Early Preimplantation Development Research Articles

Dyes illuminate live human embryogenesis

Assisted reproduction is on the rise globally. Cell morphology is commonly used for embryo selection, but the cell biology of early preimplantation development remains poorly understood. In this issue of Cell, Domingo-Muelas etal. reveal novel features of human embryos with critical implications for preimplantation genetic testing.

Involvement of linker histone variant H1a in the regulation of early preimplantation development in mice.

Linker histone variants regulate higher-order chromatin structure and various cellular processes. It has been suggested that linker histone variant H1a loosens chromatin structure and activates transcription. However, its role in early mouse development remains to be elucidated. We investigated the functions of H1a during preimplantation development using H1a gene-deleted mice. Although H1a homozygous knockout (KO) mice were born without any abnormalities, the number of offspring were reduced when the mothers but not fathers were homozygous KO animals. Maternal H1a KO compromised development during the morula and blastocyst stages, but not differentiation of the inner cell mass or trophectoderm. Thus, maternal linker histone H1a is important in early development.

The transcription factor ELF5 is essential for early preimplantation development.

During early embryonic development, the cell adhesion molecule E-cadherin encoded by the Cdh1 gene plays a vital role in providing proper cell-cell adhesion, ensuring an undifferentiated state critical for maintaining the pluripotency for the development of the preimplantation embryo. The transcriptional regulation of Cdh1 gained attention recently but is not yet fully understood. In a previous study, our team established a correlation between Elf3 and Cdh1 expression and showed its importance in the regulation of MET. Here, the regulation of Cdh1 by Ets transcription factors in early embryogenesis was investigated. A loss-of-function approach was used to study the effect of Elf5 loss on Cdh1 gene expression by small interfering RNAs in fertilized oocytes. Changes in gene expression were measured by qPCR analysis, and developing embryos were visualized by microscopy. Loss of Elf5 arrested the embryos at the 2-cell stage, accompanied by a significant downregulation of Cdh1 expression. The findings presented here illustrate the role of ELF5 in preimplantation development and in regulating the expression of Cdh1. The maintenance of the ELF5 and Cdh1 regulatory node proved essential for the proper development of the early mouse embryos, which is in agreement with the critical role of Elf5 and Cdh1 genes in regulating the early events during embryogenesis.

Viable offspring derived from single unfertilized mammalian oocytes

In mammals, a new life starts with the fusion of an oocyte and asperm cell. Parthenogenesis, a way of generating offspring solelyfrom female gametes, is limited because of problems arising fromgenomic imprinting. Here, we report live mammalian offspringderived from single unfertilized oocytes, which was achieved by tar-geted DNA methylation rewriting of seven imprinting control regions.Oocyte coinjection of catalytically inactive Cas9 (dCas9)-Dnmt3a ordCpf1-Tet1 messenger RNA (mRNA) with single-guide RNAs (sgRNAs)targeting specific regions induced de novo methylation or demethyla-tion, respectively, of the targeted region. Following parthenogeneticactivation, these edited regions showed maintenance of methylationas naturally established regions during early preimplantation develop-ment. The transfer of modified parthenogenetic embryos into fostermothers resulted in significantly extended development andfinally inthe generation of viable full-term offspring. These data demonstratethat parthenogenesis can be achieved by targeted epigenetic rewrit-ing of multiple critical imprinting control regions.

Oviduct epithelial cells-derived extracellular vesicles improve preimplantation developmental competence of in vitro produced porcine parthenogenetic and cloned embryos.

Extracellular vesicles (EVs) carry bioactive cargoes involved in the early preimplantation development. This study investigated the effects of EVs obtained from an oviductal epithelial cell (OEC) conditioned medium on the developmental competence of in parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) porcine embryos. The OEC-EV-treated group showed significant increases in blastocyst formation and hatching rates compared to the control group (40.8% ± 2.2% and 20.1% ± 2.1% vs. 24.9% ± 2.0% and 5.3% ± 1.1%; p < 0.05), respectively. The 7 day OEC-EVs treatment group significantly increased blastocyst formation rate than the 3 day and 0 day-groups (45.0 ± 0.8 vs. 33.0 ± 0.7 and 26.7 ± 0.5; p < 0.05), respectively. SCNT revealed that the OEC-EV increased blastocyst formation rate compared to that of oviductal fluid EVs (OF-EVs) (35.4% ± 1.4% vs. 29.3% ± 1.3%; p < 0.05). Reactive oxygen species levels, apoptosis, and blastocyst lipid content were significantly decreased in the OEC-EVs group compared with the control group. OEC-EV group showed a significantly decreased BAX and increased BCL2, SOD1, POU5F1, SOX2, NANOG, GATA6, PNPLA2, LIPE, and MGLL gene expression than the control group (p < 0.05). In conclusion, OEC-EVs supplementation in embryo culture media improved the quality of porcine embryos, potentially helping porcine-cloned embryonic development possibly through transfer of messenger RNA and proteins to the early embryos.

Higher preconceptional maternal body mass index is associated with faster early preimplantation embryonic development: the Rotterdam periconception cohort

BackgroundOverweight and obesity affect millions of people globally, which has also serious implications for reproduction. For example, treatment outcomes after in vitro fertilisation (IVF) are worse in women with a high body mass index (BMI). However, the impact of maternal BMI on embryo quality is inconclusive. Our main aim is to study associations between preconceptional maternal BMI and morphokinetic parameters of preimplantation embryos and predicted implantation potential. In addition, associations with clinical IVF outcomes are investigated.MethodsFrom a tertiary hospital, 268 women undergoing IVF or IVF with intracytoplasmic sperm injection (ICSI) were included; 143 normal weight, 79 overweight and 46 obese women. The embryos of these women were cultured in the EmbryoScope, a time-lapse incubator. The morphokinetic parameters of preimplantation embryos and predicted implantation potential, assessed by the KIDScore algorithm were longitudinally evaluated as primary and secondary outcomes, respectively. The tertiary outcomes included clinical outcomes, i.e., fertilization, implantation and live birth rate.ResultsAfter adjustment for patient- and treatment-related factors, we demonstrated in 938 embryos that maternal BMI is negatively associated with the moment of pronuclear appearance (βtPNa -0.070 h (95%CI -0.139, -0.001), p = 0.048), pronuclear fading (βtPNf -0.091 h (95%CI -0.180, -0.003), p = 0.043 and the first cell cleavage (βt2 -0.111 h (95%CI -0.205, -0.016), p = 0.022). Maternal BMI was not significantly associated with the KIDScore and tertiary clinical treatment outcomes. In embryos from couples with female or combined factor subfertility, the impact of maternal BMI was even larger (βtPNf -0.170 h (95%CI -0.293, -0.047), p = 0.007; βt2 -0.199 h (95%CI -0.330, -0.067), p = 0.003). Additionally, a detrimental impact of BMI per point increase was observed on the KIDScore (β -0.073 (se 0.028), p = 0.010).ConclusionsHigher maternal BMI is associated with faster early preimplantation development. In couples with female or combined factor subfertility, a higher BMI is associated with a lower implantation potential as predicted by the KIDScore. Likely due to power issues, we did not observe an impact on clinical treatment outcomes. However, an effect of faster preimplantation development on post-implantation development is conceivable, especially since the impact of maternal BMI on pregnancy outcomes has been widely demonstrated.

The Functional Role of Platelet-Activating Factor in Spermatozoa Physiology

Platelet-activating factor (alkylacetylglycerolphosphocholine; PAF) is a potent signaling phospholipid which has been found in numerous cell types in every physiological system studied to date. In reproduction, PAF is found to have a variety of roles, for example: in ovulation, sperm function, and early preimplantation development. The goal of this mini review is to highlight PAF’s synthesis, mechanism of action and its functional role in sperm physiology. PAF functions via a G protein coupled receptor mediated pathway, which ultimately increases intracellular calcium levels to enhance sperm motility required for fertilization. Exogenous PAF was also found to increase fertilization potential of spermatozoa in cases of non-male factor infertility. Finally, the mini review explores various lifestyle factors that could potentially affect PAF levels and fertility.

Downregulation of gene expression and the outcome of ICSI in severe oligozoospermic patients: A preliminary study.

Preimplantation embryo development might be influenced by a specific set of transcripts that are delivered to the oocyte by the sperm. The aim of the study was to determine the relationship between the level of selected transcripts in spermatozoa and preimplantation development of the embryos in couples with severe oligozoospermia undergoing intracytoplasmic sperm injection (ICSI) procedure. Therefore, we assessed messenger RNA (mRNA) levels of genes involved in fertilization events, oocyte activation, chromatin remodeling, and DNA repair in severe oligozoospermic compared with normozoospermic men as well as morphokinetic parameters of embryos using the time-lapse imaging system. mRNA profiling (44 genes), in mature sperm, was carried out with custom-designed 384-well TLDA Cards. The morphokinetic parameters of zygotes and embryos were recorded by using a time-lapse imaging system. The transcript levels of 21 genes were significantly decreased in the severe oligozoospermic group. Most were associated with fertilization events, oocyte activation and embryonic genome activation. Among them, mRNA of AKAP4 and PTK7 was greatly reduced, moreover, the transcripts of PLCζ and POU5F1, essential for OA and EGA, were not detected at all in patients with severe oligozoospermia. Moreover, the reduced expression of genes important for spermatogenesis, chromatin remodeling and DNA repair was also observed in this group. Time-lapse analysis revealed that fertilization failure occurred in 14% of retrieved oocytes and 90% of all degenerated embryos did not reach morula stage. This study provides preliminary results indicating a significant decrease in transcripts of genes important for spermatogenesis and early preimplantation development in the mature sperm of men with severe oligozoospermia.

Sperm Originated Chromatin Imprints and LincRNAs in Organismal Development and Cancer.

SummaryImportance of sperm-derived transcripts and chromatin imprints in organismal development is poorly investigated. Here using an integrative approach, we show that human sperm transcripts are equally important as oocyte. Sperm-specific and sperm-oocyte common transcripts carry distinct chromatin structures at their promoters correlating with corresponding transcript levels in sperm. Interestingly, sperm-specific H3K4me3 patterns at the lincRNA promoters are not maintained in the germ layers and somatic tissues. However, bivalent chromatin at the sperm-specific protein-coding gene promoters is maintained throughout the development. Sperm-specific transcripts reach their peak expression during zygotic genome activation, whereas sperm-oocyte common transcripts are present during early preimplantation development but decline at the onset of zygotic genome activation. Additionally, there is an inverse correlation between sperm-specific and sperm-oocyte lincRNAs throughout the development. Sperm-lincRNAs also show aberrant activation in tumors. Overall, our observations indicate that sperm transcripts carrying chromatin imprints may play an important role in human development and cancer.

Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development.

The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b. Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR. We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group. These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

DNAJC2 is required for mouse early embryonic development

DNAJC2 protein, also known as ZRF1 or MPP11, acts both as chaperone and as chromatin regulator. It is involved in stem cell differentiation and its expression is associated with various cancer malignancies. However, the role of Dnajc2 gene during mouse embryogenesis has not been assessed so far. To this aim, we invalidated Dnajc2 gene in FVB/Nj mice using the CrispR/Cas9 approach. We showed that this invalidation leads to the early post-implantation lethality of the nullizygous embryos. Furthermore, using siRNAs against Dnajc2 in mouse 1-cell embryos, we showed that maternal Dnajc2 mRNAs may allow for the early preimplantation development of these embryos. Altogether, these data demonstrate for the first time the requirement of DNAJC2 for early mouse embryogenesis.

Inhibition of Skp1-Cullin-F-box complexes during bovine oocyte maturation and preimplantation development leads to delayed development of embryos†

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

KDM5B is a master regulator of the H3K4-methylome in stem cells, development and cancer.

Epigenetic regulation of chromatin plays a critical role in controlling stem cell function and tumorigenesis. The histone lysine demethylase, KDM5B, which catalyzes the demethylation of histone 3 lysine 4 (H3K4), is important for embryonic stem (ES) cell differentiation, and is a critical regulator of the H3K4-methylome during early mouse embryonic pre-implantation stage development. KDM5B is also overexpressed, amplified, or mutated in many cancer types. In cancer cells, KDM5B regulates expression of oncogenes and tumor suppressors by modulating H3K4 methylation levels. In this review, we examine how KDM5B regulates gene expression and cellular fates of stem cells and cancer cells by temporally and spatially controlling H3K4 methylation levels.

Characterization of transcriptional complexity during pre‐implantation development of the yak (Bos grunniens) using RNA‐Seq

The objective of this study was to investigate the mechanism that regulates pre-implantation development of the yak (Bos grunniens). We determined the transcriptomes of in vitro-produced yak embryos at two-cell, four-cell, eight-cell stages, and morula and blastocyst using the Illumina RNA-seq for the first time. We obtained 47.36-50.86million clean reads for each stage, of which, 85.65%-90.02% reads were covered in the reference genome. A total of 17,368 genes were expressed during the two-cell stage to blastocyst of the yak, of which 7,236 genes were co-expressed at all stages, whereas 10,132 genes were stage-specific expression. Transcripts from 9,827 to 14,893 different genes were detected in various developmental stages. When |log2 ratio|≥1 and q-value <0.05 were set as thresholds for identifying differentially expressed genes (DEGs), we detected a total of 6,922-10,555 DEGs between any two consecutive stages. The GO distributions of these DEGs were classified into three categories: biological processes (23 terms), cellular components (22 terms) and molecular functions (22 terms). Pathway analysis revealed 310 pathways of the DEGs that were operative in early pre-implantation yak development, of which 32 were the significantly enriched pathways. In conclusion, this is the first report to investigate the mechanism that regulates yak embryonic development using high-throughput sequencing, which provides a comprehensive framework of transcriptome landscapes of yak pre-implantation embryos.

26 Drugs that Modify Epigenetics...What do they do to Porcine Clones?

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. In this study, we assessed whether the use the inhibitor of DNA (cytosine 5) methyltransferase 5-azacitidine (5Aza) combined with the MEK inhibitor in the MAPK pathway PD0325901 (PD) could improve SCNT efficiency in pigs. In vitro maturation of cumulus–oocyte complexes was performed in TCM for 44 h at 39°C under 5% CO2. Cumulus cells and zona pellucida was removed from matured oocytes, followed by enucleation of the metaphase plate previously stained with Hoëchst 33342. Each enucleated oocyte was attached to a donor cell by phytohemagglutinin treatment followed by an electric pulse of 80V for 30 μs. After fusion, reconstituted embryos were activated by an electric pulse followed by an incubation in 2 mM 6-DMAP for 3 h. Cloned embryos were cultured in vitro in a modified well-of-well system in SOF medium, where 3 cloned embryos were placed per microwell (3X). The experimental group 3X + drugs was exposed for the first 3 days to 1 μM PD and 1 μM 5Aza in SOF medium. After washing, embryos were cultured until Day 7 in regular SOF medium. The control group (3X) was cultured in regular SOF medium for 7 days. In vitro embryo developmental rates, gene expression, histone acetylation, and DNA methylation status were studied. The use of epigenetic modifying drugs significantly increased blastocyst rates (40.9% v. 29%; Fisher’s test, P &lt; 0.05) and embryo size (41.46% v. 28.56%; Student’s t-test, P &lt; 0.05) compared with the control group. Regarding gene expression, an increase of the relative expression of genes related to cell differentiation (Igf2 and Cdx2), antiapoptotic pathways (Bcl-xl) and DNA methylation modulation (Mapk1) was observed (P &lt; 0.05). Pluripotency genes Oct4 and Nanog did not show differences between groups. The Bax proapoptotic gene significantly decreased its expression after drug treatment, as did the Klf4 gene (P &lt; 0.05). Results were analysed by Student’s t-test. According to Histone H3K27ac, which is associated with enhancers or gene promoters, its marker was located mainly in the nuclear periphery respect to the control group with a uniform dispersion, indicating that the treatment could be activating certain genes by locating them near the periphery. Histone H3K4me1 was more uniformly localised throughout the nucleus in both groups. The intensity of the fluorescence was measured by quantitative confocal microscopy using a histogram produced by the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Regarding DNA methylation by bisulphite sequencing, the 2 genes studied (Oct4 and DNMT1) showed a higher demethylation status for the treated group. Our results indicate that the combination of 5Aza+PD during early pre-implantation development dramatically increase blastocyst rates and embryo quality. This novel combination could be used as a strategy to improve the efficiency of SCNT in pigs and potentially other animals.

Berberine impairs embryonic development in vitro and in vivo through oxidative stress-mediated apoptotic processes.

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H2 O2 -induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine-triggering or preventing apoptosis-has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5-10 μM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine-induced injury of mouse blastocysts appeared to be attributable to oxidative stress-triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.

Causes and consequences of chromosome segregation error in preimplantation embryos.

Errors in chromosome segregation are common during the mitotic divisions of preimplantation development in mammalian embryos, giving rise to so-called 'mosaic' embryos possessing a mixture of euploid and aneuploid cells. Mosaicism is widely considered to be detrimental to embryo quality and is frequently used as criteria to select embryos for transfer in human fertility clinics. However, despite the clear clinical importance, the underlying defects in cell division that result in mosaic aneuploidy remain elusive. In this review, we summarise recent findings from clinical and animal model studies that provide new insights into the fundamental mechanisms of chromosome segregation in the highly unusual cellular environment of early preimplantation development and consider recent clues as to why errors should commonly occur in this setting. We furthermore discuss recent evidence suggesting that mosaicism is not an irrevocable barrier to a healthy pregnancy. Understanding the causes and biological impacts of mosaic aneuploidy will be pivotal in the development and fine-tuning of clinical embryo selection methods.

NLRPs, the subcortical maternal complex and genomic imprinting

Before activation of the embryonic genome, the oocyte provides many of the RNAs and proteins required for the epigenetic reprogramming and the transition to a totipotent state. Targeted disruption of a subset of oocyte-derived transcripts in mice results in early embryonic lethality and cleavage-stage embryonic arrest as highlighted by the members of the subcortical maternal complex (SCMC). Maternal-effect recessive mutations of NLRP7, KHDC3L and NLRP5 in humans are associated with variable reproductive outcomes, biparental hydatidiform moles (BiHM) and widespread multi-locus imprinting disturbances. The precise mechanism of action of these genes is unknown, but the maternal-effect phenomenon suggests a function during early pre-implantation development, while biochemical and genetic studies implement them as SCMC members or interacting partners. In this review article, we discuss the role of the NLRP family members and the SCMC proteins in the establishment of genomic imprints and post-zygotic methylation maintenance, the recent advances made in the understanding of the biology involved in BiHM formation and the wider roles of the SCMC in mammalian reproduction.

Dynamic silencing of somatic L1 retrotransposon insertions reflects the developmental and cellular contexts of their genomic integration

BackgroundThe ongoing mobilization of mammalian transposable elements (TEs) contributes to natural genetic variation. To survey the epigenetic control and expression of reporter genes inserted by L1 retrotransposition in diverse cellular and genomic contexts, we engineered highly sensitive, real-time L1 retrotransposon reporter constructs.ResultsHere we describe different patterns of expression and epigenetic controls of newly inserted sequences retrotransposed by L1 in various somatic cells and tissues including cultured human cancer cells, mouse embryonic stem cells, and tissues of pseudofounder transgenic mice and their progeny. In cancer cell lines, the newly inserted sequences typically underwent rapid transcriptional gene silencing, but they lacked cytosine methylation even after many cell divisions. L1 reporter expression was reversible and oscillated frequently. Silenced or variegated reporter expression was strongly and uniformly reactivated by treatment with inhibitors of histone deacetylation, revealing the mechanism for their silencing. By contrast, de novo integrants retrotransposed by L1 in pluripotent mouse embryonic stem (ES) cells underwent rapid silencing by dense cytosine methylation. Similarly, de novo cytosine methylation also was identified at new integrants when studied in several distinct somatic tissues of adult founder mice. Pre-existing L1 elements in cultured human cancer cells were stably silenced by dense cytosine methylation, whereas their transcription modestly increased when cytosine methylation was experimentally reduced in cells lacking DNA methyltransferases DNMT1 and DNMT3b. As a control, reporter genes mobilized by piggyBac (PB), a DNA transposon, revealed relatively stable and robust expression without apparent silencing in both cultured cancer cells and ES cells.ConclusionsWe hypothesize that the de novo methylation marks at newly inserted sequences retrotransposed by L1 in early pre-implantation development are maintained or re-established in adult somatic tissues. By contrast, histone deacetylation reversibly silences L1 reporter insertions that had mobilized at later timepoints in somatic development and differentiation, e.g., in cancer cell lines. We conclude that the cellular contexts of L1 retrotransposition can determine expression or silencing of newly integrated sequences. We propose a model whereby reporter expression from somatic TE insertions reflects the timing, molecular mechanism, epigenetic controls and the genomic, cellular and developmental contexts of their integration.

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo.

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune-stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside-Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 μM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1-treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 μM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst-stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress-mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl-2 ratio, and activates caspase-9 and caspase-3, but not caspase-8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.