The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos.

At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos.

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

The impact of ovarian stimulation for IVF on the developing embryo.

The use of assisted reproductive technologies (ART) has been increasing over the past three decades, and, in developed countries, ART account for 1-3% of annual births. In an attempt to compensate for inefficiencies in IVF procedures, patients undergo ovarian stimulation using high doses of exogenous gonadotrophins to allow retrieval of multiple oocytes in a single cycle. Although ovarian stimulation has an important role in ART, it may also have detrimental effects on oogenesis, embryo quality, endometrial receptivity and perinatal outcomes. In this review, we consider the evidence for these effects and address possible underlying mechanisms. We conclude that such mechanisms are still poorly understood, and further knowledge is needed in order to increase the safety of ovarian stimulation and to reduce potential effects on embryo development and implantation, which will ultimately be translated into increased pregnancy rates and healthy offspring.

Sperm DNA damage causes genomic instability in early embryonic development.

Genomic instability is common in human embryos, but the underlying causes are largely unknown. Here, we examined the consequences of sperm DNA damage on the embryonic genome by single-cell whole-genome sequencing of individual blastomeres from bovine embryos produced with sperm damaged by γ-radiation. Sperm DNA damage primarily leads to fragmentation of the paternal chromosomes followed by random distribution of the chromosomal fragments over the two sister cells in the first cell division. An unexpected secondary effect of sperm DNA damage is the induction of direct unequal cleavages, which include the poorly understood heterogoneic cell divisions. As a result, chaotic mosaicism is common in embryos derived from fertilizations with damaged sperm. The mosaic aneuploidies, uniparental disomies, and de novo structural variation induced by sperm DNA damage may compromise fertility and lead to rare congenital disorders when embryos escape developmental arrest.

The effects of growth factors on in vitro-cultured porcine testicular cells.

Cell lines from neonate porcine testis were cultured and characterized and the effect of growth factors were investigated, in order to determine the requirements for the establishment of porcine male germ cell lines. In primary cultures, three different colony types with distinctive morphologies could be recognized. From colonies resembling mouse spermatogonial stem cells (SSCs), two cell lines were derived and maintained for nine passages after which proliferation stopped. Growth of these cell lines depended on the growth factors leukemia inhibitory factor (LIF), epidermal growth factor (EGF), glial derived neurotrophic factor (GDNF), and fibroblast growth factor (FGF). In both cell lines NANOG, promyelocytic leukemia zinc-finger (PLZF), and EPCAM, were expressed at higher levels and GFRA1, ITGA6, and THY1 at lower levels than in neonate porcine testis. Primary cultures of neonate pig testis were subjected to a factorial design of the growth factors LIF, GDNF, EGF, and FGF. EGF and FGF had a positive effect on the number and size of the SSC-like colonies. Addition of EGF and FGF to primary cell cultures of neonate pig testis affected the expression of NANOG, PLZF, POU5F1, and GATA4, whereas effects of LIF or GDNF could not be detected. FGF decreased the expression levels of NANOG, a marker for pluripotency also expressed in neonatal porcine male germ cells. FGF decreased expression of PLZF and enhanced the expression of pluripotency-related gene POU5F1 and Sertoli cell marker GATA4. EGF had a positive effect on PLZF expression levels and counteracted the positive effect of FGF on GATA4 expression. These results suggest that FGF can impede successful derivation of porcine SSCs from neonate pig testis.

Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos.

BACKGROUND: In the developing embryo, total RNA abundance fluctuates caused by functional RNA degradation and zygotic genome activation. These variations in the transcriptome in early development complicate the choice of good reference genes for gene expression studies by quantitative real time polymerase chain reaction. RESULTS: In order to identify stably expressed genes for normalisation of quantitative data, within early stages of development, transcription levels were examined of 7 frequently used reference genes (B2M, BACT, GAPDH, H2A, PGK1, SI8, and UBC) at different stages of early porcine embryonic development (germinal vesicle, metaphase-2, 2-cell, 4-cell, early blastocyst, expanded blastocyst). Analysis of transcription profiling by geNorm software revealed that GAPDH, PGK1, S18, and UBC showed high stability in early porcine embryonic development, while transcription levels of B2M, BACT, and H2A were highly regulated. CONCLUSION: Good reference genes that reflect total RNA content were identified in early embryonic development from oocyte to blastocyst. A selection of either GAPDH or PGK1, together with ribosomal protein S18 (S18), and UBC is proposed as reference genes, but the use of B2M, BACT, or H2A is discouraged.

Molecular dissection of germline chromothripsis in a developmental context using patient-derived iPS cells.

BACKGROUND: Germline chromothripsis causes complex genomic rearrangements that are likely to affect multiple genes and their regulatory contexts. The contribution of individual rearrangements and affected genes to the phenotypes of patients with complex germline genomic rearrangements is generally unknown. METHODS: To dissect the impact of germline chromothripsis in a relevant developmental context, we performed trio-based RNA expression analysis on blood cells, induced pluripotent stem cells (iPSCs), and iPSC-derived neuronal cells from a patient with de novo germline chromothripsis and both healthy parents. In addition, Hi-C and 4C-seq experiments were performed to determine the effects of the genomic rearrangements on transcription regulation of genes in the proximity of the breakpoint junctions. RESULTS: Sixty-seven genes are located within 1 Mb of the complex chromothripsis rearrangements involving 17 breakpoints on four chromosomes. We find that three of these genes (FOXP1, DPYD, and TWIST1) are both associated with developmental disorders and differentially expressed in the patient. Interestingly, the effect on TWIST1 expression was exclusively detectable in the patient's iPSC-derived neuronal cells, stressing the need for studying developmental disorders in the biologically relevant context. Chromosome conformation capture analyses show that TWIST1 lost genomic interactions with several enhancers due to the chromothripsis event, which likely led to deregulation of TWIST1 expression and contributed to the patient's craniosynostosis phenotype. CONCLUSIONS: We demonstrate that a combination of patient-derived iPSC differentiation and trio-based molecular profiling is a powerful approach to improve the interpretation of pathogenic complex genomic rearrangements. Here we have applied this approach to identify misexpression of TWIST1, FOXP1, and DPYD as key contributors to the complex congenital phenotype resulting from germline chromothripsis rearrangements.

Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure.

The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah(-/-) Il2rg(-/-) rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat.

Genomic and functional overlap between somatic and germline chromosomal rearrangements.

Genomic rearrangements are a common cause of human congenital abnormalities. However, their origin and consequences are poorly understood. We performed molecular analysis of two patients with congenital disease who carried de novo genomic rearrangements. We found that the rearrangements in both patients hit genes that are recurrently rearranged in cancer (ETV1, FOXP1, and microRNA cluster C19MC) and drive formation of fusion genes similar to those described in cancer. Subsequent analysis of a large set of 552 de novo germline genomic rearrangements underlying congenital disorders revealed enrichment for genes rearranged in cancer and overlap with somatic cancer breakpoints. Breakpoints of common (inherited) germline structural variations also overlap with cancer breakpoints but are depleted for cancer genes. We propose that the same genomic positions are prone to genomic rearrangements in germline and soma but that timing and context of breakage determines whether developmental defects or cancer are promoted.

Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation.

Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.

The different shades of mammalian pluripotent stem cells.

BACKGROUND: Pluripotent stem cells have been derived from a variety of sources such as from the inner cell mass of preimplantation embryos, from primordial germ cells, from teratocarcinomas and from male germ cells. The recent development of induced pluripotent stem cells demonstrates that somatic cells can be reprogrammed to a pluripotent state in vitro. METHODS: This review summarizes our current understanding of the origins of mouse and human pluripotent cells. We pay specific attention to transcriptional and epigenetic regulation in pluripotent cells and germ cells. Furthermore, we discuss developmental aspects in the germline that seem to be of importance for the transition of germ cells towards pluripotency. This review is based on literature from the Pubmed database, using Boolean search statements with relevant keywords on the subject. RESULTS: There are distinct molecular mechanisms involved in the generation and maintenance of the various pluripotent cell types. Furthermore, there are important similarities and differences between the different categories of pluripotent cells in terms of phenotype and epigenetic modifications. Pluripotent cell lines from various origins differ in growth characteristics, developmental potential, transcriptional activity and epigenetic regulation. Upon derivation, pluripotent stem cells generally acquire new properties, but they often also retain a 'footprint' of their tissue of origin. CONCLUSIONS: In order to further our knowledge of the mechanisms underlying self-renewal and pluripotency, a thorough comparison between different pluripotent stem cell types is required. This will progress the use of stem cells in basic biology, drug discovery and future clinical applications.

PTEN and TRP53 independently suppress Nanog expression in spermatogonial stem cells.

Mammalian spermatogonial stem cells are a special type of adult stem cells because they can contribute to the next generation. Knockout studies have indicated a role for TRP53 and PTEN in insulating male germ cells from pluripotency, but the mechanism by which this is achieved is largely unknown. To get more insight in these processes, an RNAi experiment was performed on the mouse spermatogonial stem cell line GSDG1. Lipofectaminemediated transfection of siRNAs directed against Trp53 and Pten resulted in decreased expression levels as determined by quantitative RT-PCR and immunoblotting. The effects of knockdown were examined by determining the expression levels of genes that are involved in reprogramming and pluripotency of cells, specifically Nanog, Eras, c-Myc, Klf4, Oct4, and Sox2. Additionally, the effects of TRP53 or PTEN knockdown on Plzf and Ddx4 expression were measured, which are highly expressed in spermatogonial stem cells and differentiating male germ cells, respectively. The main finding of this study is that knockdown of Trp53 and Pten independently resulted in significantly higher expression levels of the pluripotency-associated gene Nanog, and we hypothesize that TRP53 and PTEN mediated repression is important for the insulation of male germ cells from pluripotency.

A distinct expression pattern in mammalian testes indicates a conserved role for NANOG in spermatogenesis.

BACKGROUND: NANOG is a key player in pluripotency and its expression is restricted to pluripotent cells of the inner cell mass, the epiblast and to primordial germ cells. Spermatogenesis is closely associated with pluripotency, because through this process highly specialized sperm cells are produced that contribute to the formation of totipotent zygotes. Nevertheless, it is unknown if NANOG plays a role in this process. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, NANOG expression was examined in testes of various mammals, including mouse and human. Nanog mRNA and NANOG protein were detected by RT-PCR, immunohistochemistry, and western blotting. Furthermore, eGFP expression was detected in the testis of a transgenic Nanog eGFP-reporter mouse. Surprisingly, although NANOG expression has previously been associated with undifferentiated cells with stem cell potential, expression in the testis was observed in pachytene spermatocytes and in the first steps of haploid germ cell maturation (spermiogenesis). Weak expression in type A spermatogonia was also observed. CONCLUSIONS: The findings of the current study strongly suggest a conserved role for NANOG in meiotic and post-meiotic stages of male germ cell development.

Natural selection of human embryos: decidualizing endometrial stromal cells serve as sensors of embryo quality upon implantation.

BACKGROUND: Pregnancy is widely viewed as dependent upon an intimate dialogue, mediated by locally secreted factors between a developmentally competent embryo and a receptive endometrium. Reproductive success in humans is however limited, largely because of the high prevalence of chromosomally abnormal preimplantation embryos. Moreover, the transient period of endometrial receptivity in humans uniquely coincides with differentiation of endometrial stromal cells (ESCs) into highly specialized decidual cells, which in the absence of pregnancy invariably triggers menstruation. The role of cyclic decidualization of the endometrium in the implantation process and the nature of the decidual cytokines and growth factors that mediate the crosstalk with the embryo are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We employed a human co-culture model, consisting of decidualizing ESCs and single hatched blastocysts, to identify the soluble factors involved in implantation. Over the 3-day co-culture period, approximately 75% of embryos arrested whereas the remainder showed normal development. The levels of 14 implantation factors secreted by the stromal cells were determined by multiplex immunoassay. Surprisingly, the presence of a developing embryo had no significant effect on decidual secretions, apart from a modest reduction in IL-5 levels. In contrast, arresting embryos triggered a strong response, characterized by selective inhibition of IL-1beta, -6, -10, -17, -18, eotaxin, and HB-EGF secretion. Co-cultures were repeated with undifferentiated ESCs but none of the secreted cytokines were affected by the presence of a developing or arresting embryo. CONCLUSIONS: Human ESCs become biosensors of embryo quality upon differentiation into decidual cells. In view of the high incidence of gross chromosomal errors in human preimplantation embryos, cyclic decidualization followed by menstrual shedding may represent a mechanism of natural embryo selection that limits maternal investment in developmentally impaired pregnancies.

Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered "superfertile", defined by a mean TTP of 3 months or less. CONCLUSIONS: Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Differences in early lineage segregation between mammals.

Two lineage segregation events in mammalian development form the trophectoderm, primitive endoderm, and pluripotent primitive ectoderm. In mouse embryos, Oct4, Cdx2, Nanog, and Gata6 govern these events, but it is unknown whether this is conserved between mammals. Here, the expression patterns of these genes and their products were determined in porcine oocytes and embryos and in bovine embryos. CDX2 and GATA6 expression in porcine and bovine blastocysts resembled that of mouse, indicating conserved functions. However, NANOG expression was undetectable in porcine oocytes and embryos. Some inner cell mass cells in bovine blastocysts expressed NANOG protein. OCT4 protein was undetectable in porcine morulae, but present in both the trophectoderm and the inner cell mass of blastocysts, suggesting that downregulation of OCT4 in the trophectoderm does not precede trophectoderm formation. Combined, the results indicate differences in lineage segregation between mammals.