Pavol Rehák

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.

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.

Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay

The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.

Polo‐like kinase 1 is essential for the first mitotic division in the mouse embryo

Polo‐like kinase 1 (PLK1), a member of the serine/threonine protein kinases family, is involved in multiple steps of mitotic progression. It regulates centrosome maturation, mitotic spindle formation, and cytokinesis. While studied extensively in somatic cells, little is known about PLK1 activities in the mammalian preimplantation embryo. We examined the role of PLK1 in the one‐cell mouse embryo. Western blotting showed that the PLK1 protein content increased significantly during the S‐phase of the one‐cell stage and declined during the first mitotic division. Activation of PLK1 preceded nuclear envelope breakdown (NEBD) in both pronuclei at the entry to first embryo mitosis. Immunofluorescence revealed the presence of phosphorylated, active PLK1 (pThr210‐PLK1) in both male and female pronuclei, and in the microtubule‐organizing centers (MTOCs) shortly before NEBD. During the first mitotic metaphase, pThr210‐PLK1 accumulated at the spindle poles and was also associated with condensed chromosomes. Inhibition of PLK1 activity with a specific PLK1 inhibitor, BI 2536, at the one‐cell stage induced the formation of a bipolar spindle that displayed disordered microtubular arrangements and dislocated, condensed chromosomes. Although such embryos entered mitosis, they did not complete mitosis and arrested at metaphase. Time‐lapse recording revealed progressive misalignment of condensed chromosomes during first mitotic metaphase. These data indicate that PLK1 activity is not essential for entry into first mitosis, but is required for the events leading up to metaphase‐anaphase transition in the one‐cell mouse embryo. Mol. Reprod. Dev. 80: ?–?, 2013.

Nucleolus in apoptosis-induced mouse preimplantation embryos.

Apoptosis may occur in early embryos in which the execution of essential developmental events has failed. Thus the initiation of the apoptotic mechanism may be related to activation of the embryonic genome. In this way, developmentally incompetent cells or whole embryos are eliminated. It is likely that some link exists between failed resumption of rRNA synthesis and the incidence of apoptosis in cleaving embryos. In this context, decreased developmental potential in cleaving nucleotransferred embryos is consistent with cell loss, and very likely due to programmed cell death. The effects of apoptosis inducers on cleaving embryos have not been characterised in comparable detail to that in the case of somatic cells. Early embryos provide a very good model for study of these processes because of the specificity of rRNA transcription resumption after fertilization. In our experiments three apoptosis inducers (staurosporin 10 mM, actinomycin D 0.05 mg/ml and camptothecin 0.1 mg/ml) were used in a culture medium for 15 h at the 4-cell stage (day 2) of mouse embryos, followed by further development in a pure culture medium until fixation on days 3, 4 and 5. In staurosporin-induced embryos, light microscopy immunostaining of nucleolar proteins (fibrillarin, Nopp140, protein B23) did not reveal changes in nucleolar morphology on day 3. On days 4 and 5, more compact (roundish) nucleoli (in comparison with controls) were observed. The embryos treated with camptothecin displayed a similar staining pattern to those with staurosporin at each day. In actinomycin-D-treated embryos, marked changes in nucleolar appearance were visible as early as day 3. These changes in nucleolar morphology consisted of loss of the reticulation appearance and fragmentation of nucleoli. In addition to nucleolar changes, significantly decreased cell proliferation was observed. The induced embryos did not reach the blastocyst stage. The number of blastomeres was decreased, and staining with Hoechst 33342 revealed a significant percentage of apoptotic nuclei (condensed/fragmented nuclei) from day 4.

Do embryonic polar bodies commit suicide

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.