto (11) against the reference transcriptome of Ovis aries Rambouillet breed v1.0 (Ensembl release 104).Differential gene expression analysis was performed with R (v4.1.2),with the package DESeq2 (12) and collapsing transcript expression data to the gene level with tximport (13).A gene was considered as differentially expressed (DEG) between two experimental groups if its p-adj < 0.01 and its shrunken fold change > 2. To perform gene annotation of the DEGs, the mart database "oarambouillet_gene_ensembl" from the R package biomaRt (14)

Development • Supplementary information
was done to assign gene names to those ensembl IDs without an associated gene name.This step was performed with the eggNOG mapper (15,16) deploying blastx-like searches against a DIAMOND (17) database comprised of curated mammalian proteins.RNA-seq datasets generated during this study are available under GEO accession number: GSE189360.

Data and statistical analysis
Data analysis was blinded and manually performed by two different researchers with homogeneous criteria after testing for differences.Representative examples from each category (embryo survival, complete hypoblast migration, epiblast survival and embryonic disc formation) are provided in Fig. S9.Epiblast survival was scored as the presence of SOX2+ cells at D14. SOX2+ cell number, as well as TUNEL+ and total cell number, were counted manually using the multi-point counter plugin in ZEN 3.2 (Carl Zeiss, Germany).Data were analysed using the GraphPad Prism (GraphPad Software, San Diego, CA, USA) and Sigmastat (Systat Software, San Jose, CA, USA) packages and a value of P < 0.05 was considered significant.Chi-square test was used to analyse the differences in embryo survival, complete hypoblast migration, epiblast survival and embryonic disc formation between groups.Differences in area and apoptotic cells rate between groups were analysed by Student´s t-test when data distribution was normal.When normality test failed, statistical differences were analysed by Mann-Whitney Rank Sum Test.Differences in mRNA expression, embryo length, ED area and SOX2positive cell number were analysed by One-way ANOVA.When normality test failed, statistical differences were analysed by non-parametric One-way ANOVA (Kruskal-Wallis test).Additionally, SOX2-positive cell number was analysed by non-parametric bootstrapping with the R package nptest (version 1.0-3) with 1000 replicates per statistical test, and dependence on outliers was discarded (Fig. 2B: P-value for N2B27 vs. N2B27+R = 0.01; P-value for N2B27 vs. N2B27+A = 0.003.Fig. 2E: P-value for N2B27 vs. N2B27+A+R = 0.001; Fig. 3D: P-value for D14 vs. E11 = 0.0003; P-value for D14 vs. E12.5 = 0.045; P-value for D14 vs. E14 = 0.0001).

Fig. S2 .
Fig. S2.Representative Z-series of an ED without Rauber´s layer from a D14 in vitro embryo cultured in N2B27 + A + R.This figure is related to Figure 2d.Series of confocal z-sections of the D14 embryo stained for SOX2 (magenta) and SOX17 (green); nuclei were counterstained with DAPI (white).Note the absence of trophoblast cells (white) over the epiblast.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.Similar phenotype was observed in 22 out of 36 embryos.Scale bar = 50 µm.

Fig. S3 .
Fig. S3.Representative Z-series of an ED with Rauber´s layer from a D14 in vitro embryo cultured in N2B27.This figure is related to Figure 2d.Series of confocal z-sections of an embryo stained for SOX2 (magenta) and SOX17 (green); nuclei were counterstained with DAPI (white).Arrowheads indicate trophoblast cells (white) over the epiblast.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.Similar phenotype was observed in 6 out of 6 embryos.Scale bar = 50 µm.

Fig. S4 .
Fig. S4.Representative Z-series of EDs from E11 in vivo-derived embryos.Series of confocal z-sections of EDs (a,b) with Rauber´s layer, (c) with the Rauber´s layer being removed and (d) without Rauber´s layer, stained for SOX2 (magenta) and SOX17 (green); nuclei were counterstained with DAPI (white).Note epiblast cavitation in a (sections #4 and #5) and b (sections #4 to #6).Arrowheads indicate trophoblast cells (white) over the epiblast.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.Scale bar = 50 µm.

Fig. S5 .
Fig. S5.Representative Z-series of EDs from (a, c) D14 in vitro and (b, d) E11 in vivo-derived embryos.This figure is related to Figures 4a and b.Series of confocal z-sections of an EDs stained for (a, b) SOX2 (magenta) and LAMININ (green); (c, d) SOX2 (magenta) and aPKC (green); nuclei were counterstained with DAPI (merge).Arrowheads indicate (a, b) laminin accumulation in the basal side of SOX2+ epiblast cells; (c, d) apical localization of aPKC.Arrows point to hypoblast cells.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.Similar phenotype was observed in (a) 7 out of 9 embryos; (b) 2 out of 2 embryos; (c) 4 out of 4 embryos; (d) 3 out of 3 embryos.Scale bars = 10 µm for a and b, 50 µm for c and d.

Fig. S6 .
Fig. S6.Representative Z-series of (a) a D14 in vitro embryo and (b) a E12.5 in vivo embryo showing mesoderm differentiation.This figure is related to Figure 4c.Series of confocal zsections of an embryo stained for SOX2 (magenta) and BRACHYURY (T, green) showing mesoderm differentiation.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.White arrows indicate migrating T + cells.Double arrow indicates anteriorposterior (A-P) axis.Similar phenotype was observed in (a) 8 out of 18 in vitro embryos; (b) 6 out of 6 in vivo embryos.Scale bars = 50 µm.

Fig. S7 .
Fig. S7.Representative Z-series of (a) a D14 in vitro embryo and (b) a E12.5 in vivo embryo showing gastrulation.This figure is related to Figure 4d.Series of confocal z-sections of an embryo stained for SOX2 (magenta), BRACHYURY (T, green) and N-cadherin (white) showing mesoderm cells (T+) migrating from the ED and expression of the EMT marker N-cadherin.The thickness of every section was 5 μm.Numbers on the left indicate the number of sections.White arrows indicate migrating T + cells.Arrowhead indicates N-cadherin+ basement membrane.Double arrow indicates anterior-posterior (A-P) axis.Similar phenotype was observed in (a) 4 out of 9 in vitro embryos; (b) 5 out of 5 in vivo embryos.Scale bars = 50 µm.

Fig. S8 .
Fig. S8.Timeline for in vivo and in vitro embryo collection.Superovulation protocol employed to obtain in vivo derived embryos in sheep.D: days after the beginning of IVF (for in vitro embryos), E: embryonic day, days after mating (for in vivo embryos), FSH: Follicle stimulating hormone, IVM: in vitro maturation, IU: international units, IVC: in vitro culture, IVF: in vitro fertilization, M: mating.

Fig S9 .
Fig S9.Representative examples of the embryonic parameters analyzed and validation of the antibodies used to analyze lineages development.(a) Representative brightfield stereomicroscopic image of D14 embryos.All embryos are alive except for the one pointed with an arrow.Complete (b) and incomplete (c) hypoblast migration along the inner embryo surface was used.An ortholog search Development: doi:10.1242/dev.199743:Supplementary information

Table S1 .
Survival, area and development of hypoblast and epiblast lineages of surviving embryos at D14 after culture in hIVC or hIVC + ROCK inhibitor.

Table S3 .
Details of antibodies used immunostaining.

Table S4 .
Details of primers used for qPCR.