Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.     

X chromosome regulation of autosomal gene expression in bovine blastocysts

Although X chromosome inactivation in female mammals evolved to balance the expression of X chromosome and autosomal genes in the two sexes, female embryos pass through developmental stages in which both X chromosomes are active in somatic cells. Bovine blastocysts show higher expression of many X genes in XX than XY embryos, suggesting that X inactivation is not complete. Here, we reanalyzed bovine blastocyst microarray expression data from a network perspective with a focus on interactions between X chromosome and autosomal genes. Whereas male-to-female ratios of expression of autosomal genes were distributed around a mean of 1, X chromosome genes were clearly shifted towards higher expression in females. We generated gene coexpression networks and identified a major module of genes with correlated gene expression that includes female-biased X genes and sexually dimorphic autosomal genes for which the sexual dimorphism is likely driven by the X genes. In this module, expression of X chromosome genes correlates with autosome genes, more than the expression of autosomal genes with each other. Our study identifies correlated patterns of autosomal and X-linked genes that are likely influenced by the sexual imbalance of X gene expression when X inactivation is inefficient.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

Global gene expression analysis of bovine somatic cell nuclear transfer blastocysts and cotyledons

Low developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos is a universal problem. Abnormal placentation has been commonly reported in SCNT pregnancies from a number of species. The present study employed Affymetrix bovine expression microarrays to examine global gene expression patterns of SCNT and in vivo produced (AI) blastocysts as well as cotyledons from day‐70 SCNT and AI pregnancies. SCNT and AI embryos and cotyledons were analyzed for differential expression. Also in an attempt to establish a link between abnormal gene expression patterns in early embryos and cotyledons, differentially expressed genes were compared between the two studies. Microarray analysis yielded a list of 28 genes differentially expressed between SCNT and AI blastocysts and 19 differentially expressed cotyledon genes. None of the differentially expressed genes were common to both groups, although major histocompatibility complex I (MHCI) was significant in the embryo data and approached significance in the cotyledon data. This is the first study to report global gene expression patterns in bovine AI and SCNT cotyledons. The embryonic gene expression data reported here adds to a growing body of data that indicates the common occurrence of aberrant gene expression in early SCNT embryos. Mol. Reprod. Dev. 76: 471–482, 2009. © 2008 Wiley‐Liss, Inc.     

Analysis of development-related gene expression in cloned bovine blastocysts with different developmental potential

The high incidence of abnormalities in cloned calves is a most serious problem for bovine somatic cell nuclear transfer (NT) technology. Because there is little information on the differences in mRNA expression in cloned blastocysts with donor cells of different sex and origin, we compared development-related gene expression in two types of cloned bovine blastocysts with different potentials to develop into normal calves, a female adult cumulus cell line (high potential to develop into live calves) and a male fibroblast cell line (low potential to develop into live calves) to examine the correlation between the normality of cloned calves and blastocyst mRNA expression patterns. We analyzed 12 genes involved in apoptosis, growth factor signaling, metabolism, and DNA methylation in blastocysts originating from two types of donor cells and in vitro-fertilized blastocysts using quantitative real-time polymerase chain reaction. Expression of the pro-apoptotic Bax gene and anti-apoptotic Bcl-2 and Glut-1 genes in fibroblast-derived blastocysts was significantly higher than in cumulus cell-derived and in vitro-fertilized blastocysts. The high Bcl-2 and Glut-1 gene expression suggests that some embryonic cells with damaged DNA in fibroblast-derived blastocysts are not removed, and their descendants later manifest abnormal placenta or fetus formation. Transfer of pre-selected cloned blastocysts into recipients is required, however, to determine whether the expression pattern of these apoptosis-related genes reflects differences in the potential to develop into normal calves.     

Increase in DNA methylation downregulates conceptus interferon-tau gene expression

Expression of ovine interferon-tau (oIFNtau) genes, essential for the maternal recognition of pregnancy in ruminant ungulates, is restricted to the trophoblast and is not detected in any other cell types or tissues. Substantial secretion of oIFNtau starts on day 12-13 of pregnancy (day 0 = day of estrus), reaches the highest on day 16-17, and then declines rapidly. Ovine IFNtau mRNA, on the other hand, reaches the highest level on day 14 of pregnancy, 2-3 days before peak production of the protein. In this study, day 14 and 17 conceptuses treated with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, were cultured in vitro and only day 17, not day 14, conceptuses resulted in upregulation of oIFNtau gene expression. To gain insight into the molecular mechanism of oIFNtau gene downregulation, the methylation status within 1 kb of the 5'-flanking region of oIFNtau-o10 gene was investigated: CpG dinucleotides of this gene in day 14 ovine conceptuses were hypomethylated compared to day 20 conceptuses or other tissues. In vitro methylation of oIFNtau-o10-reporter constructs caused suppression of reporter activity in transient transfections. Cotransfection of methyl-CpG-binding protein (MeCP2) with the reporter construct elicited further suppression of the reporter activity. In electrophoretic mobility shift assay (EMSA), patterns of shifted bands did not show much difference between methylated and unmethylated probes in distal regions, but exhibited differences in the proximal region of upstream sequences of the oIFNtau gene. These results provide evidence that changes in the degree of DNA methylation could be one of the major mechanisms leading to downregulation of the oIFNtau-o10 gene during early gestation, and possibly its silencing in nonconceptus tissues.     

Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferrioxamine

Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.     

Differential apoptosis-related protein expression, mitochondrial properties, proteolytic enzyme activity, and DNA fragmentation between skeletal muscles

Increased skeletal muscle apoptosis has been associated with a number of conditions including aging, disuse, and cardiovascular disease. Skeletal muscle is a complex tissue comprised of several fiber types with unique properties. To date, no report has specifically examined apoptotic differences across muscles or fiber types. Therefore, we measured several apoptotic indices in healthy rat red (RG) and white gastrocnemius (WG) muscle, as well as examined the expression of several key proteins across fiber types in a mixed muscle (mixed gastrocnemius). The protein content of apoptosis-inducing factor (AIF), apoptosis repressor with caspase recruitment domain (ARC), Bax, Bcl-2, cytochrome c, heat shock protein 70 (Hsp70), and second mitochondria-derived activator of caspases (Smac) were significantly (P < 0.05) higher in RG vs. WG muscle. Cytosolic AIF, cytochrome c, and Smac as well as nuclear AIF were also significantly (P < 0.05) higher in RG compared with WG muscle. In addition, ARC protein expression was related to muscle fiber type and found to be highest (P < 0.001) in type I fibers. Similarly, AIF protein expression was differentially expressed across fibers; however, AIF was correlated to oxidative potential (P < 0.001). Caspase-3, -8, and -9 activity, calpain activity, and DNA fragmentation (a hallmark of apoptosis) were also significantly higher (P < 0.05) in RG compared with WG muscle. Furthermore, total muscle reactive oxygen species generation, as well as Ca(2+)-induced permeability transition pore opening and loss of membrane potential in isolated mitochondria were greater in RG muscle. Collectively, these data suggest that a number of apoptosis-related indices differ between muscles and fiber types. Given these findings, muscle and fiber-type differences in apoptotic protein expression, signaling, and susceptibility should be considered when studying cell death processes in skeletal muscle.     

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts

Vitrification is becoming a preferred method for pre‐implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo‐ and in vitro‐produced bovine embryos after vitrification. In vitro‐ (IVF) and in vivo‐derived (IVV) bovine blastocysts were identified as follows: in vitro‐produced fresh (IVF‐F), in vitro‐produced vitrified (IVF‐V), in vivo‐derived fresh (IVV‐F), in vivo‐derived vitrified (IVV‐V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF‐F × IVV‐F, IVF‐V × IVV‐V, IVF‐F × IVF‐V, and IVV‐F × IVV‐V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up‐regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up‐regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo‐produced blastocysts. After vitrification, however, in vitro‐produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro‐produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up‐regulation of genes that are involved in stress responses. Mol. Reprod. Dev. 79: 613–625, 2012. © 2012 Wiley Periodicals, Inc.     

Polony analysis of gene expression in ES cells and blastocysts

Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously.     

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 × 10⁶ cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 °C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced.     

cDNA cloning and expression of an apoptosis-related gene, humanTFAR15 gene

By means of cDNA-RDA method, some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these fragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1 218 nucleotides and encodes 212 amino acids. The putative protein product of TFAR15 is partially homologous toC. elegans protein C14A4.11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung, and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-1 cells after GM-CSF withdrawal.In vitro analysis showed that the recombinant TFAR15 protein could inhibit the natural cell death of 293 cells, an embryonic kidney cell line.     

DNA methylation status of bovine blastocysts obtained from peripubertal oocyte donors

In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age‐related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8‐month‐old donors was more hypermethylated, while the DNA from embryos from 11‐month‐old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor β1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.