Gamma-aminobutyric acid (GABA) can affect physiological processes in preimplantation embryos via GABAA and GABAB receptors.

Purpose: Several widely used substances (e.g., some therapeutics or food supplements) can act on gamma-aminobutyric acid (GABA) receptors, and we investigated whether the activation of these receptors could affect the preimplantation embryo. Methods: Transcripts of all GABA receptor subunits and selected proteins were examined using quantitative RT-PCR and immunohistochemistry. To analyze the effects of receptor activation, in vitro culture of mouse preimplantation embryos with natural and synthetic GABA receptor ligands was used. Results: We detected nine GABA receptor transcripts in mouse blastocysts and 14 GABA receptor transcripts in ovulated oocytes. The results of this study indicate that ionotropic GABAA receptors can be formed from α5, ß3, and γ3 (or δ, π) subunits, GABAA-ρ receptors can be formed from ρ2 subunits and metabotropic GABA receptors can be formed from GABAB1b and GABAB2 subunits in mouse blastocysts. Supplementing the culture medium with GABA at concentrations of 2-10 mM or with specific GABAA and GABAB receptor agonists (at concentrations of 10-100 µM) significantly increased the proportion of dead cells in blastocysts. The GABA-induced effects were prevented by pretreatment of embryos with GABAA and GABAB receptor antagonists. Conclusion: The results of this study indicate that GABA and synthetic GABA receptor ligands can negatively affect preimplantation embryos via GABAA and GABAB receptors.

Overweight and Fertility: What We Can Learn from an Intergenerational Mouse Obesity Model.

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.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

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.

Maternal overweight increased sensitivity of mouse preimplantation embryos to oxidative stress in vitro.

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 (P < 0.01). Similar impact was recorded in two-cell embryos obtained from control mothers only after exposure to BSO (P < 0.05). 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.

In vitro exposure to pyrethroid-based products disrupts development of mouse preimplantation embryos.

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.

Glucocorticoid receptor isoforms and effects of glucocorticoids in ovulated mouse oocytes and preimplantation embryos†.

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, GRß 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.

Fipronil causes toxicity in mouse preimplantation embryos.

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.

RX-207, a Small Molecule Inhibitor of Protein Interaction with Glycosaminoglycans (SMIGs), Reduces Experimentally Induced Inflammation and Increases Survival Rate in Cecal Ligation and Puncture (CLP)-Induced Sepsis.

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.

Exposure to neonicotinoid insecticides induces embryotoxicity in mice and rabbits.

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 (P<0.001). 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 thiacloprid>acetamiprid, thiomethoxam>clothianidin. Thiacloprid impairs development and quality of both mouse and rabbit preimplantation embryos, and shows embryotoxicity even at acute reference dose.

The Responses of Mouse Preimplantation Embryos to Leptin In Vitro in a Transgenerational Model for Obesity.

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 proapoptotic 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.

The effect of maternal stress on blastocyst quality depends on maternal physiological status.

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.

Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

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.

Mechanism of action and efficacy of RX-111, a thieno[2,3-c]pyridine derivative and small molecule inhibitor of protein interaction with glycosaminoglycans (SMIGs), in delayed-type hypersensitivity, TNBS-induced colitis and experimental autoimmune encephalomyelitis.

OBJECTIVE AND DESIGN: 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. MATERIALS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.

The effect of maternal body condition on in vivo production of zygotes and behavior of delivered offspring in mice.

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.

Amount of maternal body fat significantly affected the quality of isolated mouse preimplantation embryos and slowed down their development.

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.

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties.

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.

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.

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.

Biogenic monoamines in preimplantation development.

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).