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The aim of this study was to compare the sensitivity of mouse preimplantation embryos obtained from mothers with different body conditions to an environment with increased oxidative stress. An intergenerational dietary model based on mouse overfeeding during the intrauterine and early postnatal period was used to produce females with increased body fat content (≥ 11 %). Three different sources of oxidative stress were applied: 0.01 mM 2,2'-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), free radical-generating compound; 5 mM l-Buthionine-sulfoximine (BSO), glutathione synthesis inhibitor; and 0.01 mM Sodium nitroprusside dihydrate (SNP), nitric oxide donor. Two-cell embryos isolated from controls (with 7 %-8 % body fat content) and overweight mice were cultured in vitro with selected compounds until blastocyst formation. Development of two-cell embryos isolated from overweight dams was negatively affected by the presence of BSO and SNP (P0.01). Similar impact was recorded in two-cell embryos obtained from control mothers only after exposure to BSO (P0.05). Fluorescence analysis of blastocysts recovered from overweight dams revealed reduced total cell numbers after AAPH and BSO treatment, and increased incidence of cell death after BSO and SNP. In the controls, negative impact on blastocyst quality, represented by reduced cell number, was observed only after BSO. Immunofluorescence evaluation of freshly-recovered zygotes and two-cell embryos showed that those obtained from overweight dams displayed significantly lower fluorescence signal intensity of Glutathione peroxidase 8 than those from control dams. In conclusion, the results suggest that preimplantation embryos originating from overweight mice might be more vulnerable to oxidative stress than those originating from control females.

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

The aim of this study was to evaluate the effects of being overweight on the ability to conceive, fertilization rate, and in vivo development of embryos in regularly cycling, spontaneously ovulating, and naturally mated female mice. The study was based on statistical analysis of data collected during 14 experiments with identical design, performed on 319 control and 327 obese mice, developed in an intergenerational model of obesity induction which eliminates the impact of aging and high-fat feeding. Six-week-old mice with a vaginal sperm plug were slaughtered on embryonic days 2, 3, or 4, and the flushed contents of the oviducts and uteri were assessed by stereomicroscopy. The results showed no association between being overweight and the proportion of ovulating or fertilized females. On the other hand, a strong association was found between being overweight and ovulation yield. On embryonic day 2, significantly higher numbers of eggs were recovered from the oviducts of fertilized obese mice. Maternal overweight status was also associated with higher developmental capacities of preimplantation embryos. In conclusion, contrary to studies based on the high-fat-diet model, in female mice fed regular chow, being overweight was associated with an increased ovulation quota and higher developmental rate of fertilized oocytes. Being overweight did not impact ability to conceive. On the other hand, as documented in our previous studies, the quality of oocytes and blastocysts recovered from overweight mice developed in an intergenerational model of obesity was low.

In this study the possible toxicity of phenylpyrazole fipronil, the related commercial product FIPRON spot-on as well as FIPRON spot-on secondary ingredients on the developmental capacities and quality of mouse preimplantation embryos was evaluated. During in vitro tests, isolated two-cell stage embryos were cultured in media with addition of the listed chemicals until blastocyst formation. Stereomicroscopic evaluation of in vitro produced embryos showed that fipronil at 1 μM and higher concentration negatively affected embryonic development. Fluorescence staining revealed that the obtained blastocysts displayed lower numbers of blastomeres at 10 μM concentrations and elevated incidence of cell death from 1 μM concentration. The presence of FIPRON spot-on at a concentration equivalent to 10 μM of fipronil caused massive degeneration of all embryos. Secondary ingredients (butylhydroxyanisolum, butylhydroxytoluenum) at corresponding concentrations negatively impacted the development and quality of preimplantation embryos as well. During in vivo tests (daily oral administration of fipronil during the preimplantation period) in embryos collected from treated mouse females, significantly elevated incidence of cell death was observed even at the acute reference dose. Fipronil impaired the development and quality of mouse preimplantation embryos in both in vitro and in vivo tests. Embryotoxicity of the commercial product FIPRON spot-on was potentiated by the presence of secondary ingredients.

The objective of this study was to evaluate the potential toxicity of pyrethroids (deltamethrin, permethrin, fenvalerate, λ-cyhalothrin), commercial pyrethroid-based products DECIS EW 50 (deltamethrin mixture), TOP SPOT ON STRONGER (permethrin mixture), as well as related secondary ingredients on mouse preimplantation embryo development. Two-cell stage embryos were in vitro cultured with addition of the listed chemicals until blastocyst formation. All active pyrethroids negatively affected embryonic development at 1000 μM concentration. Decreased quality of obtained blastocysts in permethrin, fenvalerate and λ-cyhalothrin-treated embryos was revealed as well. Deltamethrin showed harmful impact on embryo development at 100 μM concentration. Lower concentrations of pyrethroids (1, 10 μM) had no effect on embryo development. The presence of DECIS EW 50 containing deltamethrin at 100 μM caused degeneration of all embryos. Similarly, TOP SPOT ON STRONGER containing 100 μM of permethrin impaired embryonic development and quality of obtained blastocysts. Evaluated secondary ingredients (butylhydroxyanisol, butylhydroxytoluen, butylparaben and cyclohexanone) at corresponding concentrations showed damaging impact on preimplantation embryo development as well. Our results indicate that the embryotoxic potential of active pyrethroids is relatively low, whereas pyrethroid-based products have relatively high potential to impair mouse preimplantation development. Embryotoxicity of commercial products is probably attributable to the presence of secondary ingredients.

The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors.

To investigate possible involvement of glucocorticoid receptor (GR) in mediating effects of maternal stress or therapeutically administered glucocorticoids on early embryo, we analyzed the expression of GR subtypes in ovulated mouse oocytes and preimplantation embryos. RT-PCR analysis results showed that GRα and GRγ transcripts are relatively highly expressed in mouse oocytes, and both transcripts are present at lower amounts in preimplantation embryos. We also detected low expression of two other splice variants, GRβ and a transcript orthologous to the human GR-P subtype, mainly at the blastocyst stage. Using western blot analysis, we detected several GR protein bands that differed in size between oocytes and preimplantation embryos. To compare the effects of corticosterone (a major endogenous glucocorticoid in rodents) and dexamethasone (a synthetic glucocorticoid) on early embryos, we cultured mouse preimplantation embryos in the presence of these glucocorticoids. Corticosterone showed a strong inhibitory effect on embryo development (starting from a 50 μM concentration), without a significant influence on apoptosis incidence. On the other hand, dexamethasone induced apoptosis in early embryo cells (starting from a 1.5 μM concentration), and its effect on embryo development was less detrimental than that found with the same dose of corticosterone. In summary, our results showed that different GR subtypes are expressed in ovulated mouse oocytes and preimplantation embryos and that the composition of GR subtypes changes during early embryo development. Moreover, we found significant differences in the effects of the two glucocorticoids on early embryo development, which might be associated with activation of different GR subtypes.

Using RT-PCR we examined expression of dopamine and adrenergic receptors in undifferentiated and spontaneously differentiating mouse embryonic stem (ES) cells. We also examined expression of dopamine receptor subtypes in mouse ovulated oocytes and preimplantation embryos. Comparing the expression of catecholamine receptors in undifferentiated mouse ES cells and in blastocysts (from which ES cells are derived), we found that transcripts of all five dopamine receptors were expressed in both cell types. In contrast, we detected eight adrenergic receptor subtypes in undifferentiated mouse ES cells, but only three subtypes were found in mouse blastocysts. In three adrenergic receptors (α1D, α2B, β1), we found higher expression in the spontaneously differentiating ES cells than in undifferentiated ES cells, and the α1B adrenoceptor was not even detectable in the undifferentiated cells. These results indicate that genes encoding all types of catecholamine receptors are transcribed in mouse ES cells, and some of them are differentially expressed during ES cell differentiation. We found several profiles of dopamine receptor mRNA expression during the preimplantation period. The DR3 transcript was present in all examined stages (oocytes, 4-cell embryos, 8- to 16-cell embryos, blastocysts). DR1 and DR4 transcripts were not found in oocytes, but we detected them in preimplantation embryos. The DR2 receptor transcript was found in all examined stages except for the 4-cell embryos, and the DR5 receptor transcript was found in all examined stages except for the 8- to 16-cell embryos. The expression profiles of dopamine receptor transcripts suggest different roles of some receptor subtypes in particular preimplantation developmental stages.

The fused quinazolinone derivative, RX-207, is chemically and functionally related to small molecule inhibitors of protein binding to glycosaminoglycans (SMIGs). Composed of a planar aromatic amine scaffold, it inhibits protein binding to glycosaminoglycans (GAGs). RX-207 reduced neutrophil migration in thioglycollate-induced peritonitis (37%), inhibited carrageenan-induced paw edema (32%) and cerulein-induced pancreatitis (28%), and increased animal survival in the mouse model of cecal ligation and puncture (CLP)-induced sepsis (60%). The mechanism of RX-207 action, analyzed by UV spectroscopy, confirmed that which was elucidated for chemically related anti-inflammatory SMIGs. RX-207 binding to cell surface GAGs can account for the inhibition of neutrophil recruitment via the micro-vasculature and as a consequence, the reduction of neutrophil mediated tissue damage in the animal models of inflammation and improved survival of mice in CLP-induced sepsis.

The potential toxicity of neonicotinoids (thiacloprid, acetamiprid, thiamethoxam and clothianidin) as well as related commercial products Calypso 480SC (thiacloprid mixture), Mospilan 20SP (acetamiprid mixture) and Agita 10WG (thiamethoxam mixture) on developmental capacities and quality of preimplantation embryos was evaluated. During in vitro tests, isolated 2-cell stage mice embryos were cultured in media with various concentrations of active compounds or commercial products until blastocyst formation. As found using stereomicroscopic examination, all neonicotinoids at highest (100μM) concentration negatively affected embryonic development (P0.001). Fluorescence staining revealed that the blastocysts obtained displayed lower numbers of blastomeres and elevated incidence of cell death. Thiacloprid and acetamiprid decreased quality of blastocysts also at 10μM concentration. From the tested products only Calypso 480SC containing 10μM of thiacloprid showed harmful impact on embryo quality. In an experiment using rabbit embryos, similar negative effect of thiacloprid in vitro was recorded. In vivo testing confirmed that blastocysts collected from thiacloprid-treated mice displayed lower total cell counts than blastocysts from controls. The sensitivity of embryonic cells to neonicotinoids is in the order of thiaclopridacetamiprid, thiomethoxamclothianidin. Thiacloprid impairs development and quality of both mouse and rabbit preimplantation embryos, and shows embryotoxicity even at acute reference dose.

The aim of the present study was to test the hypothesis that leptin can directly mediate the negative effect of maternal obesity on preimplantation embryos. As previously shown, maternal obesity retards early embryonic development in vivo and increases the incidence of apoptosis in blastocysts. When two-cell embryos isolated from control and obese mice were transferred to identical (leptin-free) conditions in vitro, no differences in any growth or quality parameters were recorded, including apoptosis incidence in blastocysts. Embryos isolated from control mice responded to transfer to environments with a high concentration of leptin (10 ng/ml) with a significant increase in arrest at the first or subsequent cell cycle. However, the majority of non-arrested embryos developed into blastocysts, showing morphology comparable to those cultured in the leptin-free group. On the other hand, the exposure of embryos isolated from obese mice to high leptin concentration in vitro did not retard their development. Furthermore, these embryos developed into blastocysts, showing a lower incidence of apoptosis. In vivo-developed blastocysts recovered from obese mice showed elevated expression levels of the pro-apoptotic gene BAX and the insulin-responsive glucose transporter gene SLC2A4. In conclusion, elevated leptin levels have both positive and negative effects on preimplantation embryo development in vitro, a response that likely depends on the body condition of the embryo donor. Moreover, these results suggest that leptin acts as a survival factor rather than an apoptotic inductor in embryonic cells. Since no elevations in the expression of the leptin receptor gene (LEPR) or fat metabolism-associated genes (PLIN2, SLC27A4) were recorded in blastocysts recovered from obese mice, the role of leptin in mediating the effects of obesity on embryos at the peripheral level is likely lower than expected.

Catecholamines play an important role in embryogenesis, and data obtained in the rodent model indicate that they can act even during the preimplantation period of development. Using RT-PCR with specific oligonucleotide primers distinguishing among all members of the adrenergic receptor family, we examined expression of adrenergic receptors in bovine and rabbit oocytes, morulas and blastocysts. We found several profiles of adrenoceptor mRNA expression. Transcripts for some receptor subtypes (bovine alpha 2 receptors, rabbit α2A, α2C, β1 and β2 receptors) were detected at all examined stages, which suggests receptor expression throughout (or at most stages) the preimplantation developmental period. Expression in oocytes but not at later stages was found in only one adrenoceptor subtype (rabbit α1B). In contrast, mRNA for several adrenoceptors was found in embryos but not in oocytes (bovine beta adrenoceptors and rabbit α1A). Nucleotide sequences of our PCR products amplified in rabbit oocytes, and preimplantation embryos represent the first published mRNA sequences (partial sequences coding at least one transmembrane region) of rabbit α2C, β1 and β2 adrenoceptors. Our results suggest that the expression of adrenergic receptors can be a general feature of mammalian oocytes and preimplantation embryos. On the other hand, comparison of three mammalian species (cattle, rabbit and mouse) revealed possible interspecies differences in the expression of particular adrenoceptor subtypes. Our results support the opinion that stress mediators can act directly in cells of preimplantation embryos.

SummaryThe 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.

BACKGROUND The involvement of biogenic monoamines in early ('preneural') embryogenesis has been well documented in lower vertebrates, but much less information is available about the role of these molecules in the earliest stages of development in mammals, including humans. METHODS Databases (PubMed, ISI Web of Knowledge and Scopus) were searched for studies relating to biogenic monoamines functioning in early embryos. The available data on the expression of histamine, serotonin and adrenergic receptors during mammalian preimplantation development were summarized, and the potential roles of biogenic monoamines in very early pregnancy were discussed. RESULTS The roles of biogenic monoamines in mammalian preimplantation embryo development can be diverse, depending on the embryo developmental stage, and the physiological status of the maternal organism. Several receptors for biogenic monoamines are expressed and biologically functional in cells of preimplantation embryos. Activation of histamine receptors can play a role in embryo implantation and trophoblast invasion. Activation of adrenergic and serotonin receptors can influence proliferation and survival of early embryonic cells. CONCLUSIONS Biogenic monoamines can play an important role in physiological conditions, contributing to embryo-maternal interactions, or can influence the early embryo under unfavorable or pathological conditions (e.g. in maternal stress, or in women taking certain antidepressants, anti-migraine or anti-ulcer drugs).

The aim of this study was to evaluate the possible effect of non-specific acute inflammation localized outside the reproductive tract on the quality of preimplantation embryos. In fertilized female mice two experimental models of inflammation were used-trinitrobenzene sulfonic acid colitis and carrageenan paw oedema. Inflammation was induced during the cleavage period of embryo development and embryos were collected at 92 h post hormonal synchronization. Stereomicroscopical evaluation of in vivo derived embryos showed that the presence of inflammation in the maternal body did not affect their basic developmental abilities, i.e. there were no significant differences in the proportion of early blastocysts, morulas, slowly developing embryos and degenerates between embryonic pools obtained from mothers with induced inflammation and control mothers. In the next step, non-degenerated embryos from all mothers were cultured in vitro under standard conditions for another 24 h, and the average cell number (fluorescence DNA staining) and the incidence of cell death (fluorescence viability staining combined with TUNEL assay) were evaluated. The majority of cultured embryos reached expanded blastocyst stage. There were no significant differences in the average cell numbers of blastocysts, but blastocysts derived from mothers with induced inflammation showed a significantly higher incidence of dead cells in both experiments. The majority of dead cells were of apoptotic origin. These results show that non-specific inflammation localized outside the reproductive tract has no detrimental effect on the preimplantation embryo growth; however it can affect the embryo quality.

Zusammenfassung Der Einflus von Insulin und Methionin auf den Aminosauren-Stoffwechsel von saugenden und wiederkauenden Lammern Es wurden die Unterschiede in der Konzentration von freien Aminosauren im Blutserum bei saugenden und wiederkauenden Lammern nach der Injektion von 4 U/kg s. c. bzw. 4 U/kg i. p. Insulin und Methionin (300 mg/kg) untersucht. Die Kontrollgruppe erhielt statt Methionin 0,9% NaCl-Losung. Die Analyse der freien Aminosauren im Blutplasma wurde durch Saulen-Chromatographie mit Hilfe des automatischen Aminosauren-Analysators HD-1200 durchgefuhrt. Wahrend der Aufnahme von Milch konnte ein Abfall in der Konzentration der essentiellen Aminosauren im Blutserum 2,5 Std. nach s.c. und i.p.-Insulin-Injektion und ebenso nach Methion-Injektion beobachtet werden. Dieser Abfall betraf hauptsachlich Lysin, Isoleucin, Phenylalanin und die nicht essentielle Aminosaure Tyrosin. Beim wiederkauenden Lamm konnten die gleichen Beobachtungen 7,5 Std. nach intraperitonealer Verabreichung von Insulin gefunden werden. Nach der Verabreichung von Methionin waren diese Unterschiede starker ausgepragt. Die Unterschiede in der Blutserum-Konzentration der Aminosauren bei Lammern mit entwickeltem Vormagensystem zeigt, das eine intraperitoneale Verabreichung des Insulins biologisch effektiver ist als eine subcutane Injektion und, das Insulin den Aminosauren-Stoffwechsel nicht direkt beeinflust, da signifikante Unterschiede erst nach 7,5 Std. nach der Injektion beobachtet wurden, wahrend eine neutrale Zn-Insulin-Injektion innerhalb von 5–6 Std. wirksam wird.

SummaryThe present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were culturedin vitroand treated with chemicals that cause developmental arrest and apoptosis (α-amanitin, actinomycin D, TNF-α). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

The objective of this study was to determine the effects of bovine growth hormone (bGH) on body growth of suckling lambs. Suckling Merino lambs (fed on milk replacer from 4th day of life) were daily injected from 10th to 38th day of life with 80 micrograms bGH/kg b.w. Only insignificant increase of body weight was observed after 4 weeks of GH treatment. After 31st day of life body weight gain in the experimental lambs was significantly higher. It can be concluded that bGH increases body weight gain of lambs in 2nd month of life. It is probable that growth rate of sucklings is limited only by biological and biochemical capabilities of the organism and it can be hardly influenced by exogenous hormonal manipulation.

The objective of this study was to determine the effects of growth hormone on body growth and the content of RNA, DNA and protein in liver, brain and heart muscle in rat pups from reduced nests. Suckling rats raised in litters of 4 youngs were daily injected from 3rd to 28th day of life with 1 microgram of bGH/g body weight. Our results show that GH administration evoked higher body weight gain and increase of tail length after 23rd day of life. There was significant increase of liver content of nucleic acids and protein. This GH-responsivity of pups from reduced nests might be related to differences in their metabolic profile and accelerated somatic and psychosocial development in comparison to normal youngs.

The experiment was conducted to investigate the effects of Cinnamomum zeylanicum essential oil on antioxidant status of chickens. Thirty-two female Ross 308 hybrid broilers were fed one of four diets supplemented with 0%, 0.1%, 0.05% and 0.025% of essential oil for 38 days. Blood, liver, kidney and duodenal epithelium were collected for the subsequent evaluation of antioxidant status. Feeding of adiet supplemented with 0.1% of essential oil significantly decreased the concentration of malondialdehyde (MDA) in plasma and duodenal mucosa in comparison with the control group (0%). The activities of glutathione peroxidase (GPx) were significantly higher in blood of chicks fed the diet containing 0.1% of essential oil. Diets containing 0.05% and 0.025% of essential oil reduced alanine amino transferase (ALT) activity in plasma in comparison with the control group. Blood phagocytic activity significantly increased in chickens fed the diet supplemented with 0.1% and the index of phygocytic activity was affected by the diet containing 0.025% of essential oil in comparison with the control group. The present investigation shows that Cinnamomum zeylanicum essential oil exhibits a significant antioxidant activity in fattening chickens and can be used as a source of antioxidant in dietary supplement.

Plant essential oils are plant secondary metabolites possessing various pharmacological properties, primarily anti-oxidative, antimicrobial or immunomodulatory ones. The aim of this work was to study the effects of thyme essential oil dietary administration in murine DTH/ CHS reaction, carrageenan paw oedema and TNBS colitis. Thyme essential oil was added to the murine diet at three concentrations (5000, 2500 and 1250 ppm) and fed to Balb/c mice. The extent of ear swelling in DTH/CHS reaction and paw oedema induced by carrageenan application was measured using the Mitutoyo thickness gauge. In the model of TNBS colitis we evaluated the changes in body weight, the colon weight : body weight ratio, bacterial translocation to mesenteric lymph nodes, and macroscopical and histological scores. IL-1β and IL-6 messenger RNA expression in colonic samples of one experimental group were assessed using quantitative real-time reverse transcriptase PCR. Dietary supplementation with 5000 ppm of thyme essential oil significantly decreased paw oedema and ear swelling. This thyme essential oil concentration caused a significant inhibition of total mRNA IL-1β expression in the mouse colon, and markedly decreased the macroscopic and microscopic scores of colitis. On the other hand, the 1250 ppm of thyme essential oil in diet increased ear oedema induced by oxazolone application in mice. Our study indicates that thyme essential oil is able to affect murine experimental inflammatory models depending on the concentration used. It is concluded that the anti-inflammatory effects of thyme essential oil should be interpreted with a caution due to its contradictory, dose-related effects.

Domarack˘ M., P. Rehak, J. Legath, J. Koppel: Effects of Fungicide Euparen Multi (Tolylfluanid) on Development of Preimplantation Embryos in Mouse. Acta Vet. Brno 2007: 76: 209-214. The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) on the development of mouse preimplantation embryos was evaluated. Euparen Multi was daily administered per os to female mice (ICR strain) at four different doses of 118, 294, 588 and 1177 mg/kg b.m., beginning on day 1 of pregnancy. Embryos obtained on day 4 of pregnancy were stained by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM), and the number of nuclei, blastocyst formation, distribution of embryos according to the nucleus number and cell death incidence were determined. Embryos in the experimental groups (except for the lowest dose 118 mg/kg b.m.) showed a highly significant dose-dependent reduction in total cell numbers corresponding to the lower proportion of blastocysts. The occurrence of cell death was significantly increased in all experimental groups, indicating that Euparen Multi is able to cause cell death at relatively low doses. Our data demonstrate that Euparen Multi could induce significant alterations in the preimplantation embryo development.

The effect of maternal stress on blastocyst quality, with respect to maternal metabolic status, was investigated in this study. We exposed female mice with different amounts of body fat to restraint stress and examined their blastocyst quality. Blood concentrations of corticosterone, leptin, adiponectin, insulin and glucose were measured in these females. Significantly lower stress-induced corticosterone elevations were observed in females with high and low amounts of body fat, indicating that the stress response was altered in these females. The basal leptin concentrations were significantly higher in females with high amounts of body fat than in females with low amounts of body fat, and stress induced different responses in these two groups of females. Our results showed that maternal stress can significantly increase the proportion of blastocysts that contain dead (apoptotic) cells in females with high and medium amounts of body fat. In females with low amounts of body fat, the proportion of blastocysts containing dead (apoptotic) cells was already increased before the stress exposure, and application of stress did not significantly change this parameter. Our results showed that the effects of maternal stress on early embryos can depend on the actual physiological status of the maternal organism exposed to stress.

Elucidate the mechanism of action of the small molecule inhibitor of protein binding to glycosaminoglycans, RX-111 and assay its anti-inflammatory activity in animal models of inflammatory disease. The glycosaminoglycan, heparin, was used in the mechanism of action study of RX-111. Human T lymphocytes and umbilical vein endothelial cells were used to assay the in vitro activity of RX-111. Mouse and rat models of disease were used to assay the anti-inflammatory activity of RX-111 in vivo. Circular dichroism and UV/Vis absorption spectroscopy were used to study the binding of RX-111 to the glycosaminoglycan, heparin. T lymphocyte rolling on endothelial cells under shear flow was used to assay RX-111 activity in vitro. Delayed-type hypersensitivity (DTH) and tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in mice and experimental autoimmune encephalomyelitis (EAE) in rats were used to assay anti-inflammatory activity of RX-111 in vivo. RX-111 was shown to bind directly to heparin. It inhibited leukocyte rolling on endothelial cells under shear flow and reduced inflammation in the mouse model of DTH. RX-111 was efficacious in the mouse model of inflammatory bowel disease, TNBS-induced colitis and the rat model of multiple sclerosis, EAE. RX-111 exercises its broad spectrum anti-inflammatory activity by a singular mechanism of action, inhibition of protein binding to the cell surface GAG, heparan sulfate. RX-111 and related thieno[2,3-c]pyridine derivatives are potential therapeutics for the treatment of inflammatory and autoimmune diseases.

The paper provides a review of the current state of knowledge on apoptosis during normal preimplantation development based on the literature and on the authors' own findings. Information is focused on the occurrence and the characteristics of spontaneous apoptotic processes. Reports concerning the chronology and the incidence of programmed cell death in mouse, cow, pig and human embryos in early preimplantation stages up to the blastocyst stage are summarized. In addition, specific attributes of the apoptotic process in mammalian preimplantation development are provided, including the description of both morphological and biochemical features of cell death.

Accumulating evidence indicates the role of endogenous catecholamines in mammalian embryogenesis. We searched public databases containing nucleotide sequences derived from mouse preimplantation cDNA libraries and found a partial sequence homology between a cDNA clone from mouse blastocysts and the mouse beta 2-adrenergic receptor sequence. No significant sequence homology was found for other mouse adrenergic and dopamine receptors. Using RT-PCR, we showed that beta 2-adrenoceptor is transcribed not only at blastocyst stage but also at earlier stages of preimplantation development as well as in oocytes. Moreover, we demonstrated that transcripts encoding both isoforms of the beta 3-adrenoceptor (beta 3a- and beta 3b-) are expressed in mouse oocytes and preimplantation embryos as well. We did not detect the beta 1-adrenoceptor transcript either in oocytes or in preimplantation embryos. Using an antibody against the mouse beta 2-adrenergic receptor, we showed that the receptor protein is expressed in oocytes and preimplantation embryos; in blastocysts, the immufluorescence labeling was stronger in the inner cell mass than in throphectodermal cells. The cell number of the in vitro cultured mouse preimplantation embryos exposed to isoproterenol (a potent beta adrenoceptor agonist) was lower than in control embryos, suggesting that activation of beta adrenergic receptors by appropriate agonist concentration can influence cell proliferation in mouse pre-implantation embryos. Thus, our results indicate that beta adrenergic receptors are expressed in mouse oocytes and preimplantation embryos and that ligands for the receptors can affect the mouse embryo even in the very early stages of development. Mol. Reprod. Dev. 71: 145–153, 2005. © 2005 Wiley-Liss, Inc.

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 μM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 μM and 0.01 μM) 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.

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.

This study investigated the effects of maternal body condition on oocyte quality and zygote production. Additionally, we examined the possible consequences on somatic parameters and behavior of naturally delivered offspring. We used an experimental model based on overfeeding of outbred mice during intrauterine and early postnatal development to produce the following four types of females: physiological (7%-8%), slightly increased (8%-11%), highly increased (11%), and low (7%) body fat content (Echo Magnetic Resonance Imaging). The fertilized females with slightly increased body fat showed increased numbers of spontaneously ovulated oocytes and an increased fertilization index compared with control animals. On the contrary, mice with slightly and highly increased body fat showed increased numbers of isolated immature oocytes and degenerates. Furthermore, animals with increased body fat had significantly decreased deposits of neutral lipids in the cytoplasm of mature oocytes (Nile red staining) and showed lower reduction in DNA cytosine methylation signal in parental pronuclei (5-methylcytosine immunohistochemistry). The highly increased amount of body fat in mothers was accompanied with lower weights in newborn pups and 5-week-old offspring. We also observed several deviations from normal behavior (open-field test and forced swimming test). The females with low body fat displayed a lower fertilization index, a lower percentage of zygotes at pronuclear stage 4 with demethylated DNA cytosine in parental pronuclei, and lower newborn weights. Although delivered offspring were able to gain normal weight by the fifth week of life, there were several deviations from normal behavior observed. Our results show that periconceptional status of maternal body condition adversely affects the quality of oocytes and might be correlated with significant changes during postnatal offspring development. The data documenting later onset of DNA demethylation in zygotes and decreased amounts of neutral lipids in oocytes suggest that the observed alterations in offspring might originate in modifications established at the earliest stages of conceptus development.

We report the cloning and sequence of a novel gene, BALGR, which is coding for a candidate G protein-coupled receptor (GPCR) that is distantly related to the histamine, adrenergic, serotonin and dopamine receptors. The coding region of the human BALGR gene predicts a seven transmembrane domain receptor of 451 amino acids. BALGR has 42% amino acid identity to a Medaka fish ‘orphan’ GPCR. BALGR gene has been conserved throughout the mammalian evolution as indicated by Southern blot analysis. BALGR gene has been assigned to chromosome 1 by typing a panel of somatic cell hybrids and its exon/intron organization has been predicted. As determined by semiquantitative RT-PCR, expression of BALGR is relatively the highest in the human brain. A high level of BALGR transcript is also detected in testes. Within the brain, Northern blot analysis revealed relatively high expression in frontal and temporal lobes, occipital pole, amygdala and hippocampus. The preferential expression of BALGR in the areas of the human brain associated with cognition, learning and memory, and its conservation in evolution, indicate a potentially important biological function for this biogenic amine-like receptor and its putative neurotransmitter ligand.

The effects of intraperitoneal insulin on the food intake have been determined in infant rats up to weaning. It was found that intraperitoneal (IP) insulin reduced the milk intake of 13 and 17 day-old pups for three hours after treatment. In 5, 9 and 24 day-old pups the food intake was not significantly changed after IP insulin administration. Only in 28 day-old rat pups IP insulin induced an increase of food intake. Since subcutaneously (SC) administered insulin gave rise to short-term hyperphagia in 24 day-old rat pups we assessed the effects of SC versus IP insulin on the blood glucose level. Blood glucose was lower 3 hours after SC administration compared to the IP route. Results indicate that IP insulin causes a short lasting hypoglycaemia and consequently IP insulin increases food intake only after the 28th day of life.

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT-PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two-cell and later four- to eight-cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation.

Background Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified. Methods Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models. Results Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity. Conclusions A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events. General significance SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.

The aim of our study was to investigate the effect of maternal obesity on the quality and developmental capabilities of in vivo-derived preimplantation embryos. A two-generation dietary model, based on mice overfeeding during intrauterine and early postnatal development, was used to produce four types of female animals: with physiological (7%-8%), slightly elevated (8%-11%), highly elevated (11%), and low (7%) amounts of body fat. Spontaneously ovulating females (5-6 weeks old) were mated with male animals and subjected to embryo isolation at Day 4. Stereomicroscopical evaluation of collected embryos showed that the amount of maternal body fat did not affect the average number of collected embryos per dam. However, significant differences were found in the stage-distribution of isolated embryos: dams with highly elevated body fat and dams with low fat delivered decreased numbers of blastocysts and increased numbers of lower-stage or degenerated embryos compared with dams with physiological or slightly elevated fat value. Fluorescence staining showed that blastocysts isolated from dams with high and low percentage of body fat contained significantly higher numbers of dead cells. Most of such dead cells were of apoptotic origin. In contrast, the amount of maternal body fat did not affect blastocyst growth-the average numbers of cells per blastocyst were comparable in all groups. In conclusion, highly elevated or decreased amount of maternal body fat slowed down the development and negatively affected the quality of naturally in vivo-derived preimplantation embryos. No negative effect of slightly elevated fat was observed.

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.

The localization of fibrillarin and nucleolin in the nuclei of mouse two-cell, four-cell, and eight-cell embryos has been studied using immunofluorescent staining with specific antibodies. In all of these cleavage stages, both antigens were associated exclusively with the peripheral region of the nucleolus precursor bodies (NPBs). The original speckled fluorescent staining pattern in the early two-cell stage was progressively changed into a continuous fluorescent-positive layer localized in the cortex of the NPBs in the four-cell embryos. The compact central area of NPBs was never stained. Both proteins were colocalized in the same substructures of developing nucleoli. In order to analyze the interaction of chromatin, with NPBs, DNA structures were specifically immunolbelled. At the time of resumption of nucleolar transcription (in the two-cell mouse embryo), DNA was detected at the periphery of, but not penetrating into, NPBs. Our results confirm the view that the cortical region of NPBs could represent a nucleolonemal area involved in the resumption of nucleolar transcription in the early mouse embryo. © 1995 Wiley-Liss, Inc.

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.

The aim of this study was to evaluate the possible effect of maternal poisoning by BASTA-15 on developmental capacities and quality of preimplantation embryos in a mouse model. During in vivo tests, fertilized mice were fed with various doses of BASTA-15 for several days. During in vitro tests, isolated embryos were cultured in a medium with the addition of herbicide or its main compound glufosinate ammonium. Stereomicroscopic evaluation of embryonic pools obtained from treated dams showed that BASTA-15 at dose 58 μl/kg bw negatively affected their ability to reach the blastocyst stage. Moreover, as shown by morphological evaluation, based on cell counting and cell death assay, even the application of herbicide at the lowest dose (approx. 1/100 LD50) had a negative effect on obtained embryo quality. In vitro tests proved the direct ability of BASTA-15 to negatively affect embryo growth and quality. On the other hand, the addition of glufosinate ammonium at equivalent concentrations (from 0.015 to 15 μg/ml) had almost no damaging effect on embryos. It was harmful only at very high doses. Results show that maternal intoxication with BASTA-15 might affect the development of preimplantation embryos and suggest that the responsibility for this effect lies probably not solely with glufosinate ammonium, but in combination with the herbicide's secondary compounds.

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.

SummaryThis study was undertaken to obtain information about characteristics of different types of induced apoptosis in preimplantation embryos. Freshly isolated mouse blastocysts were culturedin vitrowith the addition of two apoptotic inductors – TNFα 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 TNFα. 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 TNFα 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.

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-1βand 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.

This study was undertaken to obtain specific information on the characteristics of spontaneous and induced apoptosis during preimplantation development of rabbit in vivo and in vitro developed embryos and mouse in vitro embryos. After reaching appropriate developmental stages, embryos were transferred into culture media with or without apoptotic inductor (actinomycin D 500 ng/mL) and cultured for 10 h. The identification of apoptotic cells was based on morphological assessment of nuclei and on detection of specific DNA degradation, phosphatidylserine redistribution and active caspase-3 under fluorescence microscope. Our experiments proved that apoptosis is a frequent physiological event occurring during normal preimplantation development. A high number of untreated rabbit and mouse blastocysts contained at least one apoptotic cell. Rabbit embryos showed a lower incidence of spontaneous apoptosis. Treated blastocysts of both species responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and significant increase in the incidence of apoptotic cell death. The occurrence of spontaneous apoptosis during earlier preimplantation development was sporadic and its presence was observed only at stages following embryonic genome activation (at 4-cell stage and later in mouse, at 16-cell and morula stage in rabbit). The susceptibility of embryos at early stages to the apoptotic inductor was much lower. The presence of actinomycin D did not increase the incidence of apoptotic embryos or apoptotic cells. Nevertheless, it slowed down embryo growth and triggered earlier appearance of some apoptotic features (at the 6-cell stage in rabbit). The results show that the occurrence of both spontaneous and induced apoptosis in preimplantation embryos is stage- and species-specific.

Accumulating evidence indicates the role of endogenous catecholamines in mammalian embryogenesis. We searched public databases containing nucleotide sequences derived from mouse preimplantation cDNA libraries and found a partial sequence homology between a cDNA clone from mouse blastocysts and the mouse beta 2-adrenergic receptor sequence. No significant sequence homology was found for other mouse adrenergic and dopamine receptors. Using RT-PCR, we showed that beta 2-adrenoceptor is transcribed not only at blastocyst stage but also at earlier stages of preimplantation development as well as in oocytes. Moreover, we demonstrated that transcripts encoding both isoforms of the beta 3-adrenoceptor (beta 3a- and beta 3b-) are expressed in mouse oocytes and preimplantation embryos as well. We did not detect the beta 1-adrenoceptor transcript either in oocytes or in preimplantation embryos. Using an antibody against the mouse beta 2-adrenergic receptor, we showed that the receptor protein is expressed in oocytes and preimplantation embryos; in blastocysts, the immufluorescence labeling was stronger in the inner cell mass than in throphectodermal cells. The cell number of the in vitro cultured mouse preimplantation embryos exposed to isoproterenol (a potent beta adrenoceptor agonist) was lower than in control embryos, suggesting that activation of beta adrenergic receptors by appropriate agonist concentration can influence cell proliferation in mouse pre-implantation embryos. Thus, our results indicate that beta adrenergic receptors are expressed in mouse oocytes and preimplantation embryos and that ligands for the receptors can affect the mouse embryo even in the very early stages of development.

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, P0.001; actinomycin D, P0.001). When IGF-I was added to the culture medium in the presence of an apoptosis inductor, apoptosis incidence was significantly decreased (P0.001). 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 (P0.05). Our data suggest a primary role for IGF-I as an apoptotic survival factor in mouse preimplantation embryos in specific conditions.