Juraj Koppel

Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis

BackgroundFluorescent data obtained from real-time PCR must be processed by some method of data analysis to obtain the relative quantity of target mRNA. The method chosen for data analysis can strongly influence results of the quantification.ResultsTo compare the performance of six techniques which are currently used for analysing fluorescent data in real-time PCR relative quantification, we quantified four cytokine transcripts (IL-1β, IL-6 TNF-α, and GM-CSF) in an in vivo model of colonic inflammation. Accuracy of the methods was tested by quantification on samples with known relative amounts of target mRNAs. Reproducibility of the methods was estimated by the determination of the intra-assay and inter-assay variability. Cytokine expression normalized to the expression of three reference genes (ACTB, HPRT, SDHA) was then determined using the six methods for data analysis. The best results were obtained with the relative standard curve method, comparative Ct method and with DART-PCR, LinRegPCR and Liu & Saint exponential methods when average amplification efficiency was used. The use of individual amplification efficiencies in DART-PCR, LinRegPCR and Liu & Saint exponential methods significantly impaired the results. The sigmoid curve-fitting (SCF) method produced medium performance; the results indicate that the use of appropriate type of fluorescence data and in some instances manual selection of the number of amplification cycles included in the analysis is necessary when the SCF method is applied. We also compared amplification efficiencies (E) and found that although the E values determined by different methods of analysis were not identical, all the methods were capable to identify two genes whose E values significantly differed from other genes.ConclusionOur results show that all the tested methods can provide quantitative values reflecting the amounts of measured mRNA in samples, but they differ in their accuracy and reproducibility. Selection of the appropriate method can also depend on the design of a particular experiment. The advantages and disadvantages of the methods in different applications are discussed.

Inhibitory effect of IGF-I on induced apoptosis in mouse preimplantation embryos cultured in vitro

Insulin-like growth factor I (IGF-I) has been shown to promote mammalian early embryo development. Increased cell division or decreased cell death have been proposed as two main possible mechanisms in its effect. Here we examine the nature of this promoting effect in a model situation. Camptothecin (0.01 microg/ml) and actimomycin D (0.005 microg/ml) were used to induce apoptosis. Four-cell mouse embryos were cultured in vitro to blastocyst stage in the temporary (15 h) presence or absence of apoptotic inductors and in the permanent presence or absence of IGF-I (100 ng/ml). Embryos were assessed by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM) and comet assay on Day 5, 120 h after administration of hCG. The number of nuclei, the blastocyst formation, the proportion of embryos containing fragmented DNA and the percentage of apoptotic and secondary necrotic nuclei were assessed. Both inductors of apoptosis significantly increased the percentage of apoptotic and secondary necrotic cells and reduced total cell counts (camptothecin, P>0.001; actinomycin D, P>0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P<0.001). The addition of IGF-I into control samples also decreased the percentage of apoptotic and secondary necrotic cells. In contrast, IGF-I addition had no significant influence on embryo development (P>0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.

Effects of a Combination of Thyme and Oregano Essential Oils on TNBS-Induced Colitis in Mice

We examined the anti-inflammatory effects of the combination of thyme and oregano essential oil dietary administered at three concentrations (0.4% thyme and 0.2% oregano oils; 0.2% thyme and 0.1% oregano oils; 0.1% thyme and 0.05% oregano oils) on mice with TNBS-induced colitis. Treatment of colitic animals with the essential oils decreased the mRNA levels of pro-inflammatory cytokines IL-1β, IL-6, GM-CSF, and TNFα, especially after application of the medium dose. The medium dose of the essential oils significantly lowered the amount of IL-1β and IL-6 proteins too. Moreover, administration of the medium dose decreased the mortality rate, accelerated the body weight gain recovery, and reduced the macroscopic damage of the colonic tissue. Our results indicate that combined treatment with appropriate concentrations of thyme and oregano essential oils can reduce the production of proinflammatory cytokines, and thereby attenuate TNBS-induced colitis in mice.

Transformation of real-time PCR fluorescence data to target gene quantity

Gene quantification has been significantly simplified by the development of the real-time polymerase chain reaction (PCR) technique, which has become the most sensitive method for detection and quantification of specific nucleic acid sequences. Quantitative real-time PCR is widely used in basic biological and medical research as well as for diagnostics and monitoring of the disease process [for reviews see 1–6]. Fluorescence data obtained from real-time PCR must be transformed by some method of real-time PCR data analysis to obtain the target DNA or RNA quantity. Two methods are widely used for the quantification—the standard curve method and the ‘‘2 ” method [7,8]. Recently, other techniques for real-time PCR data analysis have been developed and have become alternatives to the two classical methods. This review describes and compares the traditional and the newly developed techniques used for quantification of specific DNA and RNA sequences.

Induction of stathmin expression during liver regeneration

Stathmin is a 19 kDa cytoplasmic phosphoprotein proposed to act as a relay for signals activating diverse intracellular regulatory pathways. After two‐thirds partial hepatectomy, the concentration of stathmin reached a peak between 48 and 72 hours, comparable to the levels observed in neonatal liver, at about 10 times the basal adult level. Stathmin then decreased to basal levels within 7 days, more rapidly than during postnatal tissue development (7 weeks), with no detectable change in its phosphorylation state. Interestingly, the mRNA for stathmin reached a peak much earlier than the protein, at 24 hours posthepatectomy, and decreased to a still detectable level until 96 hours after hepatectomy. Altogether, the present results further support the generatility of the implication of stathmin in regulatory pathways of cell proliferation and differentation during normal tissue development and posttraumatic regeneration.

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.

Expression of adrenergic receptors in mouse preimplantation embryos and ovulated oocytes

Epinephrine and norepinephrine can play an important role in basic developmental processes such as embryogenesis and morphogenesis, regulating cell proliferation, differentiation and migration. We showed that beta-adrenergic receptors can mediate the effects of catecholamines on preimplantation embryos in our previous work. In the present study, we designed specific oligonucleotide primers which can distinguish among all members of the alpha-adrenergic receptor family, and showed (using RT-PCR) that the alpha2C-adrenergic receptor is transcribed in ovulated oocytes, 8- to 16-cell morulae and expanded blastocysts. We did not detect the alpha2C-adrenoceptor transcript in 4-cell embryos. Our immunohistochemical study showed the presence of alpha-2C-adrenoceptor protein in ovulated oocytes, 8- to 16- cell embryos and blastocysts, but the signal in 4-cell embryos was weak, and probably represents remaining protein of maternal origin. We did not detect any other alpha-adrenergic receptor in preimplantation embryos and oocytes. Exposure of mouse preimplantation embryos to the alpha2-adrenergic agonist UK 14 304 led to significant reduction of the embryo cell number, and the effect was dose dependent. Our results suggest that epinephrine and norepinephrine could affect the embryo development in the oviduct via adrenergic receptors directly and support the opinion that maternal stress can influence the embryo even in very early pregnancy.

Expression of adiponectin receptors and effects of adiponectin isoforms in mouse preimplantation embryos

BACKGROUND Adiponectin, a pleiotropic hormone secreted from adipose tissue, can mediate some negative effects of obesity on female health, and can participate in the impaired reproductive performance of obese women. Using a mouse model, we investigated expression of adiponectin receptors in ovulated oocytes and in vivo derived preimplantation embryos, and tested effects of different adiponectin isoforms on development of preimplantation embryos in vitro. METHODS AND RESULTS Using RT-PCR and immunohistochemistry, we found expression of adiponectin receptors AdipoR1 and AdipoR2, at the mRNA and protein level, in mouse ovulated oocytes and preimplantation embryos. Quantitative real-time RT-PCR analysis showed a decrease in the amount of AdipoR1 and AdipoR2 mRNA after fertilization, which was followed by an increase in mRNA at the morula and blastocyst stage; mRNA for adiponectin was detected only at the blastocyst stage. Administration of full-length adiponectin significantly changed the distribution in numbers of cells of cultured preimplantation embryos, increasing the proportion of embryos with high cell numbers (>128 cells) and decreasing the proportion of embryos with lower cell numbers (<65 cells). Blastocysts possessed significantly higher cell numbers after full-length adiponectin treatment. Mutated trimeric adiponectin had the opposite effect, a significant decrease in the proportion of embryos with higher cell numbers (>96 cells) and increase in the proportion of embryos with lower cell numbers (<65 cells). Trimeric adiponectin also significantly decreased the cell number and increased cell death in blastocysts. Truncated globular adiponectin had no significant effect on development of mouse preimplantation embryos. CONCLUSIONS Our results indicate that adiponectin can directly influence the development of the preimplantation embryo, and the effects are isoform dependent.

Dose- and time-dependent effects of TNFα and actinomycin D on cell death incidence and embryo growth in mouse blastocysts

This study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were cultured in vitro with the addition of two apoptotic inductors--TNFalpha and actinomycin D--at various doses and times. The average number of nuclei and the percentage of dead cells were evaluated in treated embryos. Classification of dead cells was based on morphological assessment of their nuclei evaluated by fluorescence microscopy, the detection of specific DNA degradation (TUNEL assay), the detection of active caspase-3 and cell viability assessed by propidium iodide staining. The addition of both apoptotic inductors into culture media significantly increased cell death incidence in blastocysts. Their effects were dose and time dependent. Lower concentrations of inductors increased cell death incidence, usually without affecting embryo growth after 24 h culture. Higher concentrations of inductors caused wider cell damage and also retarded embryo development. In all experiments, the negative effect of actinomycin D on blastomere survival and blastocyst growth was greater than the effect of TNFalpha. Furthermore, the addition of actinomycin D into culture media increased cell death incidence even after 6 h culture. Differences resulted probably from diverse specificity of apoptotic inductors. The majority of dead cells in treated blastocysts were of apoptotic origin. Morphological and biochemical features of apoptotic cell death induced by both TNFalpha and actinomycin D were similar and had homologous profile. In blastomeres, similarly to somatic cells, the biochemical pathways of induced apoptosis included activation of caspase-3 and internucleosomal DNA fragmentation.

Maternal restraint stress negatively influences growth capacity of preimplantation mouse embryos.

In our study we investigated the effect of maternal restraint stress on preimplantation embryo development using a mouse model. We exposed hormonally stimulated (superovulated) and unstimulated (i.e. spontaneously ovulating) mouse females to restraint stress for 30 min three times a day during the preimplantation period. The stress exposure caused significant increase in blood plasma corticosterone concentration. Microscopical evaluation of embryos isolated from spontaneously ovulating females showed that maternal stress significantly increased the proportion of embryos with lower cell numbers (≤32 cells) and decreased the proportion of embryos with higher cell numbers (65-96 cells and 97-128 cells). Moreover maternal restraint stress decreased the cell counts per embryo and per blastocyst. After an additional 24 h in vitro culture we did not find any difference in the embryo distribution or in the cell counts per embryo/blastocyst between embryos isolated from stressed and control mothers. The exposure to restraint stress did not affect the incidence of apoptosis in blastocysts isolated from spontaneously ovulated dams. In gonadotropin stimulated dams, the hormonal treatment itself notably changed embryo distribution (increasing the proportion of degenerated embryos) and increased the occurrence of apoptotic cells. Our results indicate that psychical stress exposure in very early pregnancy can significantly influence the developmental capacity of preimplantation embryos.