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.

Dietary Supplementation of Flaxseed (Linum usitatissimum L.) Alters Ovarian Functions of Xylene-Exposed Mice.

The aim of the performed study was to examine the ability of xylene, flaxseed, and their combinations to affect morphological and endocrine indexes of murine ovaries. The 72 indexes of secondary and tertiary follicular cells, oocytes, corpora lutea, and ovarian stroma have been quantified: diameter, markers of proliferation PCNA and apoptosis caspase 3, receptors to FSH, oxytocin, estrogen (alpha and beta), and progesterone. In addition, concentrations of the ovarian hormones progesterone, estradiol, and IGF-I in the blood, as well as their production by isolated ovaries cultured with and without gonadotropins (FSH + LH mixture), were determined using histological, immunohistochemical, and immunoassay analyses. The character of xylene and flaxseed effects on ovarian functions in mice depended on the stage of ovarian folliculogenesis. It was shown that flaxseed could mitigate and prevent the major (63%) effects of xylene on the ovary. In addition, the ability of gonadotropins to affect ovarian hormone release and prevent its response to xylene has been shown. The effects of these additives could be mediated by changes in the release and reception of hormones. These observations suggest that flaxseed and possibly gonadotropins could be natural protectors of a female reproductive system against the adverse effects of xylene.

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 High-Energy Diet during Pregnancy and Lactation Impairs Neurogenesis and Alters the Behavior of Adult Offspring in a Phenotype-Dependent Manner.

Obesity is one of the biggest and most costly health challenges the modern world encounters. Substantial evidence suggests that the risk of metabolic syndrome or obesity formation may be affected at a very early stage of development, in particular through fetal and/or neonatal overfeeding. Outcomes from epidemiological studies indicate that maternal nutrition during pregnancy and lactation has a profound impact on adult neurogenesis in the offspring. In the present study, an intergenerational dietary model employing overfeeding of experimental mice during prenatal and early postnatal development was applied to acquire mice with various body conditions. We investigated the impact of the maternal high-energy diet during pregnancy and lactation on adult neurogenesis in the olfactory neurogenic region involving the subventricular zone (SVZ) and the rostral migratory stream (RMS) and some behavioral tasks including memory, anxiety and nociception. Our findings show that a maternal high-energy diet administered during pregnancy and lactation modifies proliferation and differentiation, and induced degeneration of cells in the SVZ/RMS of offspring, but only in mice where extreme phenotype, such as significant overweight/adiposity or obesity is manifested. Thereafter, a maternal high-energy diet enhances anxiety-related behavior in offspring regardless of its body condition and impairs learning and memory in offspring with an extreme phenotype.

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.

Dietary flaxseed's protective effects on body tissues of mice after oral exposure to xylene.

Xylene is a common pollutant in the environment that enters the body of animals and humans in various ways, but most often through the respiratory tract and adversely affects their overall health. However, xylene effects after oral exposure have not been sufficiently studied. This study aimed to investigate the effects of xylene exposure on the mouse organism and to identify possible beneficial effects of flaxseed on such exposure. Eighty mice were divided into four groups: control group C (basal diet + no xylene exposure), group X (oral exposure by 400 mg/kg/day xylene), group F (10% flaxseed supplementation of basal diet), and group XF (10% dietary flaxseed + oral exposure by xylene). Experimental trial took 14 days. Clinical examination, spectroscopic analysis of tissue aminotransferases, total lactate dehydrogenase (TLDH), and acetylcholinesterase (AchE) activities, electrophoretic analysis of LDH isoenzymes, western blot and immunohistochemical analysis of apoptosis as well as routine histology of the kidneys and jejunum, and transmission electron microscopy of the liver were performed. Marked restlessness in group X and high weight losses in mice of all groups were recorded during the experiment. Xylene promoted apoptosis (caspase-3 expression) without causing marked structural changes in the liver and jejunum, although renal cortex structure was affected adversely. In the brain, liver, and kidney of mice, xylene increased levels of liver transaminases, LDH, and decreased AchE activities, reflecting cell membrane damage. Flaxseed feeding improved animal behaviour, leakage of enzymes and prevented selected tissue toxic damage induced by xylene by protecting cell membrane integrity and fluidity and by suppressing apoptosis. These results point at the protective effect of flaxseed consumption on mice.

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.

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.

Body fat affects mouse reproduction, ovarian hormone release, and response to follicular stimulating hormone.

We investigated the effects of body fat content on mouse fecundity, ovarian hormone release, and their response to follicle stimulation hormone (FSH). 4 types of females were produced: lean (group 1), normal (group 2), slightly fat (group 3), and significantly fat (group 4). The body weights, fat content, fertility rate, embryo number produced, retarded and degenerated embryo percentage, the release of progesterone (P4), testosterone (T), and insulin-like growth factor I (IGF-I) by isolated ovaries cultured with and without FSH (1.0IU/mL medium) were evaluated. A gradual increase in body weight and fat contents from groups 1 to 4 was observed. Group 2 had higher fertility rate than those from the other groups. Groups 2 and 3 had fewer retarded and degenerated embryos that those from groups 1 and 4. Embryo production rate was not different among the groups. P4 and T secretion was higher from group 4 than in those from groups 1-3; secretion of IGF-I of group 3 was less than that of groups 1, 2, and 4. FSH promoted ovarian T output in all groups and stimulated ovarian P4 release in groups 1, 3, and 4, but not in group 2. FSH did not affect IGF-I release in any group. Therefore, both malnutrition and overfeeding can affect body weight and fat content in female mice, reducing embryo quality or developmental capacity, but not fertility and embryo production. Excess weight or fat can have stimulatory effects on ovarian P4 and T, but inhibitory effects on ovarian IGF-I release. Both leanness and excess weight or fat can induce the stimulatory action of FSH on ovarian P4.

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.

Metabolic state can define the ovarian response to environmental contaminants and medicinal plants.

Environmental contaminants and medicinal plants can affect reproductive processes. The aim of this study was to investigate the effect of maternal metabolic status on the response of mouse ovaries to the environmental contaminants benzene and xylene, as well as to extracts of the medicinal plant yucca. Ovaries isolated from normal-lean and slightly obese mice were cultured with or without 0.1% benzene or xylene for 24 h. Similarly, ovaries isolated from normal-lean, slightly obese, and significantly obese mice were cultured for 24 h with or without an extract of Yucca shidigera (YS, 10 µ g/mL). We found that the metabolic status did not influence the release of basal progesterone (P4), testosterone (T), or insulin-like growth factor I (IGF-I), but obesity influenced the effects of the environmental contaminants and YS. Benzene reduced P4 output in ovaries from obese but not normal-lean mice; it also reduced IGF-I (but not T) release from ovaries irrespective of the metabolic status. Xylene dramatically increased P4 and T (but not IGF-I) release by ovaries from normal-lean mice, but there were no changes in P4 and only small increases in T output in obese mice. YS increased P4 (but not T or IGF-I) release in normal-lean and slightly obese animal ovaries, whilst significant obesity was associated with a lack of P4 response to YS. Obesity might affect the basal ovarian release of T or IGF-I and increases the sensitivity of ovaries to the action of benzene but decreases their responsiveness to xylene and YS.

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.