Survivin acts as anti-apoptotic factor during the development of bovine pre-implantation embryos

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos.     

Differential expression pattern of key regulatory developmental genes in pre-implant zona free cloned vs in vitro fertilized goat embryos

The success of Somatic cell nuclear transfer (SCNT) primarily depends on the extent of reprogramming of donor cells genome. The error of reprogramming may lead to inappropriate expression of embryonic genes at any stage of development. Under the present study the relative expression of different genes related to pluripotency (Oct-4 and Nanog), growth factors (IGF-2 and IGF-2R) and DNA methyltransferase gene (Dnmt-1) was evaluated in SCNT embryos at 8–16 cells, morula and blastocyst stages as compared to IVF group. In SCNT, significantly higher degree of relative expression was observed for Oct-4 in morula (1.41) and blastocysts (1.14) as compared to 8–16 cells (referral stage) whereas in IVF, a lower expression was observed at morula (0.82) stage. The expression of Nanog in SCNT embryos was increased significantly in morula (2.23) and decreased subsequently in blastocyst (0.56), whereas it was increased significantly from 8 to 16 cells to morula (1.62) and blastocyst (4.5) of IVF group. The IGF-2 and IGF-2R showed significantly higher expression rates in morula and blastocysts of SCNT (6.56, 5.90 and 1.11, 1.4) and IVF (8.69, 8.25 and 2.96, 3.91) embryos, respectively as compared to referral stage. The expression of Dnmt-1 was significantly higher in SCNT morula (1.29) and blastocyst (1.15) however in IVF, it was similar in 8–16 cells stage and morula but, higher in blastocyst (1.58). The dissimilar pattern of gene expression of SCNT might be a consequence of incomplete reprogramming of donor nucleus which resulted into lower blastocyst rate of SCNT as compared to IVF embryos.     

Allele‐specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre‐implantation bovine embryo development by characterizing the allele‐specific expression pattern of the X chromosome‐linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4‐, 8‐ to 16‐cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT‐PCR‐RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele‐specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4‐, 8‐ to 16‐cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele‐specific expression of an X‐linked gene that is subject to XCI in in vitro bovine embryos from the 4‐cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. 77: 615–621, 2010. © 2010 Wiley‐Liss, Inc.     

Gene expression and in vitro development of inter-species nuclear transfer embryos

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.     

RNA interference as a tool to study gene function in bovine oocytes

RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.     

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.     

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.     

Knockdown of IGF-IR by siRNA injection during bovine preimplantation embryonic development

This study aimed to assess the efficiency and effects of insulin-like growth factor receptor-1 (IGF-IR) siRNA knockdown during bovine preimplantation embryonic development. In oocytes injected with IGF-IR siRNA, the relative IGF-IR mRNA levels compared to controls were 28% and 46% at 6 and 24 h after injection, respectively. With respect to the injection of IGF-IR siRNA in zygotes, 24 h after injection the relative levels of IGF-IR mRNA and protein in the two-cell embryos were 74% and 78% of those in the controls, respectively. IGF-IR siRNA reduced blastocyst formation (23.2%) compared to siRNA controls (33.0%) and uninjected oocytes (35.4%; P < 0.05) and the number of viable cells per IGF-IR siRNA-treated blastocyst (64 ± 3) was significantly reduced, compared to control siRNA and uninjected blastocysts (81 ± 3 and 116 ± 4; P < 0.01). In conclusion, IGF-IR siRNA knockdown reduces the development of bovine embryos, and microinjection in zygotes can decrease blastocyst cell number.     

RNA interference for the organizer-specific gene Xlim-1 in Xenopus embryos

Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in Xenopus embryos. First, expression of an exogenous luciferase gene as a reporter in embryos was reduced by coinjection with dsRNA corresponding to the luciferase gene. Next, injection of dsRNA for Xlim-1, a homeobox gene suggested to be involved in Spemann organizer functions, reduced the endogenous level of Xlim-1 mRNA and produced embryos with reduced eyes or anterior truncation at high efficiency. In addition, injection of an antisense expression construct of Xlim-1 elicited phenotypes very similar to those of Xlim-1 dsRNA-injected embryos. These results indicate the effectiveness of RNAi for loss of function studies in Xenopus embryos, and the importance of Xlim-1 in head formation.     

Gene expression regulating blastocyst formation

Development of embryos to the blastocyst stage is a critical event in the early lives of all eutherian mammalian species. Blastocyst formation is essential for implantation and is the principal morphological determinant of embryo quality prior to embryo transfer. The physiological events and roles of specific gene families that regulate blastocyst formation are subjects of intense research Recent findings have demonstrated that bovine embryos express multiple members of the Na/K-ATPase ion transporter gene family. Two members of this family have been co-localized to bovine trophectoderm, but each becomes largely confined to opposing cell membrane margins. Bovine blastocysts display a greater sensitivity to ouabain (potent inhibitor of the Na/K-ATPase) than murine blastocysts, and enzyme activity (ouabain sensitive 86Rb+ uptake) undergoes a 9-fold increase from the bovine morula to the blastocyst stage. Disruption of Na/K-ATPase gene expression by antisense oligodeoxynucleotide inhibition abolishes blastocyst formation. These results have implicated the Na/K-ATPase as a key regulator of bovine blastocyst formation and have provided insights necessary for the production of healthy bovine embryos by the application of in vitro maturation, in vitro fertilization and in vitro culture methods.     

RNA interference‐mediated knockdown of IAP in Lygus lineolaris induces mortality in adult and pre‐adult life stages

In recent years, RNA interference (RNAi) has been validated as a viable approach for functional genetic studies in non‐model organisms. In this report we demonstrate the efficacy of RNAi in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Miridae: Hemiptera). A L. lineolaris inhibitor of apoptosis gene (LlIAP) has been identified and cloned. The translated sequence encodes a 381 amino acid protein similar to other insect IAPs and contains two conserved baculovirus inhibitor of apoptosis protein repeat (BIR) domains. Microinjection of double stranded RNA (dsRNA) corresponding to two disparate portions of the gene resulted in decreased LlIAP mRNA quantities relative to controls. Both nymphs and adult specimens injected with IAP dsRNA exhibited significantly reduced lifespan compared with those injected with non‐insect dsRNA (eGFP). Thus, RNAi‐mediated knockdown of LlIAP expression has been correlated with a lethal phenotype in adults and nymphs.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.