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.

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.

Apoptotic cells in mouse blastocysts are eliminated by neighbouring blastomeres.

Apoptosis is a physiological process that occurs commonly during the development of the preimplantation embryo. The present work examines the ability of apoptotic embryonic cells to express a signal promoting their phagocytosis, and quantifies the ability of neighbouring, normal embryonic cells to perform that task. Microscopic analysis of mouse blastocysts revealed phosphatidylserine externalization to be 10 times less common than incidence of apoptotic cells (as detected by TUNEL). In spite of the low frequency of phosphatidylserine-flipping (in inner cell mass, no annexin V staining was recorded), fluorescence staining of the plasma membrane showed more than 20% of apoptotic cells to have been engulfed by neighbouring blastomeres. The mean frequency of apoptotic cells escaping phagocytosis by their extrusion into blastocyst cavities did not exceed 10%. Immunochemically visualised RAC1 (an enzyme important in actin cytoskeleton rearrangement) was seen in phagosome-like structures containing a nucleus with a condensed morphology. Gene transcript analysis showed that the embryonic cells expressed 12 receptors likely involved in phagocytic process (Scarf1, Msr1, Cd36, Itgav, Itgb3, Cd14, Scarb1, Cd44, Stab1, Adgrb1, Cd300lf, Cd93). In conclusion, embryonic cells possess all the necessary mechanisms for recognising, engulfing and digesting apoptotic cells, ensuring the clearance of most dying blastomeres.

Different response of embryos originating from control and obese mice to insulin in vitro.

The aim of the present work was to investigate the impact of maternal obesity on DNA methylation in ovulated oocytes, and to compare the response of in vitro-developing preimplantation embryos originating from control and obese mice to insulin. An intergenerational, diet-induced obesity model was used to produce outbred mice with an increased body weight and body fat. Two-cell and eight-cell embryos recovered from obese and control mice were cultured in a medium supplemented with 1 or 10 ng/ml insulin until blastocyst formation. In the derived blastocysts, cell proliferation, differentiation, and death rates were determined. The results of immunochemical visualization of 5-methylcytosine indicated a slightly higher DNA methylation in ovulated metaphase II oocytes recovered from obese females; however, the difference between groups did not reach statistical significance. Expanded blastocysts developed from embryos provided by control dams showed increased mean cell numbers (two and eight-cell embryos exposed to 10 ng/ml), an increased inner-cell-mass/trophectoderm ratio (two-cell embryos exposed to 1 ng/ml and eight-cell embryos exposed to 10 ng/ml), and a reduced level of apoptosis (two and eight-cell embryos exposed to 10 ng/ml). In contrast, embryos originating from obese mice were significantly less sensitive to insulin; indeed, no difference was recorded in any tested variable between the embryos exposed to insulin and those cultured in insulin-free medium. Real-time RT-PCR analysis showed a significant increase in the amount of insulin receptor transcripts in blastocysts recovered from obese dams. These results suggest that maternal obesity might modulate the mitogenic and antiapoptotic responses of preimplantation embryos to insulin.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

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.

A Diabetic Pregnancy Alters the Expression of Stress-Related Receptors in Gastrulating Rabbit Blastocyst and in the Reproductive Tract.

The incidence of diabetes mellitus for young people rises since years. A preconceptional diabetes mellitus leads to subfertility. Most of the causes for a diabetic subfertility are still unknown. Stress can significantly deteriorate glycemic control in diabetes. Several mechanisms by which "stress hormones", like adrenaline and cortisol or corticosterone, can contribute to the regulation of glucose homeostasis have been identified. Using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR, we examined the expression of adrenergic receptors and the glucocorticoid receptor transcripts in the female rabbit reproductive tract and in gastrulating blastocysts developed in normoinsulinemic mothers and in mothers with experimentally induced diabetes mellitus type 1. The glucocorticoid receptor expression was detected in the reproductive tract as well as in gastrulating blastocysts at a high level. In maternal endometrium, α1D-, α2A-, ß1-, and ß2-adrenergic receptors were expressed, whereby ß1 transcript was not detectable in the endometrium from diabetic mothers. In preimplantation embryos, all 9 adrenergic receptors were expressed, most of them predominantly in the embryoblast. A maternal diabetes mellitus altered α2A-adrenergic receptor expression in the blastocyst and reversed the ratio of α2A transcript quantity between embryoblast and trophoblast. Our results show that the maternal reproductive tract and the preimplantation embryo express a distinct pattern of the stress response system. Alterations in the pattern and/or in functionality are likely linked to subfertility in diabetes mellitus.

Overweight negatively affects outcome of superovulation treatment in female mice.

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

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.

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.

Intestinal ischemia-reperfusion injury mediates expression of inflammatory cytokines in rats.

The small intestine is an organ with very well developed immunological activity, responsible for synthesis of specific inflammatory mediators that participate in causing the systemic inflammation that can occur after ischemia-reperfusion injury. The aim of our work was to study mRNA expression and protein concentration of inflammatory cytokines IL-10 and TNFα in the jejunal wall after intestinal ischemia-reperfusion injury (IRI). Cytokine concentration levels confirmed the direct effect of IRI on the inflammation process. The results refer to the changes in balance between pro-inflammatory and anti-inflammatory mediators and indicate that the predominant disturbance of homeostasis after intestinal IRI is present after 1 hour of reperfusion.

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.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds.

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

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.

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.