Interleukin-6 promotes primitive endoderm development in bovine blastocysts

Does primordial germ cell (PGC) activation start before mouse embryo implantation, and does the possible regulation of the DNA (cytosine-5-)-methyltransferase 3-like (Dnmt3l) by transcription factor AP-2, gamma (TCFAP2C) have a role in this activation and in the primitive endoderm (PE)-epiblast (EPI) lineage specification? A burst of expression of PGC markers, such as Dppa3/Stella, Ifitm2/Fragilis, Fkbp6 and Prdm4, is observed from embryonic day (E) 3.25, and some of them, together with the late germ cell markers Zp3, Mcf2 and Morc1, become restricted to the EPI subpopulation at E4.5, while the dynamics analysis of the PE-EPI transitions in the single-cell data suggests that TCFAP2C transitorily represses Dnmt3l in EPI cells at E3.5 and such repression is withdrawn with reactivation of Dnmt3l expression in PE and EPI cells at E4.5. In the mouse preimplantation embryo, cells with the same phenotype take different fates based on the orchestration between topological clues (cell polarity, positional history and division orientation) and gene regulatory rules (at transcriptomics and epigenomics level), prompting the proposal of positional, stochastic and combined models explaining the specification mechanism. PGC specification starts at E6.0-6.5 post-implantation. In view of the important role of DNA methylation in developmental events, the cross-talk between some transcription factors and DNA methyltransferases is of particular relevance. TCFAP2C has a CpG DNA methylation motif that is not methylated in pluripotent cells and that could potentially bind on DNMT3L, the stimulatory DNA methyltransferase co-factor that assists in the process of de novo DNA methylation. Chromatin-immunoprecipitation analysis has demonstrated that Dnmt3l is indeed a target of TCFAP2C. We aimed to assess the timing of early preimplantation events and to understand better the segregation of the inner cell mass (ICM) into PE and EPI. We designed a single-cell transcriptomics dynamics computational study to identify markers of the PE-EPI bifurcation in ICM cells through searching for statistically significant (using the Student's t-test method) differently expressed genes (DEGs) between PE and EPI cells from E3.5 to E4.5. The DEGs common for E3.5 and E4.5 were used as the markers defining the steady states. We collected microarray and next-generation sequencing transcriptomics data from public databases from bulk populations and single cells from mice at E3.25, E3.5 and E4.5. The results are based on three independent single-cell transcriptomics data sets, with a fold change of 3 and P-value <0.01 for the DEG selection. The dynamics analysis revealed new transitory E3.5 and steady PE and EPI markers. Among the transitory E3.5 PE markers (Dnmt3l, Dusp4, Cpne8, Akap13, Dcaf12l1, Aaed1, B4galt6, BC100530, Rnpc3, Tfpi, Lgalsl, Ckap4 and Fbxl20), several (Dusp4, Akap13, Cpn8, Dcaf12l1 and Tfpi) are related to the extracellular regulated kinase pathway. We also identified new transitory E3.5 EPI markers (Sgk1, Mal, Ubxn2a, Atg16l2, Gm13102, Tcfap2c, Hexb, Slc1a1, Svip, Liph and Mier3), six new stable PE markers (Sdc4, Cpn1, Dkk1, Havcr1, F2r/Par1 and Slc7a6os) as well as three new stable EPI markers (Zp3, Mcf2 and Hexb), which are known to be late stage germ cell markers. We found that mouse PGC marker activation starts at least at E3.25 preimplantation. The transcriptomics dynamics analyses support the regulation of Dnmt3l expression by TCFAP2C. Since the regulation of Dnmt3l by TCFAP2C is based on computational prediction of DNA methylation motifs, Chip-Seq and transcriptomics data, functional studies are required to validate this result. We identified a collection of previously undescribed E3.5-specific PE and EPI markers, and new steady PE and EPI markers. Identification of these genes, many of which encode cell membrane proteins, will facilitate the isolation and characterization of early PE and EPI populations. Since it is so well established in the literature that mouse PGC specification is a post-implantation event, it was surprising for us to see activation of PGC markers as early as E3.25 preimplantation, and identify the newly found steady EPI markers as late germ cell markers. The discovery of such early activation of PGC markers has important implications in the derivation of germ cells from pluripotent cells (embryonic stem cells or induced pluripotent stem cells), since the initial stages of such derivation resemble early development. The early activation of PGC markers points out the difficulty of separating PGC cells from pluripotent populations. Collectively, our results suggest that the combining of the precision of single-cell omics data with dynamic analysis of time-series data can establish the timing of some developmental stages as earlier than previously thought. Not applicable. This work was supported by grants DFG15/14 and DFG15/020 from Diputación Foral de Gipuzkoa (Spain), and grant II14/00016 from I + D + I National Plan 2013-2016 (Spain) and FEDER funds. The authors declare no conflict of interest.

Cell fate specification begins in preimplantation embryo with the emergence of trophectoderm (TE). Primitive endoderm (PE) is the second extraembryonic lineage to form from cells within the inner cell mass (ICM) during embryogenesis. Ruminant conceptuses elongate dramatically prior to uterine attachment and extensive migration and proliferation of TE and PE is crucial for normal conceptus development and elongation. Work described here provides evidence that FGF2 and potentially other FGFs impact PE development during bovine embryonic development. Bovine blastocyst outgrowths were generated from in vitro-produced embryos using a feeder-layer free culture system containing a Matrigel-coated surface. Individually cultured bovine blastocysts were supplemented with FGF2 at the beginning of culture (day 8 post-IVF) and identification of PE-like cells was evaluated microscopically on days 13 and 15. In controls, only a single blastocyst outgrowth contained what appeared morphologically as PE cells (1.3% of all outgrowths). By contrast, 10.5±6.7%, 19.7±0.28% and 22.4±0.26% of outgrowths contained PE colonies on day 15 when embryos were exposed to 0.5, 5 and 50ng/ml FGF2, respectively. A subset of outgrowths (n=6 TE only and 6 PE-containing) were selected and cultured to day 20, then lineage-specific markers were evaluated using qRT-PCR. Transcripts for IFNT and CDX2 (TE markers) were present in TE outgrowths. Trace amounts of IFNT and CDX2 mRNA were detected in PE colonies (P<0.05) likely because residual TE cells still existed in some of these outgrowths. PE-containing outgrowths contained ample quantities of GATA4 and GATA6 mRNA (putative PE markers) whereas TE outgrowths contained no GATA4 and low amounts of GATA6 mRNA. None of the outgrowths contained NANOG mRNA whereas all TE and PE outgrowths contained OCT4 mRNA. Western blot analysis determined PE-containing outgrowths produced transferrin, a PE product. Moreover, immunoreactive GATA4 localized to nuclei of PE cells but was not detected in TE cells. By contrast, CDX2 localized to TE nuclei but was not detected in PE cells. Continued cultivation and selection for TE and PE yielded pure cell populations (n=6 of each). Differences in relative abundance of FGF receptors (FGFRs) and selective alternatively spliced receptor isoforms were evident between the cell types. Specifically, transcripts for FGFR2b and FGFR3 predominated in TE cells while FGFR1b represented the primary transcript in PE cells. TE and PE cells responded differently to FGF2 treatment. FGF2 supplementation did not affect the mitotic index of TE cells whereas the mitotic index of PE cells increased (P<0.05) following exposure to 5 or 50ng/ml FGF2 (35.1±0.09% and 34.7±0.11%, respectively versus 19.9±0.06% for controls). These observations indicate that the non-TE cells formed with blastocyst exposure to FGF2 are PE, and these cells respond differently to FGF2 than TE. Collectively, this work provides new insight into another vital activity for FGF2 and potentially other FGFs during early embryonic development in bovine. This project was supported by NRICGP number 2008-35203-19106 from the USDA-CSREES. (platform)

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.

Specification of the epiblast (EPI) and primitive endoderm (PE) in the mouse embryo involves fibroblast growth factor (FGF) signaling through the RAS/MAP kinase pathway. FGFR1 and FGFR2 are thought to mediate this signaling in the inner cell mass (ICM) of the mouse blastocyst and BMP signaling can also influence PE specification. In this study, we further explored the dynamics of FGFR2 expression through an enhanced green fluorescent protein (eGFP) reporter mouse line (FGFR2-eGFP). We observed that FGFR2-eGFP is present in the late 8-cell stage; however, it is absent or reduced in the ICM of early blastocysts. We then statistically correlated eGFP expression with PE and EPI markers GATA6 and NANOG, respectively. We detected that eGFP is weakly correlated with GATA6 in early blastocysts, but this correlation quickly increases as the blastocyst develops. The correlation between eGFP and NANOG decreases throughout blastocyst development. Treatment with FGF from the morula stage onwards did not affect FGFR2-eGFP presence in the ICM of early blastocysts; however, late blastocysts presented FGFR2-eGFP in all cells of the ICM. BMP treatment positively influenced FGFR2-eGFP expression and reduced the number of NANOG-positive cells in late blastocysts. In conclusion, FGFR2 is not strongly associated with PE precursors in the early blastocyst, but it is highly correlated with PE cells as blastocyst development progresses, consistent with the proposed role for FGFR2 in maintenance rather than initiating the PE lineage.

Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

Programmed Cell Death (Apoptosis) Differs in IVF Versus ICSI Blastocysts from Non-Human Primates

At the pre-implantation blastocyst stage of development, the mammalian embryo is composed of a unique collection of cells of which three major populations predominate. The outermost layer the trophectoderm (TE) gives rise to the placenta, which acts to sustain the developing fetus connecting it to the mother host. The next is a cluster of cells known as the inner cell mass (ICM) these cells are said to be pluripotent (Fig. 1). A third group of cells known as the primitive endoderm, surrounds the ICM cells at the epiblast stage. As development proceeds the ICM cells rapidly divide and eventually begin to differentiate forming the three embryonic germ layers (ectoderm, mesoderm and endoderm). Effectively these pluripotent ICM cells are the precursors of all adult tissues. As these pluripotent cells commit to a specific cellular lineage, they lose their pluripotency. Embryonic stem (ES) cells are euploid pluripotent cell lines isolated directly from cultured preimplantation embryos. The first stable ES cell lines were isolated by immunosurgery from the ICM of implantationdelayed, mouse blastocysts (Martin, 1981; Evans and Kaufman, 1981). Mouse ES cells are very closely related to early ICM cells in terms of their developmental potential (Beddington and Robertson, 1989). This chapter will focus on mouse ES cells (mES) unless otherwise stated. Three features characterize mES cells; 1. They are isolated directly from the embryo (Robertson, 1987). 2. They can colonize the germ line when introduced to the embryo. 3. They possess unrestricted proliferative potential (Suda et al., 1987). These features effectively mean that under appropriate conditions, a karyotype stable selfrenewing, pluripotent population of cells can be propagated indefinitely in vitro. mES cells have other characteristics, which prove useful when comparing embryo derived stem cells to their differentiated progenies. mES cells have a euploid (2n) chromosome complement, a feature that allows their participation in germ cell development and the formation of chimeras (Bradley et al., 1984; Evans, 1994). The functional demonstration of mES cell developmental potential through chimera formation is the definitive proof of the pluripotent nature of the cell population in question. Biomarkers are often used as indicators of the stem cell state due to the time consuming and technically more difficult nature of getting functional proof of stemness. Many of the common markers are transcription factors expressed in the ICM and mES cells and have been shown to have functional roles in selfrenewal and in the maintenance of pluripotency, in both isolated stem cells or the ICM. The

The number of trophectoderm (TE) and inner cell mass (ICM) cells was determined by complementmediated lysis and differential staining in rat embryos collected at different times during in vivo preimplantation development. At 90 h after fertilization, two groups of morulae were discriminated according to the presence or absence of detectable ICM cells, and the analysis of their total cell number indicated that acquisition of a permeability seal between TE cells begins at the 14-cell stage. On the other hand, our data confirmed that blastocoele formation occurs after the fourth cleavage division in the rat. The total cell number increased exponentially with time in blastocysts recovered between 90 h and 127 h but the cell kinetics of TE and ICM cells were different. The proportion of ICM cells consequently varied throughout blastocyst development, with a peak value for expanded blastocysts at 103 h. Finally, a linear-quadratic relationship was found between the numbers of TE and ICM cells when all the embryos with a detectable ICM were analysed together.

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

The morphology of the inner cell mass (ICM) cells and the proportion of dead ICM cells in frozen-thawed bovine preimplantation embryos were investigated by differential fluorochrome staining. Embryos at the blastocyst stage of development were frozen and thawed by two different techniques (three-step and one-step) in two different basic salt solutions (PBS and TCM 199) containing 1.36M glycerol. After thawing and glycerol removal, embryos were co-cultured in a cumulus cells monolayer in TCM 199 for 48 hr (morula) or 24 hr (blastocysts). Differential cell counts of the ICM and trophectoderm were then done using differential fluorochrome staining. Overall, there was no significant difference in the viability of embryos frozen in the two basic salt solutions. Low proportions of dead ICM cells were observed in embryos frozen at the morula stage in both PBS (19.1%) or TCM 199 (18.0%). However, blastocyst stage embryos frozen by the three-step technique had a higher (P < 0.05) proportion of dead ICM cells in TCM 199 (37.7%) than in PBS (18.2%). Blastocysts frozen by the one-step technique had a higher (P < 0.05) proportion of dead ICM cells (42.2%) than those frozen by the three-step technique (18.2%), regardless of basic salt solutions. Results indicate that freezing and thawing damages ICM cells in morphologically normal embryos and that the degree of damage depended on the basic salt solution and the freezing method.

Hox proteins are transcription factors known to be essential for embryo patterning. The detection of some Hox transcripts in oocytes and early embryos suggests that they could play a role before gastrulation. We previously demonstrated Hoxb9 expression in oocytes and from the zygote to the blastocyst stage in the mouse and the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436). The protein is present at all stages and in all cells with a strong nuclear staining in both species. The objective of this study was to perform an in-depth study at the blastocyst stage to compare the level of the nuclear protein between the inner cell mass (ICM) and the trophectoderm (TE) from the early to the expanded blastocyst stage. In vitro produced bovine blastocysts were collected at Day 6, Day 7.5, and Day 8 post-insemination. Hoxb9 proteins were detected by whole-mount immunofluorescence. TE nuclei were strongly stained at all stages while from D6 but especially from D7.5, the level of HOXB9 seemed to decrease in ICM nuclei with an increasing heterogeneity of staining between ICM nuclei. A light and apparently stable staining was also observed in the cytoplasm. Confocal images were quantified (Nis-element 3.1, Nikon). For each cell of TE or ICM, the ratio between the mean intensity of the nucleus and the mean intensity of the corresponding total cytoplasm was calculated. Whatever the stages, TE ratios were significantly (Mann–Whitney test; P &lt; 0.0001) higher than ICM ratios, suggesting that HOXB9 is present in higher amounts in TE than in ICM cells. This observation could be correlated with the reduced HOXB9 relative expression observed in blastocysts. Moreover, the proportion of blastocysts showing a reduction of HOXB9 staining in at least one nucleus significantly increased from Day 6 to Day 7.5 blastocysts and Day 8 blastocysts (from 26% to 74% or 85%, chi-squared test; P &lt; 0.001). Mouse zygotes, collected from superovulated mice, were cultured in vitro and embryos were collected 72 h, 80 h, 92 h and 100 h post-hCG injection. A similar nuclear staining was observed in all cells until 80 h post-hCG injection, while heterogeneity of staining appeared in ICM cells 92 h post-hCG, but especially in 100 h post-hCG embryos. The quantitative study was performed only on this latest stage and confirmed the stronger staining in TE than in ICM nuclei (Mann–Whitney test; P &lt; 0.0001) observed in the bovine. At this stage, 82% of blastocysts presented a reduced Hoxb9 staining in some or all ICM nuclei. In conclusion, Hoxb9 protein is detected in all blastocyst nuclei both in the mouse and in the bovine. However, the protein seems globally less abundant in the ICM than in the TE cells. Moreover, the percentage of bovine blastocysts showing a reduction in HOXB9 staining intensity in ICM nuclei increases with blastocyst expansion. These results suggest an involvement of Hoxb9 in cell lineage differentiation in mammals.C. S. holds a FRIA PhD grant from the FRS-FNRS (Belgium). This study is supported by the FRS-FNRS and by an Action de Recherche Concertée.

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Postthaw Viability of the Inner Cell Mass of in Vitro-Matured/in Vitro-Fertilized Bovine Embryos Frozen in Various Cryoprotectants

Live imaging endogenous transcription factor dynamics reveals mechanisms of epiblast and primitive endoderm fate segregation

An efficient method of inducing differentiation of mouse embryonic stem cells into primitive endodermal cells