Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

Autophagy is required for proper meiosis of porcine oocytes maturing in vitro.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

Inhibition of cathepsin B activity prevents deterioration in the quality of in vitro aged porcine oocytes.

The activity of cathepsin B, a member of the lysosomal protease family, directly correlates with oocyte quality and subsequent embryonic development. However, its biological function during the progression of in vitro aging of oocytes in pigs has not been demonstrated. Here, we showed that cathepsin B activity was dramatically increased during in vitro aged oocytes. The inhibition of cathepsin B activity prevented the decline of the quality of aged oocytes and improved their subsequent developmental competence. Moreover, the inhibition of cathepsin B activity reduced aging-induced mitochondrial dysfunction and attenuated oxidative stress. The inhibition of cathepsin B activity also markedly decreased early apoptosis levels and the frequency of spindle anomalies during in vitro aging of oocytes. These results demonstrate that in vitro aging of oocytes induces cathepsin B activity, which is associated with a decline in oocyte quality. The inhibition of cathepsin B activity has a beneficial effect on oocytes during the process of in vitro aging.

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress.

Laminarin (LMA), a ß-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 µg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.

Methyl-CpG-Binding Protein 2 Improves the Development of Mouse Somatic Cell Nuclear Transfer Embryos.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

Continuous passages accelerate the reprogramming of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming somatic cells through transduction with a transcription factor cocktail. However, the low efficiency of this procedure has kept iPSCs away from the study of the clinical application of stem cell biology. Our research shows that continuous passage increases the efficiency of reprogramming. Compared with conventional method of establishment of iPSCs, more embryonic stem cell (ESC)-like clones are generated by continuous passage during early reprogramming. These inchoate clones, indistinguishable from genuine ESC clones, are closer to fully reprogrammed cells compared with those derived from classical iPSC induction, which increased the expression of pluripotent gene markers and the levels of demethylation of Oct4 and Nanog. These results suggested that full reprogramming is a gradual process that does not merely end at the point of the activation of endogenous pluripotency-associated genes. Continuous passage could increase the pluripotency of induced cells and accelerate the process of reprogramming by epigenetic modification. In brief, we have provided an advanced strategy to accelerate the reprogramming and generate more nearly fully reprogrammed iPSCs efficiently and rapidly.

Generation of neural progenitors from induced Bama miniature pig pluripotent cells.

Pig pluripotent cells may represent an advantageous experimental tool for developing therapeutic application in the human biomedical field. However, it has previously been proven to be difficult to establish from the early embryo and its pluripotency has not been distinctly documented. In recent years, induced pluripotent stem (iPS) cell technology provides a new method of reprogramming somatic cells to pluripotent state. The generation of iPS cells together with or without certain small molecules has become a routine technique. However, the generation of iPS cells from pig embryonic tissues using viral infections together with small molecules has not been reported. Here, we reported the generation of induced pig pluripotent cells (iPPCs) using the iPS technology in combination with valproic acid (VPA). VPA treatment significantly increased the expression of pluripotent genes and played an important role in early reprogramming. We showed that iPPCs resembled pig epiblast cells in their morphology and pluripotent markers, such as OCT4, NANOG, and SSEA1. It had a normal karyotype and could form embryoid bodies, which express three germ layer markers in vitro. In addition, the iPPCs might directly differentiate into neural progenitors after being induced with the retinoic acid and extracellular matrix. Our study established a reasonable method to generate pig pluripotent cells, which might be a new donor cell source for human neural disease therapy.

Identifying MicroRNA and mRNA expression profiles in embryonic stem cells derived from parthenogenetic, androgenetic and fertilized blastocysts.

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

Autophagy influences maternal mRNA degradation and apoptosis in porcine parthenotes developing in vitro.

Autophagy, an essential process for cellular maintenance, cell viability, and development, is the bulk degradation of proteins and organelles. This study investigated the expression levels of autophagy-related genes and the effect of 3-methyladenine (3-MA, an autophagy inhibitor) or rapamycin (an autophagy inducer) on maternal gene degradation and apoptosis in porcine parthenotes developing in vitro. LC3, which is essential for the formation of autophagosomes, was widely expressed in porcine parthenotes. High levels of autophagy-related genes, Atg5, Beclin1 and Lc3 transcripts were expressed in the 1-cell (1C) stage and gradually decreased through the 2-cell (2C) to blastocyst stages. The mRNA expression of Gdf9, c-mos and cyclin B maintained high levels in 2C and 4-cell (4C) embryos treated with 3-MA compared with the control. The Bmp15 and cyclin B mRNA levels were significantly reduced in embryos treated with rapamycin compared with the control. These results suggest that autophagy influences the degradation of these maternal genes. Furthermore, 3-MA-treated embryos exhibited significantly reduced developmental rates, decreased total cell numbers and increased rates of apoptosis. Expression of Atg5, Beclin1 and Lc3 and synthesis of LC3 protein were significantly reduced at the blastocyst stage. Although rapamycin treatment did not affect the developmental rate, it decreased the cell number and increased the rate of apoptosis, and the expression of Atg5, Beclin1 and Lc3 and LC3 protein synthesis were increased. Finally, blastocysts derived following treatment with 3-MA or rapamycin exhibited significantly decreased expression of selected transcription factors, including Pou5f1, Sox2 and Nanog. In conclusion, our results demonstrate that autophagy influences maternal mRNA degradation and apoptosis at the blastocyst stage and suggest that autophagy plays an important role in early embryo development in the pig.

rRNA genes are not fully activated in mouse somatic cell nuclear transfer embryos.

The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.

Interleukin-6 enhances porcine parthenote development in vitro, through the IL-6/Stat3 signaling pathway.

Signal transducer and activator of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. The Stat3 pathway is essential for embryonic development. The aim of this study was to determine the effects of recombinant IL-6 on the viability and development of porcine diploid parthenotes cultured in vitro. Four-cell parthenotes, derived in vitro, were cultured to the blastocyst stage, with or without recombinant IL-6. The addition of 10 or 100 ng/ml of recombinant swine IL-6 into PZM3 medium increased the development rate of parthenotes to the blastocyst stage (P<0.05). When supplemented with 10 ng/ml of recombinant swine IL-6, the number of parthenotes at the blastocyst stage increased (P<0.05) and apoptosis decreased (P<0.05). Real-time RT-PCR experiments revealed that the addition of recombinant swine IL-6 decreased the mRNA expression of the pro-apoptotic gene Caspase3 (P<0.01) but increased the expression levels of the anti-apoptotic genes Bcl2l1 and Survivin. IL-6 receptors and Stat3 mRNA expression were upregulated after treatment with 10 ng/ml recombinant swine IL-6. Immunoblots and fluorescence labeling experiments showed that the levels of phosphorylated Stat3 were upregulated. These results suggest that recombinant swine IL-6 prevents apoptosis of porcine parthenotes and enhances porcine embryo viability through the IL-6/Stat3 signaling pathway in vitro.

Aggregation of pre-implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes.

Parthenogenetic embryonic stem cells (PgES) might advance cell replacement therapies and provide a valuable in vitro model system to study the genomic imprinting. However, the differential potential of PgES cells was limited. It could result from relative low heterology of PgES cells compared with ES cells from fertilization (fES), which produce different expression of most imprinted genes. Here, we described the establishment of PgES cells by aggregating parthenogenetic embryos at the 8-cell stage (aPgES cells), which may increase heterozygy. We found that derivation of aPgES cells in association with an increased number of inner cell mass cells by aggregating was more efficient than that of PgES cells from a single parthenogenetic blastocyst. The aPgES cells have normal karyotype, stain positive for alkaline phosphatase, express high levels of ES cell markers and can differentiate into teratomas composed of the three germ layers. Moreover, compared with PgES cells, the more highly upregulated paternally expressed imprinted genes were observed in aPgES cells, the same change was not shown in aPg blastocysts. This suggested that the aggregation induced effect could modify the expression of paternally expressed imprinted genes. Our studies showed that aPgES cells, the expression of imprinted genes in which more closely resemble fES cells than PgES cells, would contribute to all organs and avoiding immuno-rejection, which may provide invaluable material for regeneration medicine.

Cytochalasin B treatment of mouse oocytes during intracytoplasmic sperm injection (ICSI) increases embryo survival without impairment of development.

Intracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 µg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos.

Trichostatin A modulates apoptotic-related gene expression and improves embryo viability in cloned bovine embryos.

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transferred nuclei into oocytes. Trichostatin A (TSA), a histone deacetylase inhibitor, has been used to enhance nuclear reprogramming following SCNT. However, the molecular mechanism of TSA for the improvement of the preimplantation embryo and fetal development following SCNT is not known. The present study investigates embryo viability and gene expression of cloned bovine preimplantation embryos in the presence and absence of TSA compared to embryos produced by in vitro fertilization or parthenogenetic activation. Our results indicated that TSA treatment significantly improved total and inner cell mass (ICM) cell number and ratio of ICM:trophectoderm (TE) and also decreased the apoptotic index including total, ICM, and ratio of ICM:TE. Four apoptotic-related genes, Bcl-xL, survivin, Bcl2-associated X protein (Bax), and caspase 3 (Casp3), and four pluripotency/differentiation related genes, Oct4, SRY (sex determining region Y)-box 2 (Sox2), Cdx2, and colony-stimulating factor 1 receptor (Csf1r), were measured by real-time RT-PCR. TSA treatment resulted in the high expression of antiapoptotic gene Bcl-xL and low expression of pro-apoptotic gene Bax compared to untreated NT embryos, fertilized embryos, or parthenotes. Furthermore, mRNA expression of Cdx2 was higher in NT-TSA embryos than in NT and in vitro fertilization (IVF) counterparts. Additionally, low expression of microRNA (mir)-21 in NT embryos was enhanced following TSA treatment. These results suggest that TSA positively regulates nuclear reprogramming, and TSA may increased resistance or reduced signal for induction of apoptosis.

Ago2 and GW182 expression in mouse preimplantation embryos: a link between microRNA biogenesis and GW182 protein synthesis.

MicroRNA-mediated RNA interference appears to play a role in early development and differentiation processes in preimplantation embryos. However, the expression of its key effectors, including Ago2, a key component of the RNA-induced silencing complex, and GW182, a critical component of GW bodies (GWBs), has not been assessed in preimplantation embryos. To characterise the roles of Ago2 and GW182 in early embryo development, we determined their transcription and protein synthesis in mouse embryos. Transcript levels of Ago2 and GW182 increased steadily from the one-cell stage through to the blastocyst stage when data were not normalised against an internal reference. However, when normalised against the internal standard, transcript levels for both genes were highest in four-cell stage embryos and decreased steadily through to the blastocyst stage. Indirect immunocytochemistry showed that both AGO2 and GW182 proteins were expressed in each stage in the early embryo and were observed to colocalise in the morula and blastocyst stages. Specific silencing of mRNA expression by short interference (si) RNA against Ago2 or Dicer1 decreased the expression of selected apoptosis- and development-related microRNAs, but did not inhibit development up to the blastocyst stage. However, transcription levels of Oct3/4, Nanog and Sox2 were decreased in both Ago2- and Dicer1-knockdown embryos at the blastocyst stage. Furthermore, although knockdown of these genes did not change transcript levels of GW182, GW182 protein synthesis was decreased in blastocyst stage embryos. These results suggest that Ago2 and Dicer1 regulate GW182 protein expression in mouse embryos, which is linked to microRNA biogenesis and likely to be important for differentiation in the blastocyst stage.

Involvement of ER-calreticulin-Ca2+ signaling in the regulation of porcine oocyte meiotic maturation and maternal gene expression.

Calcium is one of the most ubiquitous signaling molecules, and controls a wide variety of cellular processes. It is mainly stored in the endoplasmic reticulum (ER), bound to lumenal proteins. Calreticulin is the major Ca(2+)-binding chaperone in oocytes, and is integral to numerous cellular functions. To better understand the role of the ER- calreticulin-Ca(2+) pathway in oocyte maturation and early embryogenesis, we characterized the porcine calreticulin gene and investigated its expression profile during oocyte maturation and early embryonic development. Calreticulin was widely expressed in pig tissues and its transcripts were downregulated during maturation, especially at 44 hr, and were undetectable at the blastocyst stage. We also investigated the effect of increased cytosolic Ca(2+) induced by the Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on pig oocyte maturation and maternal gene expression. CPA at 10 microM did not inhibit germinal vesicle breakdown, but did result in the arrest of 38.6% oocytes at or before the MI stage. In addition, expression of the maternal genes C-mos, BMP15, GDF9, and Cyclin B1 was significantly increased in CPA-treated MII oocytes compared with control groups. These data were supported by the results of poly(A)-test PCR, which revealed that the cyclin B1 short isoform (CB-S), GDF9, and C-mos underwent more intensive polyadenylation modification in CPA-treated oocytes than control oocytes, suggesting that polyadenylation may influence Ca(2+)-modulated changes in gene expression. Furthermore, CPA treatment decreased the percentage of four-cell parthenotes that developed into blastocysts, suggesting the need for functional SR/ER Ca(2+)-ATPase pumps or Ca(2+) signals during early embryo development after zygotic genome activation. Together, these data indicate that ER-calreticulin-associated Ca(2+) homeostasis plays a role in oocyte and embryo development, and that alterations in maternal gene expression may contribute to the underlying molecular mechanism, at least partially, via polyadenylation.

Hyperglycemia reduces mitochondrial content and glucose transporter expression in mouse embryos developing in vitro.

The objective of this research was to examine the effects of high concentrations of glucose on mouse embryos developing in vitro by studying embryo viability, mitochondrial content and expression of glucose transporters. Addition of 55 mM glucose to the culture medium of two-cell stage embryos significantly reduced the formation of morulae and blastocysts, resulting in fewer cells in the blastocyst stage embryos and increased levels of apoptosis. Quantitative reverse transcriptase (RT) PCR analysis revealed that the expression levels of the pro-apoptotic genes Bax and Casp3 at the blastocyst stage were increased significantly by the addition of either 25 or 55 mM glucose to the culture medium. However, addition of 25 or 55 mM glucose to the culture medium did not change the copy numbers of the apoptosis-related miRNAs mmu-mir-15a, mmu-mir-16 and mmu-mir-21. MitoTracker Green fluorescence revealed a decrease in the mitochondrial mass. The expression levels of the mitochondrial DNA-encoded genes Cox1 and Cox2 decreased sharply with the addition of 25 or 55 mM glucose to the culture medium. Both transcripts and protein synthesis of the glucose transporters Glut1 and Glut3 were reduced in blastocysts cultured in the presence of either 25 or 55 mM glucose. These results suggest that hyperglycemia reduces both mitochondrial content and expression levels of glucose transporters in mouse embryos developing in vitro and that this may result in apoptosis in these embryos.

A link between the interleukin-6/Stat3 anti-apoptotic pathway and microRNA-21 in preimplantation mouse embryos.

Signal transducers and activators of transcription-3 (Stat3) plays a central role in interleukin-6 (IL-6)-mediated cell proliferation by inhibiting apoptosis in a variety of cell types. MicroRNA-21 (miRNA-21), a ubiquitous miRNA, acts as an anti-apoptotic factor that seems to be indirectly but strictly linked to Stat3. In order to determine whether the IL-6 induced Stat3 anti-apoptosis pathway is linked with miRNA-21, we first determined the effects of recombinant mouse IL-6 on Stat3 expression, mouse embryo viability, and the mRNA levels of apoptosis related genes and miRNA-21 during mouse embryo development in vitro. Addition of 10 or 100 ng/ml of recombinant IL-6 to the culture medium did not affect the developmental ability of 2-cell stage embryos into blastocysts. However, total cell number was significantly increased and apoptosis was reduced in blastocyst stage embryos cultured in the presence of 100 ng/ml of recombinant IL-6. Furthermore, addition of recombinant IL-6 to the culture medium significantly increased the copy numbers of anti-apoptotic miRNA-21, up-regulated Bcl2l1, and down-regulated casp3. Similarly, the injection of mature miRNA-21 into cells up-regulated Bcl2l1 and down-regulated casp3. These results suggest that the induction of the Stat3 anti-apoptotic pathway by IL-6 is linked to miRNA-21 expression, which possibly results in the regulation of cell apoptosis in early mouse embryo development.

High mobility group box 1 (HMGB1) is implicated in preimplantation embryo development in the mouse.

High mobility group box 1 (HMGB1) regulates multiple cell functions, including transcription, DNA repair, differentiation, and apoptosis. In order to obtain insight into the role of HMGB1 in embryo development, we first evaluated its gene expression levels in mouse preimplantation embryos. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed high expression levels in zygotes, and expression steadily increased after zygotic genome activation when normalized to the rabbit Globin mRNA. Indirect immunocytochemistry showed that the HMGB1 protein was also produced in mouse embryos. Injection of a small interfering RNA (siRNA) specific for HMGB1 into zygotes specifically reduced both mRNA expression (P < 0.001) and protein synthesis of HMGB1 in early embryos developed in vitro. Injection of siRNA into the zygote did not affect development to the blastocyst stage, but significantly decreased cell numbers (P < 0.01) in the blastocyst and increased caspase3 (Casp3, P < 0.05) gene expression and apoptosis (P < 0.005). Addition of recombinant HMGB1 (Sigma, H-4652) into the culture medium enhanced the development of zygote stage mouse embryos to blastocysts, in the absence of BSA supplementation. These findings suggest that endogenous and exogenous HMGB1 are implicated in preimplantation embryo development in the mouse.

Transcription profile in mouse four-cell, morula, and blastocyst: Genes implicated in compaction and blastocoel formation.

To gain insight into early embryo development, we utilized microarray technology to compare gene expression profiles in four-cell (4C), morula (MO), and blastocyst (BL) stage embryos. Differences in spot intensities were normalized, and grouped by using Avadis Prophetic software platform (version 3.3, Strand Genomics Ltd.) and categories were based on the PANTHER and gene ontology (GO) classification system. This technique identified 622 of 7,927 genes as being more highly expressed in MO when compared to 4C (P < 0.05); similarly, we identified 654 of 9,299 genes as being more highly expressed in BL than in MO (P < 0.05). Upregulation of genes for cytoskeletal, cell adhesion, and cell junction proteins were identified in the MO as compared to the 4C stage embryos, this means they could be involved in the cell compaction necessary for the development to the MO. Genes thought to be involved in ion channels, membrane traffic, transfer/carrier proteins, and lipid metabolism were also identified as being expressed at a higher level in the BL stage embryos than in the MO. Real-time RT-PCR was performed to confirm differential expression of selected genes. The identification of the genes being expressed in here will provide insight into the complex gene regulatory networks effecting compaction and blastocoel formation.