Expression In Preimplantation Embryos Research Articles

P-187 Profiling secreted proteins in human embryos: Time-specific discrimination of ploidy and confirmation of enhanced post-implantation development with maternal co-culture

Abstract Study question Can proteins released by embryos into culture medium between 7- and 14-days post-fertilization (d.p.f.) potentially function as biomarkers indicating the ploidy status of the embryos? Summary answer Combined secreted levels of calreticulin (CALR), human Placental Lactogen (hPL), Placental Growth Factor (PlGF) and human Pappalysin-1 (PAPP-A) differentiate euploid from chromosome 21-aneuploid embryos. What is known already Exploring post-implantation embryo development is vital due to potential errors impacting early pregnancy or postnatal health during this period. Trisomy 21 (T21) is a common aneuploidy that can lead to live births but with postnatal health concerns, while chromosome 21 monosomy (M21) often results in embryo developmental arrest. Understanding post-implantation development and secreted products into human post-implantation niche is crucial for unraveling embryo-maternal interactions. Building on prior studies indicating ploidy-related gene and metabolite expression in pre-implantation embryos (DOI:10.1016/j.fertnstert.2019.01.023), this study aims to assess variations in the secretome of embryos with different ploidy during post-implantation development. Study design, size, duration Comparative study involving 48 science-donated human 6 d.p.f. blastocysts initiating hatching coming from in-vitro fertilization cycles. Preimplantation genetic testing identified Euploid (n = 20), pure T21 (n = 14), and pure M21 (n = 14) embryos. Two extended in-vitro culture protocols were compared: co-culturing of human embryos in a 3D system with endometrial cells or in a culture medium designed for post-implantation development but lacking maternal cells. Participants/materials, setting, methods Euploid, T21 and M21 blastocysts were thawed and cultured in-vitro until 14 d.p.f. in both culture systems described above. Levels of CALR, hPL, PlGF and PAPP-A in conditioned embryo media were quantified by ELISA immunoassays at 7, 10, 12 and 14 d.p.f. Absolute secreted protein concentrations (ng/mL) in each case were compared by two-way ANOVA followed by Tukey’s test for multiple comparisons. P < 0.05 was consider as statistically significant. Main results and the role of chance In both culture systems tested, at 7 d.p.f. secreted CALR, hPL and PlGF by M21 embryos were diminished when compared to euploid embryos (p < 0.01). At 10 d.p.f., euploid embryos displayed a significant increase in secreted PAPP-A, surpassing levels in T21 (p < 0.05) and M21 embryos (p < 0.01). Embryo co-culture in 3D endometrial systems led to progressive increase in secreted PAPP-A and PlGF by euploid embryos at 14 d.p.f. compared to earlier timepoints (7 d.p.f.,p<0.01; 10d.p.f.,p<0.05). PAPP-A secretion did not increase in T21 embryos during extended culture. T21 embryos demonstrated decreased PAPP-A secretion at 10, 12, and 14 d.p.f. compared to euploid embryos (p < 0.01 in all cases). Secreted PlGF in T21 embryos rose significantly at 12 (p < 0.05) and 14 d.p.f. (p < 0.05), despite consistently lower levels than euploid embryos (p < 0.01). All studied proteins showed significantly diminished secretions during post-implantation development in M21 embryos compared to euploid ones. Comparing culture systems, the absence of maternal cells led to a significant decrease in secreted protein levels and viability impairment beyond 11 d.p.f. Consequently, only two timepoints (7- 10d.p.f.) were considered for analysis in this culture system. The findings underscore the potential of these secreted biomarkers for distinguishing embryo ploidy, with maternal co-culture supporting enhanced developmental outcomes. Limitations, reasons for caution This is an in-vitro pilot study that should be validated in larger cohorts. Wider implications of the findings Secretions of CALR, hPL, and PlGF at 7 d.p.f. effectively distinguish M21 embryos, while PAPP-A and PlGF secretions at 10-12 d.p.f. are indicative of embryo ploidy. Additionally, co-culturing with maternal cells notably promotes proper embryo development, particularly enhancing trophoblast differentiation and secretion of trophoblast-related proteins. Funding: ISCII(PI20/00405;PI23/00860;FI18/00009;MV21/00086); GVA (CIAICO/2022/203;CIAPOT/2022/18) and IDI-20210916 Trial registration number Not Applicable

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells

BackgroundGenome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs.ResultsWe show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine.ConclusionsThe results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Single-cell analysis of isoform switching and transposable element expression during preimplantation embryonic development.

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.

Glufosinate-Ammonium Induced Aberrant Histone Modifications in Mouse Sperm Are Concordant With Transcriptome in Preimplantation Embryos.

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its male reproductive toxicity. Abnormalities in sperm histone modification induced by GLA exposure observed in our previous study aroused our interest in whether such alterations could further affect embryonic gene expression. Here we administered adult male mice with 0.2 mg/kg⋅day of GLA for 5 weeks to collect their sperm or 4-cell embryos after copulation. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing showed alterations of sperm H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac), which are active histone modification marks involved in embryo development, while RNA sequencing identified differentially expressed genes in 4-cell embryos. Differentially H3K4me3 and H3K27ac occupied regions were mainly distributed at the gene promoters and putative enhancers, and were enriched in pathways related to the immune system and nervous system. Integrative analysis of these sequencing data showed that genes such as Mgl2 with increased H3K4me3 and H3K27ac in sperm were up-regulated in embryos, and vice versa for genes such as Dcn. Additionally, differentially occupied H3K4me3 and H3K27ac in sperm were linked to gene expression changes in both paternal and maternal alleles of 4-cell embryos. In conclusion, GLA-induced changes in sperm H3K4me3 and H3K27ac are concordant with gene expression in preimplantation embryos, which might further affect embryo development and offspring health.

Eutherian-Specific Gene TRIML2 Attenuates Inflammation in the Evolution of Placentation.

Evolution of highly invasive placentation in the stem lineage of eutherians and subsequent extension of pregnancy set eutherians apart from other mammals, that is, marsupials with short-lived placentas, and oviparous monotremes. Recent studies suggest that eutherian implantation evolved from marsupial attachment reaction, an inflammatory process induced by the direct contact of fetal placenta with maternal endometrium after the breakdown of the shell coat, and shortly before the onset of parturition. Unique to eutherians, a dramatic downregulation of inflammation after implantation prevents the onset of premature parturition, and is critical for the maintenance of gestation. This downregulation likely involved evolutionary changes on maternal as well as fetal/placental side. Tripartite-motif family-like2 (TRIML2) only exists in eutherian genomes and shows preferential expression in preimplantation embryos, and trophoblast-derived structures, such as chorion and placental disc. Comparative genomic evidence supports that TRIML2 originated from a gene duplication event in the stem lineage of Eutheria that also gave rise to eutherian TRIML1. Compared with TRIML1, TRIML2 lost the catalytic RING domain of E3 ligase. However, only TRIML2 is induced in human choriocarcinoma cell line JEG3 with poly(I:C) treatment to simulate inflammation during viral infection. Its knockdown increases the production of proinflammatory cytokines and reduces trophoblast survival during poly(I:C) stimulation, while its overexpression reduces proinflammatory cytokine production, supporting TRIML2’s role as a regulatory inhibitor of the inflammatory pathways in trophoblasts. TRIML2’s potential virus-interacting PRY/SPRY domain shows significant signature of selection, suggesting its contribution to the evolution of eutherian-specific inflammation regulation during placentation.

Differential integrin expression in pre-implantation embryos developing under in vivo and in vitro conditions

Implantation failure is a major problem in human assisted reproduction, which persists regardless the optimization of endometrial receptivity and selection of genetically and morphologically healthy embryos. Since embryo-endometrium interaction depends on cell junctional, cell adhesion and cell-substratum adhesion molecules, the present study inquired whether in vitro growing murine embryos display similar to the in vivo growing embryos patterns of adhesion molecules. To this extend aVb3 expression and distribution in zygotes and 2-cell stage embryos were studied. The results demonstrated that only the in vivo growing embryos displayed specifically polarized aVb3 distribution, indicating their potential successful interaction with endometrium. Based on previous studies showing that L-carnitine (L-Cn) could affect embryonic development, it was demonstrated that the addition of L-Cn to the culture medium, could lead the in vitro growing embryos to acquire aVb3 expression and distribution similar to the in vivo growing embryos. Visualization of the effect of L-Cn using third harmonic generation imaging showed decreased lipid droplet levels in 2-cell-stage embryos, observation that correlates with an active energetic state of the growing embryos. Thus, the application of L-Cn to the culture medium could assist pre-implantation-state embryos to acquire aVb3 expression and distribution similar to the in vivo developing conditions.

Interleukin-6 regulates expression of Fos and Jun genes to affect the development of mouse preimplantation embryos.

AimWe investigated whether recombinant mouse interleukin‐6 (IL‐6) affects the development of preimplantation embryos and induces the ‐signal transducers and activators of transcription (JAK–STAT) signaling pathway by binding IL‐6 signal transducer (IL‐6st) and regulating Fos and Jun gene expression, thereby accounting for the negative effect of superovulation on embryo development.MethodsWe compared rates of blastocyst formation from embryos cultured with different concentrations of IL‐6 or/and anti‐interleukin 6 receptor antibody (anti‐IL‐6RAb) in superovulated experimental and normal control groups. IL‐6 expression in preimplantation embryos was determined by immunofluorescence identification. Fos, Jun and IL‐6st messenger RNA expression was detected by PCR and microarray experiments.ResultsRates of blastocyst formation significantly decreased in superovulated embryos, whether or not they were incubated in 0.1, 1, 25 or 50 pg/mL IL‐6, (P < 0.01) compared to embryos from naturally ovulated controls, whereas incubation in 5 and 10 pg/mL IL‐6 reversed the negative effects of superovulation. The addition of anti‐IL‐6RAb to naturally ovulated embryos reduced blastocyst rates to those of superovulated embryos. Gene chip analysis indicated that the JAK–STAT signaling pathway contained differentially expressed IL ‐6, IL ‐6st, Jun and Fos genes. Both anti‐IL‐6RAb or ovarian stimulation downregulated IL‐6st, Jun, and Fos messenger RNA expression, but expression of the same three genes increased in 5 pg/mL IL‐6.ConclusionOvarian stimulation negatively impacts the development of preimplantation embryos by reducing IL‐6 release. IL‐6 affects the rate of development of zygotes to blastocyst by regulating IL ‐6st, F os and J un expression in the JAK–STAT signaling pathway.

Zygotic Genome Activation Revisited: Looking Through the Expression and Function of Zscan4.

Zygotic genome activation (ZGA, a.k.a. zygotic gene activation) is a critical event in development, when the paternally derived genome and maternally derived genome begin to be activated and transcribed after fertilization. Major ZGA occurs at the two-cell stage in mice and the four- to eight-cell stage in human preimplantation embryos. It has been thought that ZGA exists to provide RNAs and proteins supporting embryonic development after supplies stored in oocytes are used up; however, this paradigm does not seem to explain recent findings. For example, many ZGA genes-once activated-are quickly turned off, and thus ZGA forms a transient wave of transcriptional activation. In addition, ZGA genes are not evolutionarily conserved. In this review, we address these issues by focusing on Zscan4 (zinc finger and SCAN domain-containing 4), which was identified for its specific expression in preimplantation embryos during ZGA. Detailed molecular analyses of Zscan4 expression and function have revealed common features of Zscan4-associated events (Z4 events) in mouse embryonic stem cells and ZGA in preimplantation embryos. One feature is a rapid derepression and rerepression of constitutive heterochromatin, which includes pericentromeric major satellites and telomeres, and facultative heterochromatin, which includes retrotransposons and Z4 event-associated genes. We propose that the Z4 event superimposed on ZGA plays a critical role in the maintenance of genome and chromosome integrity in preimplantation embryos by promoting correction of DNA damage and chromosome abnormalities.

Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.

Maternal Exposure to High Temperatures Disrupts OCT4 mRNA Expression of Rabbit Pre‐Implantation Embryos and Endometrial Tissue

We examined the effect of prolonged high heat stress on reproductive performance and its relationship with gene expression in pre-implantation embryos and endometrial tissue. In experiment 1, primiparous rabbit does were divided into two environments: control does (maintained between 14 and 22°C) and heat-treated does housed in a climatic chamber (maintained between 25 and 35°C). Females were reproducing, and the litter size and live born kits were assessed at 2nd and 3rd partum. In heat-treated does, lower litter size (9.7±0.48 and 11.4±0.50) and fewer live born kits (7.2±0.55 and 10.2±0.57) were observed, although similar ovulation rates and numbers of pre-implantation embryos were noted. In experiment 2, after 3rd partum multiparous non-lactating does from each experimental group were used to obtain pre-implantation embryos and endometrial tissue. mRNA transcripts from OCT-4, VEGF, erbB3, Ifn-ɣ, HSP70 and HSP90 were analysed by real-time qPCR. Higher values of OCT-4 expression were observed in embryos and endometrial tissue in females reproduced under heat conditions. Moreover, elevated temperatures have been shown to up-regulate VEGF in embryos and down-regulate Ifn-ɣ in endometrial tissue. The findings suggest a deleterious temperature effect on litter size and live born kits as a consequence of variation in gene expression pattern of the pre-implantational embryo and the endometrium associated with proliferation and differentiation and probably with implantation and uterine and foetal development during gestation.

V-ATPase upregulation during early pregnancy: a possible link to establishment of an inflammatory response during preimplantation period of pregnancy

Various mechanisms exist to prevent a potentially deleterious maternal immune response that results in compromising survival of semiallogeneic fetus. In pregnancy, there is a necessary early preimplantation inflammatory stage followed by a postimplantation anti-inflammatory stage. Thus, there is a biphasic 'immune response' observed during the course of pregnancy. We provide the evidence that capacitation of sperm induced the expression of a2 isoform of V-ATPase (ATP6V0A2 referred to as a2V), leukemia inhibitory factor (Lif), Il1b, and Tnf in the sperm. Capacitated sperm also released cleaved N-terminal domain of a2V-ATPase (a2NTD), which upregulates the gene expression of Lif, Il1b, Tnf, and monocyte chemotactic protein-1 (Ccl2 (Mcp1)) in the uterus. Unfertilized eggs had low a2V expression, but after fertilization, the expression of a2V increased in zygotes. This increased level of a2V expression was maintained in preimplantation embryos. Seminal plasma was necessary for upregulation of a2V expression in preimplantation embryos, as mating with seminal vesicle-deficient males failed to elicit an increase in a2V expression in preimplantation embryos. The infiltration of macrophages into the uterus was significantly increased after insemination of both sperm and seminal plasma during the preimplantation period of pregnancy. This dynamic infiltration into the uterus corresponded with the uterine a2V expression through the induction of Ccl2 expression. Furthermore, the polarization ratio of M1:M2 (pro-inflammatory/anti-inflammatory) macrophages in the uterus fluctuated from a ratio of 1.60 (day 1) to 1.45 (day 4) when female mice were inseminated with both sperm and seminal plasma. These data provide evidence that exposure to semen may initiate an inflammatory milieu by inducing a2V and cytokine/chemokine expression, which triggers the influx of macrophages into the preimplantation uterus during the onset of pregnancy and ultimately leads to successful pregnancy outcome.

Association of Rex-1 to target genes supports its interaction with Polycomb function

Rex-1/Zfp42 displays a remarkably restricted pattern of expression in preimplantation embryos, primary spermatocytes, and undifferentiated mouse embryonic stem (ES) cells and is frequently used as a marker gene for pluripotent stem cells. To understand the role of Rex-1 in selfrenewal and pluripotency, we used Rex-1 association as a measure to identify potential target genes, and carried out chromatin-immunoprecipitation assays in combination with gene specific primers to identify genomic targets Rex-1 associates with. We find association of Rex-1 to several genes described previously as bivalently marked regulators of differentiation and development, whose repression in mouse embryonic stem (ES) cells is Polycomb Group-mediated, and controlled directly by Ring1A/B. To substantiate the hypothesis that Rex-1 contributes to gene regulation by PcG, we demonstrate interactions of Rex-1 and YY2 (a close relative of YY1) with Ring1 proteins and the PcG-associated proteins RYBP and YAF2, in line with interactions reported previously for YY1. We also demonstrate the presence of Rex-1 protein in both trophectoderm and Inner Cell Mass of the mouse blastocyst and in both ES and in trophectoderm stem (TS) cells. In TS cells, we were unable to demonstrate association of Rex-1 to the genes it associates with in ES cells, suggesting that association may be cell-type specific. Rex-1 might fine-tune pluripotency in ES cells by modulating Polycomb-mediated gene regulation.

Granzyme Gis expressed in the two-cell stage mouse embryo and is required for the maternal-zygotic transition

BackgroundDetailed knowledge of the molecular and cellular mechanisms that direct spatial and temporal gene expression in pre-implantation embryos is critical for understanding the control of the maternal-zygotic transition and cell differentiation in early embryonic development. In this study, twenty-three clones, expressed at different stages of early mouse development, were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). One of these clones, which is expressed in 2-cell stage embryos at 48 hr post-hCG injection, shows a perfect sequence homology to the gene encoding the granzyme G protein. The granzyme family members are serine proteases that are present in the secretory granules of cytolytic T lymphocytes. However, the pattern of granzyme G expression and its function in early mouse embryos are entirely unknown.ResultsUpon the introduction of an antisense morpholino (2 mM) against granzyme G to knock-down endogenous gene function, all embryos were arrested at the 2- to 4-cell stages of egg cleavage, and the de novo synthesis of zygotic RNAs was decreased. The embryonic survival rate was dramatically decreased at the late 2-cell stage when serine protease-specific inhibitors, 0.1 mM 3,4-dichloroisocoumarin (3,4-DCI), and 2 mM phenyl methanesulphonyl fluoride (PMSF), were added to the in vitro embryonic culture medium. Survival was not affected by the addition of 0.5 mM EDTA, a metalloproteinase inhibitor.ConclusionWe characterized for the first time the expression and function of granzyme G during early stage embryogenesis. Our data suggest that granzyme G is an important factor in early mouse embryonic development and may play a novel role in the elimination of maternal proteins and the triggering of zygotic gene expression during the maternal-zygotic transition.

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.

Stage-specific Integration of Maternal and Embryonic Peroxisome Proliferator-activated Receptor δ Signaling Is Critical to Pregnancy Success

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.

194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan� MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 � 10(8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 � 4.5% v. buffer 94.6 � 5.8%); however, it did induce a significant degree of apoptosis (P &lt; 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 � 1223 v. 3098 � 592; miR-16, 196216 � 958 v. 133514 � 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 � 0.12), the pro-apoptotic gene Bax (1.418 � 0.12), and Caspase 3 (1.314 � 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H2O2 induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P &lt; 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.

Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

110 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE IVF EMBRYOS

Lamin A/C expression is generally associated with terminally differentiated cell types; however, numerous conflicting reports in the literature demonstrate the presence of lamins A/C in pluripotent cells of pre-implantation embryos. This study characterized lamin A/C expression in bovine pre-implantation embryos using two monoclonal lamin A/C antibodies: anti-A/C IgM (A/C1) and anti-A/C IgG (A/C2) (Santa Cruz, California). Bovine embryos were produced as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255–262) and collected at various stages for immunofluorescence staining. Embryos were fixed in 100% methanol at −20°C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Samples were then incubated overnight at 4°C with mouse lamin A/C antibodies or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with rabbit anti-mouse immunoglobulins (1:20) (DAKO, Denmark) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were counter-stained with 4',6-diamidino-2-phenylindole (2 μg/mL). Bovine fetal fibroblasts (BFF1) and human embryonic teratocarcinoma cells (EC1 and EC2) were processed identically to the embryos and used as positive and negative controls, respectively (Stewart and Burke 1987 Cell 51, 383–392). Images were viewed using epifluorescence (Leica DMR, Germany) and confocal microscopy (Leica TCS). BFF1 cells reacted with both lamin A/C1 and A/C2 antibodies. EC1 and EC2 stained positively for A/C2 whereas A/C1 was negative in both. All germinal vesicle (GV)-stage oocytes stained strongly for A/C2; however, for A/C1 only 67.5% were positive, and staining intensity was variable. Metaphase II oocytes stained negatively for both antibodies. One-cell zygotes exhibited a variable staining pattern similar to that of GV-stage oocytes. In contrast, all embryos from the 2-cell to blastocyst stage were negative for A/C1 but positive for A/C2. Our observations in embryos and EC cells indicate that the mouse anti-lamin A/C1 is specifically binding to lamin A/C whereas A/C2 is cross-reacting with other nuclear envelope proteins, possibly lamin B1/B2. The cross-reactivity of A/C2 has led to contradicting results in previous reports on lamin A/C expression in pre-implantation embryos. Our results with A/C1 show that lamin A/C is present in GV oocytes and 1-cell zygotes, suggesting that lamin A is important for pronuclear formation after fertilization. These results suggest that active remodelling of the nuclear envelope occurs during the early stages of bovine embryo development. RK is supported by a BBSRC postgraduate studentship. RA is a Marie-Curie Fellow.

A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos

MacroH2A histones are variants of canonical histone H2A that are conserved among vertebrates. Previous studies have implicated macroH2As in epigenetic gene-silencing events including X chromosome inactivation. Here we show that macroH2A is present in developing and mature mouse oocytes. MacroH2A is localized to chromatin of germinal vesicles (GV) in both late growth stage (lg-GV) and fully grown (fg-GV) stage oocytes. In addition, macroH2A is associated with the chromosomes of mature oocytes, and abundant macroH2A is present in the first polar body. However, maternal macroH2A is lost from zygotes generated by normal fertilization by the late 2 pronuclei (2PN) stage. Normal embryos at 2-, 4-, and 8-cell stages lack macroH2A except in residual polar bodies. MacroH2A protein expression reappears in embryos after the 8-cell stage and persists in morulae and blastocysts, where nuclear macroH2A is present in both the trophectodermal and inner cell mass cells. We followed the loss of macroH2A from pronuclei in parthenogenetic embryos generated by oocyte activation. Abundant macroH2A is present upon the metaphase II plate and persists through parthenogenetic anaphase, but macroH2A is progressively lost during pronuclear decondensation prior to synkaryogamy. Examination of embryos generated by intracytoplasmic sperm injection (ICSI) revealed that macroH2A is associated exclusively with female pronuclei prior to loss in late pronucleus stage embryos. These results outline a surprising finding that a maternal store of macroH2A is removed from the maternal genome prior to synkaryogamy, resulting in embryos that execute three to four mitotic divisions in the absence of macroH2A prior to the onset of embryonic macroH2A expression.

Lineage-specific effects of polychlorinated biphenyls (PCB) on gene expression in the rabbit blastocyst

The ubiquitous toxic environmental contaminants polychlorinated biphenyls (PCBs) change gene expression in preimplantation embryos. Cell lineage-specific effects of PCB are not known. Rabbit day 6 blastocysts were exposed in vitro to low (0.1 ng/congener/mL medium) and high (1 μg) PCB levels of coplanar (PCB 77, 126, and 169) or non-coplanar PCBs (PCB 28, 52, 101, 118, 138, 153, and 180). Embryoblast (ICM) and trophoblast cells (TE) were separated and analyzed for transcriptional changes of PCB-and implantation-associated genes by semiquantitative RT-PCR. PCBs increased CYP 1A1 mRNA only in the ICM. CYP 1B1, VEGFR2, and COX-2 transcript numbers were elevated in both ICM and TE. Transcripts for HIF-1α were decreased in the ICM. No obvious differences in gene expression following exposure to coplanar and non-coplanar PCBs were detected. Our results show that transcriptional responses to PCBs differ between the cell lineages of the rabbit blastocyst, indicating that PCBs can influence the highly sensitive process of early mammalian development.