Genes In Preimplantation Embryos Research Articles

Constitutive heterochromatin propagation contributes to the X chromosome inactivation.

Imprinted X chromosome inactivation (iXCI) balances the expression of X-linked genes in preimplantation embryos and extraembryonic tissues in rodents. Long noncoding Xist RNA drives iXCI, silencing genes and recruiting Xist-dependent chromatin repressors. Some domains on the inactive X chromosome include repressive modifications specific to constitutive heterochromatin, which show no direct link to Xist RNA. We explored the relationship between Xist RNA and chromatin silencing during iXCI in vole Microtus levis. We performed locus-specific activation of Xist transcription on the only active X chromosome using the dCas9-SAM system in XO vole trophoblast stem cells (TSCs), which allow modeling iXCI events to some extent. The artificially activated endogenous vole Xist transcript is truncated and restricted ~ 6.6kb of the exon 1. Ectopic Xist RNA accumulates on the X chromosome and recruits Xist-dependent modifications during TSC differentiation, yet is incapable by itself repressing X-linked genes. Transcriptional silencing occurs upon ectopic Xist upregulation only when repressive marks spread from the massive telomeric constitutive heterochromatin to the X chromosome region containing genes. We hypothesize that the Xist RNA-induced propagation of repressive marks from the constitutive heterochromatin could be a mechanism involved in X chromosome inactivation.

Silencing XIST on the future active X: Searching human and bovine preimplantation embryos for the repressor.

X inactivation is the means of equalizing the dosage of X chromosomal genes in male and female eutherian mammals, so that only one X is active in each cell. The XIST locus (in cis) on each additional X chromosome initiates the transcriptional silence of that chromosome, making it an inactive X. How the active X in both males and females is protected from inactivation by its own XIST locus is not well understood in any mammal. Previous studies of autosomal duplications suggest that gene(s) on the short arm of human chromosome 19 repress XIST on the active X. Here, we examine the time of transcription of some candidate genes in preimplantation embryos using single-cell RNA sequencing data from human embryos and qRT-PCR from bovine embryos. The candidate genes assayed are those transcribed from 19p13.3-13.2, which are widely expressed and can remodel chromatin. Our results confirm that XIST is expressed at low levels from the future active X in embryos of both sexes; they also show that the XIST locus is repressed in both sexes when pluripotency factors are being upregulated, during the 4-8 cell and morula stages in human and bovine embryos - well before the early blastocyst (E5) when XIST on the inactive X in females starts to be upregulated. Our data suggest a role for DNMT1, UHRF1, SAFB and SAFB2 in XIST repression; they also exclude XACT and other 19p candidate genes and provide the transcriptional timing for some genes not previously assayed in human or bovine preimplantation embryos.

SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development.

ObjectivesGene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.Materials and methodsTo analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.ResultsOur results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.

Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). An exception are human pre-implantation embryos, where dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities with naïve pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X-chromosome are active and express the long non-coding RNAs XACT and XIST, the master regulator of XCI, which are silenced upon entry into meiosis. These findings uncover XACT as hPGC-marker, describe XCD associated with XIST-expression in hPGCs, and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Additionally, we found a unique X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.

Effect of vitrification on biogenesis pathway and expression of development-related microRNAs in preimplantation mouse embryos

Vitrification of embryos has been known as the most efficient cryopreservation method in assisted reproductive technology clinics. Vitrification of preimplantation embryo might be associated with altered gene expression profile and biochemical changes of vitrified embryos. Stringent regulation of gene expression in early embryonic stages is very critical for normal development. In the present study, we investigated the effect of vitrification on the canonical miRNA biogenesis pathway, and also the expression of developmental related miRNAs, in 8-cell and blastocyst mouse embryos. Although the expression pattern of the miRNA biogenesis pathway genes differed between 8-cell and blastocyst mouse embryos, vitrification did not affect the expression level of these genes in preimplantation embryos. The expression levels of miR-21 and let-7a were significantly decreased in vitrified 8-cell embryos and fresh blastocysts when compared with fresh 8-cell embryos. The expression of Stat3 was significantly reduced in blastocysts after vitrification. The alteration in the expression pattern of miRNAs, due to their mode of action, can affect broad downstream key developmental signaling pathways. Therefore, the blastocyst stage is the preferred point for embryo vitrification as they are less susceptible to cryo-damages regarding the stability of miRNAs related to the developmental and implantation competence of embryo.

TOCOTRIENOL-RICH FRACTION MODULATE THE PHOSPHOINOSITIDE 3-KINASES/AKT SIGNALING PATHWAY GENES AND PREVENT OXIDATIVE STRESS IN NICOTINE-INDUCED PRE-IMPLANTATION EMBRYOS

Objective: This study aimed to determine the effects of the tocotrienol-rich fraction (TRF) on the regulations of phosphoinositide 3-kinases (PI3K)/Aktpathways related genes in preimplantation embryos induced by nicotine (Nic).Methods: Twenty-four female BALB/c mice were divided into four groups with Nic and TRF supplementation for 7 consecutive days. Animalswere superovulated before mating with fertile males. Plasma malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase weredetermined and analyzed accordingly. Embryos with two and eight blastomeres were assessed for gene expression analysis.Results: The levels of endogenous antioxidative enzymes for the group with TRF intervention and TRF only group showed no significant changes whencompared to the control group. The level of oxidative stress (OS) biomarkers was also significantly decreased when compared to the Nic-induced group.At 2-cell stage, Pten gene was significantly upregulated while Akt1, GSK3β, and Mapk1 were significantly downregulated almost similar to the baseline(control) in the Nic-induced mice. Intervention with TRF resulted in a significant downregulated of Pten gene followed by a significant upregulationof other genes. The same pattern was shown at the 8-cell stage.Conclusion: This showed that TRF evidently has OS protection capacity and it could be through modulating the PI3K/Akt signaling pathway.

Blastocyst recovery and multifactorial gene expression analysis in the wild guinea pig (Cavia aperea)

The expression of specific developmentally important genes in preimplantation embryos is an accepted marker for unraveling the influence of single factors in studies that are mostly related to artificial reproduction techniques. Such studies, however, often reveal high levels of heterogeneity between single embryos, independently of the influence of factors of interest. A possible explanation for this variation could be the large variety of physiological and environmental factors to which early embryos are exposed and their ability to react to them. Here, we investigated several potentially important parameters of development at the same time, in blastocysts of the wild guinea pig (Cavia aperea) generated in vivo after natural mating. The optimal time for flushing fully developed blastocysts was between 123 and 126 hours after mating. The abundance of POU5F1 (P = 0.042), BAX (P < 0.001), SLC2A1 (P = 0.017), and DNMT3A (P < 0.001) mRNA changed significantly over time after mating. The number of sibling embryos present influenced STAT3 levels significantly (P = 0.02). Levels of BAX and POU5F1 were significantly affected by season (P = 0.03 and 0.04). The temporal pattern of SLC2A1 levels was significantly altered both after feeding a protein-deficient diet (P = 0.04) and temperature treatment (P = 0.04) of the sire. In addition, the identity of the father had a significant influence on POU5F1 (P = 0.049) and STAT3 (P < 0.001) mRNA abundances. These data report that the expression of specific genes in early embryos reflects the entire heterogeneity of their surroundings and that it is a plastic reaction toward a multifactorial environment.

Selection and expression profiles of reference genes in mouse preimplantation embryos of different ploidies at various developmental stages.

Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Manystudies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos.

Expression patterns of sirtuin genes in porcine preimplantation embryos and effects of sirtuin inhibitors on in vitro embryonic development after parthenogenetic activation and in vitro fertilization

We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1–3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1–3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

BackgroundWe have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers.ResultsHere, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo.ConclusionOn the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

118 EXPRESSION PROFILES OF CIRCADIAN CLOCK GENES IN MOUSE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Matsuo et al. reported that circadian clock genes regulate the timing of cell division in mouse regenerating liver cells (2003). Their results suggested the importance of circadian clock genes for organs or tissues for which functions are characterized by cell division, such as pre-implantation embryos. To obtain basic information on the molecular functions of circadian clock genes in pre-implantation embryos, we investigated the expression profiles of transcripts and proteins of some circadian clock genes, clock, bmal1, cry1, and per2, in mouse germinal vesicle oocytes (GV), MII oocytes (MII), and pre-implantation embryos using real-time PCR and immunocytochemistry (ICC). Germinal vesicle oocytes were collected from ICR females at 48 h after PMSG priming. The mouse at 48 h after PMSG priming was primed with hCG, and MII were collected at 15 h after hCG priming. The pre-implantation embryos were collected at 6, 12, 24, 36, 48, 60, 72, 84, and 96 h after insemination, and they corresponded to early 1-cell, late 1-cell, early 2-cell, late 2-cell, 4-cell, 8-cell, early morula, late morula, and blastocyst stages, respectively. cDNA was produced by mRNA isolated from 20 oocytes or embryos using oligo dT and was subjected to real-time PCR using a TaqMan Probe system (ABI). Three sets of 20 oocytes or embryos at each developmental stage were applied to mRNA extraction and real-time PCR analysis to ensure equal mRNA extraction efficiency between samples. The level of mRNA of each clock gene contained in 3 samples from each developmental stage was almost the same. Statistical analysis of the transcripts of each gene were done by ANOVA. Germinal vesicles, MII and embryos collected at each time point were subjected to ICC using antibodies of CLOCK, BMAL1, CRY1, and PER2. The oocytes or embryos treated with only secondary antibody did not produce any signal. All of the examined genes except per2 were expressed in oocytes and pre-implantation embryos. The transcript level of clock, bmal1, and cry1 in MII were significantly lower than those in GV (P &lt; 0.05). After fertilization, transcript levels of clock, bmal1, and cry1 significantly decreased from early 1-cell stage to late 2-cell stage (P &lt; 0.05). These decreased transcript levels were maintained until the blastocyst stage after the late 2-cell stage. Immunocytochemistry analysis showed the nuclear localization of CLOCK and BMAL1 in early and late 2-cell embryos and of CRY1 in early 2-cell embryos but no signals of PER2 in oocytes or pre-implantation embryos. Because mouse oocytes and 1- to 2-cell embryos are transcriptionally inert, the abundant transcripts of clock, bmal1, and cry1 in these stages seemed to indicate that they were synthesized and stored during the oocyte growth phase. Moreover, the nuclear localization of CLOCK, BMAL1, and CRY1 in the oocytes and 1- to 2-cell stage embryos suggested that some clock genes were translated and worked for oocyte maturation and early embryogenesis. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

Paternal exposure to cyclophosphamide induces DNA damage and alters the expression of DNA repair genes in the rat preimplantation embryo

Chronic low dose treatment of male rats with cyclophosphamide, an anticancer alkylating agent, damages male germ cells, resulting in greater than 80% peri-implantation progeny loss. Little transcription or repair takes place in the DNA of post-meiotic male germ cells. The spermatozoal genome regains its transcriptional capacity in the fertilized oocyte. We hypothesized that as a consequence of exposure of male rats to cyclophosphamide DNA damage to the male genome is transmitted to the conceptus; furthermore, this damage leads to alterations in the expression profiles of DNA repair genes during preimplantation development. Male rats were treated with either saline or cyclophosphamide (6mg/kg/day, 4–6 weeks) and mated to control females; 1–8 cell stage embryos were collected. The alkaline comet assay was used to assess DNA damage in 1-cell embryos. A significantly higher percentage (68%) of the embryos fertilized by cyclophosphamide-exposed spermatozoa displayed a comet indicative of DNA damage, compared to those sired by control males (18%). The in situ transcription/antisense RNA approach was used to determine if DNA damage alters the expression of DNA repair genes in early embryos. Dramatic increases in the transcripts for selected members of the nucleotide excision repair family (XPC, XPE and PCNA), mismatch repair family (PMS1), and recombination repair family (RAD50) were found in 1-cell stage embryos sired by cyclophosphamide-treated males compared to controls, while decreases in the expression of base excision repair family members (UNG1, UNG2, and XRCC1) and in recombination repair transcripts (RAD54) were observed. By the 8-cell stage, transcripts for specific members of the nucleotide excision repair family (XPC) and mismatch repair family (MSH2, PMS2) were elevated greatly in control embryos compared to embryos sired by drug-treated males; in contrast, transcripts for other members of the nucleotide excision repair family (XPE, PCNA), as well as some of the base excision repair family (UNG1), were elevated in embryos sired by drug-treated males. Therefore, DNA damage incurred in spermatozoa, following cyclophosphamide exposure is associated with alterations in the expression profiles of DNA repair genes in preimplantation embryos as early as the 1-cell stage. Genotoxic stress may disturb the nuclear remodeling and reprogramming events that follow fertilization and precede zygotic genome activation.

Differential expression of Ped gene candidates in preimplantation mouse embryos.

The Ped (preimplantation embryonic development) gene influences the rate of mouse preimplantation embryonic development and subsequent survival. Four similar tandem genes in the Q region of the major histocompatibility complex-Q6, Q7, Q8, and Q9-were identified as Ped gene candidates. In this study, expression of these genes during preimplantation development was examined and quantitated by reverse transcription-polymerase chain reaction and single nucleotide primer extension assays in order to investigate their contribution to the Ped gene phenotype. The Q7/Q9 gene pair was found to be transcribed in preimplantation mouse embryos, whereas transcription of the Q6/Q8 gene pair was undetectable. Both Q7 and Q9 are expressed in embryos from one Ped fast strain, C57BL/6, while only the Q9 gene is expressed in another Ped fast strain, B6.K2. These results suggest that both the Q7 and Q9 genes can function as the Ped gene in the mouse. Interestingly, the expression pattern of the Q7 and Q9 genes in preimplantation embryos is the same as in splenic lymphocytes. However, the Q6 and Q8 genes are expressed in splenic lymphocytes but not in preimplantation embryos. Treatment of mouse preimplantation embryos with interferon gamma (gamma-IFN) did not induce expression of the Q6/Q8 genes but enhanced expression of the Q7/Q9 genes. The mechanism of this differential transcription pattern is currently under investigation.

Differential mRNA expression of the phosphoprotein p19/SCG10 gene family in mouse preimplantation embryos, uterus, and placenta.

The p19/SCG10 gene family encodes two structurally related cellular proteins that are implicated in signal transduction during differentiation of mammalian cells. Previous evidence suggests that both genes are expressed in a stage-specific manner but that expression of p19 is widespread, whereas that of SCG10 is restricted to developing neurons. To determine at which developmental stage these two genes are first expressed, we have probed for mRNA transcripts in preimplantation embryos and the utero-placental unit of the mouse. As determined by polymerase chain reaction (PCR) to amplify reverse-transcribed RNA, expression of both genes was detected in preimplantation embryos, although the temporal pattern was distinct. p19 mRNA appeared transiently in 2-cell embryos, was undetectable in morulae and early blastocysts and reappeared in expanded blastocysts. In contrast, embryonic expression of SCG10 mRNA commenced in morulae and was maintained through to the blastocyst stage. Interestingly, only SCG10 expression could be detected in blastocysts derived from cultures of 2-cell embryos. During the post-implantation period, SCG10 transcripts were only detected in the uterus and placenta by reverse transcriptase-PCR, whereas p19 mRNA could be detected by Northern blotting and showed stage-specific expression in both tissues. The data confirm that, at later developmental stages, expression of p19 is widespread while that of SCG10 is more restricted. The expression of both genes in preimplantation embryos suggests distinct but possibly overlapping roles for p19 and SCG10 in early mammalian development.