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1.
Gastrointestinal glutathione peroxidase (GI-GPx) is an antioxidant enzyme that has been known to be restricted to the gastrointestinal tract in rodents. In an effort to determine the expression pattern of GI-GPx mRNA during organogenesis, quantitative real-time PCR and in situ hybridization for GI-GPx mRNA were conducted in whole embryos or each developing organ of mice. GI-GPx mRNA was expressed more abundantly in the extraembryonic tissues, including placenta than in embryos on embryonic days (EDs) 7.5-18.5 (P < 0.05). When compared with the expression levels of cytosolic GPx (cGPx) mRNA, GI-GPx mRNA levels were low in the embryos, but relatively high in the extraembryonic tissues (P < 0.05). According to the results of whole mount in situ hybridizations, GI-GPx mRNA was principally expressed in the ectoplacental cone, neural tube and fold, and primitive heart at EDs 7.5-8.5. At EDs 9.5-12.5, GI-GPx mRNA was abundantly expressed in nervous tissues such as the telencephalon, mesencephalon and dorsal neural tube and was also detected in the forelimb and hindlimb at EDs 10.5-12.5. In the sectioned embryos after ED 13.5, GI-GPx mRNA levels were high in the cerebral cortex, metanephric corpuscle, pancreatic ducts, surface epithelia of the skin, inner ear, and nasal conchae, gastrointestinal tract, liver, urinary bladder, airway passages of lung, and whisker follicles. These findings indicate that GI-GPx is not only spatiotemporally expressed in a variety of embryonic organs during organogenesis but also may perform a mutual compensatory role with the cGPx in the protection of embryos and extraembryonic tissues against the reactive oxygen species generated in ontogenetic periods.  相似文献   

2.
Macrolides are considered to be one of the safest anti-infective groups in clinical use, with severe adverse reactions being rare. However, there are limited data about their embryotoxicity and teratogenicity. We aimed to investigate and compare the effects of these agents on embryonic growth and development. Rat embryos were cultured in vitro for 48 h in rat serum. Whole rat serum was used as a culture medium for the control group while different concentrations of spiramycin and azithromycin (1.25-6.25 microg/ml), and clarithromycin (2.5-30 microg/ml) were added to rat serum for the experimental groups. Dose-dependent effects of macrolides on embryonic developmental parameters were compared using morphological methods. Embryos were evaluated for the presence of any malformations. After morphological examination of the embryos, total DNA was extracted from the cells using standard procedures to determine fragmentation of nuclear DNA of embryonic cells. When compared with the control embryos, the macrolides significantly decreased all growth and developmental parameters dose dependently. While clarithromycin was found to cause more developmental toxicity than spiramycin and azithromycin, azitromycin was determined to have more teratogenicity potential. Compared with controls, there was no difference regarding the fragmentation of nuclear DNA of all the agents used. According to these results, when the toxic and teratogenic potential of the used agents compared, because of the lower toxic and teratogenic effects observed with spiramycin, this agent may be preferred for parturients.  相似文献   

3.
The development of cleavage stage preimplantation embryos is disrupted by exposure to heat shock, such as high temperatures in the summer season. In this study, we investigated whether addition of anthocyanins, which are strong scavengers of reactive oxygen species (ROS), improves development and intracellular redox status of heat-exposed bovine preimplantation embryos by reduction of heat shock-derived oxidative stress. After in vitro fertilization (IVF), embryos were cultured at 38.5 C through Day 8 (Day 0=day of IVF) with 0, 0.1, 1 and 10 microg/ml anthocyanins (non-heat-shocked group). On Day 2, embryos were cultured at 41.5 C for 6 h with 0, 0.1, 1 and 10 microg/ml anthocyanins followed by culture at 38.5 C until Day 8 (HS group). After exposure to heat shock, the intracellular ROS and glutathione (GSH) contents of individual embryos were measured in the non-heat-shocked and HS groups using fluorescent probes. On Day 8, the blastocysts formation rates of the embryos and total cell numbers of blastocysts were evaluated. Embryos exposed to heat shock without anthocyanins showed a significant decrease in blastocyst formation rate and GSH content (P<0.05) and an increase in intracellular ROS (P<0.05) compared with non-heat-shocked embryos. In contrast, addition of 0.1 microg/ml anthocyanins significantly (P<0.05) improved the blastocyst formation rate of the heat-shocked embryos. Addition of any dose of anthocyanins produced a significant decrease in the ROS levels (P<0.05) and tended to increase the GSH levels under heat-shock conditions. However, addition of higher concentrations (1 and 10 microg/ml) of anthocyanins to the culture media under heat shock did not improve the development of embryos. These results indicate that anthocyanins maintain the intracellular redox balance of heat-shocked bovine embryos by reducing intracellular oxidative stress and increasing the GSH levels. Thus, alterations of the redox state using natural antioxidative polyphenols is a useful approach for reducing heat shock-derived oxidative stress.  相似文献   

4.
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5.
The objective of this study was to determine if levels of mRNA encoding cytosolic glutathione peroxidase (cGPx) and thioredoxin reductase (TrxR-1) change during fetal development, and if maternal Se intake during gestation affects the mRNA levels of these proteins. Prepubertal gilts (n = 24) were randomly assigned to either Se-adequate (0.39 ppm of Se; n = 12) or Se-deficient (0.05 ppm of Se; n = 12) diets, 6 wk before breeding. Maternal liver was collected at d 10, 45, 70, and 114 of pregnancy, and fetal liver samples were collected at the same times except d 10. Complementary DNA sequences encoding cGPx and TrxR-1 were cloned and sequenced. Quantitative real-time PCR analysis indicated that levels of mRNA for cGPx in fetal liver decreased more than 3-fold between d 45 and 114 of gestation. Although the gilts were only marginally deficient in Se, and maternal Se intake did not affect cGPx mRNA levels in fetal liver, the low-Se diet tended (P = 0.1) to reduce fetal TrxR-1 mRNA levels. In the liver of the dams, the low Se intake did not affect mRNA levels for either cGPx or TrxR-1. Compared with the liver of the dams, mRNA levels for cGPx were about 3.5 times lower in fetal liver. Results of this study support the hypothesis that neonatal pigs are born with reduced cGPx corresponding to reduced cGPx mRNA levels during late gestation.  相似文献   

6.
Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.  相似文献   

7.
Heparin and low molecular weight heparins (LMWHs) are used to reduce the incidence of venous thromboembolism in pregnancy. Although, these agents have been shown to be safe when used during pregnancy, the studies about direct toxic and teratogenic effects of these drugs on embryonic development are limited. In this study, the effects of heparin and LMWHs on rat embryonic development were investigated by using in vitro embryo culture and micronucleus (MN) assay methods. Rat embryos were cultured in vitro in the presence of different concentrations of heparin (5-40 IU/ml), dalteparin (2.5-20 IU/ml), enoxaparin (25-100 microg/ml) and nadroparin (1-4 IU/ml). Effects of anticoagulants on embryonic developmental parameters were compared and embryos were evaluated for the presence of any malformations. After culturing the embryos, classic MN assay was performed. Anticoagulants significantly decreased all growth and developmental parameters dose-dependently. Dalteparin and enoxaparin were found to cause more developmental toxicity than heparin and nadroparin. Along with haematoma in general, heparin and nadroparin caused maxillary deformity, situs inversus and oedema most frequently, while neural tube defects were observed with dalteparin and enoxaparin. All agents also significantly induced MN formation in rat embryonic blood cells. These results indicate the possible genotoxic effects of anticoagulant agents on the developing rat embryo when applied directly.  相似文献   

8.
Recently, developmental exposure to clothianidin (CLO) has been shown to cause reproductive toxicity in male mice, but the effects in female mice remain to be clarified. Pregnant C57BL/6N mice were given a no-observed-adverse-effect-level (NOAEL) dose of CLO until weaning. We then examined ovaries of 3- or 10-week-old female offspring. In the CLO-administered group, morphological changes, a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 (GPx4), and activation of genes in the steroid hormone biosynthesis pathway were observed in 3-week-old mice, and decreases of GPx4 immunoreactivity, 17OH-progesterone and corticosterone levels were observed in 10-week-old mice, along with high rates of infanticide and severe neglect, providing new evidence that developmental exposure to CLO affects juvenile and adult mice differently.  相似文献   

9.
One of the major difference between the in vivo and in vitro embryonic environments is the stiffness of the culture substrate. Xanthan gum (XG) and locust bean gum (LBG) are natural materials that are safe, inexpensive and easy to handle. In this study, we investigated the effects of using a polysaccharide culture substrate made from 1% XG and 1% LBG (XG-LBG gel) on bovine embryonic development. Oocytes collected from bovine ovaries were subjected to maturation, and fertilization to generate embryos at an early developmental stage (>4 cell stage). Cleaved embryos were further cultured in a well of 96-well cell culture plate coated with or without XG-LBG gel for 5 days. While the developmental rate up to the blastocyst stage did not differ between the two culture systems (control, 38.0 vs. gel, 38.6%), blastocysts developed on the XG-LBG gel produced significantly high cell numbers and ATP content. Embryos cultured on XG-LBG gels for 24 hr had high expression levels of F-actin and a highly even distribution of E-cadherin. In addition, embryos developed on XG-LBG gel demonstrated increased translocation of YAP to the nucleus and increased connective tissue growth factor (CTGF) protein levels (downstream of Hippo signalling). These findings suggest that soft culture substrates improve embryonic development by enhancing mechanotransduction, including YAP-CTGF signalling.  相似文献   

10.
To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r2 = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose.  相似文献   

11.
Development competence and pregnancy rate of in vitro-produced (IVP) dromedary embryos were studied in two culture systems: (i) semi-defined modified medium (mKSOMaa) and (ii) co-culture using camel epithelial oviducal cells. Five hundred and three cumulus-oocytes complexes (COCs) were selected, allowed to mature, fertilized and cultured in vitro (38.5 degrees C; 5% CO2, maximum humidity > 95%, with concentration of oxygen of 5% for semi-defined medium and 20% for co-culture cells). Maturation was accomplished by incubation in TCM-199 medium supplemented with 10% heat-treated foetal calf serum (FCS), 10 ng/ml epidermal growth factor, 1 microg/ml follicle-stimulating hormone, 1 microg/ml oestradiol and 500 microM cysteamine for 30 h. In vitro fertilization (IVF) was performed using fresh semen (0.5 x 10(6) spermatozoa/ml in modified TALP solution). Fertilized COCs were denuded by vortexing, then cultured in either mKSOMaa (10% heat-treated FCS was added 24 h post-IVF), under 5% O2 and 90% N2 (group 1; n = 249) or with dromedary epithelial oviducal cell monolayers in TCM-199 with 10% heat-treated FCS under 20% O2 (group 2; n = 254). The rate of cleavage was significant higher (p < 0.05) for group 1 (63%, 156/249) than for group 2 (51%, 130/254). No significant difference was found between the two groups in the rate of development to blastocyst (21% vs 16.5%) and their hatchability (21% vs 14%). Pregnancy rates were similar for the first 60 days. However, all pregnancies were lost after 60 days with the exception of two of six (33%) from recipients of hatched blastocysts from group 1. We conclude that both systems support in vitro production of dromedary embryos by in vitro maturation (IVM)/IVF of oocytes. However, embryos obtained by culture in the semi-defined medium (mKSOMaa) appear to have a better in vivo development ability.  相似文献   

12.
We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.  相似文献   

13.
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14.
The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro . Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8–16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.  相似文献   

15.
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.  相似文献   

16.
In the present study, 1‐cell stage mouse embryos were cultured with or without glucose, and their development to the blastocyst stage was compared. Embryos cultured in a glucose‐free medium had a higher percentage of development to the 8‐cell stage, and they had higher developmental speeds compared with those cultured in a glucose‐containing medium. The percentages of embryos that developed to the early blastocyst stage, blastocyst stage and expanded blastocyst stage were much lower than those developed in a glucose‐containing medium. This suggests that the culture of 1‐cell stage embryos in a glucose‐containing medium inhibits development at the 8‐cell stage, and that glucose is necessary for blastocyst formation. Previous reports indicate that, from the 1‐cell stage to the 4‐cell stage, glucose inhibits embryo development. In the present study, exposure of early embryos to a glucose‐free medium improved subsequent embryo development, and there was no difference in the percentage of development to the stages ranging from the 8‐cell stage to the expanded blastocyst stage between embryos cultured in a glucose‐free medium from the 1‐cell stage to the 2‐cell, 4‐cell and 8‐cell stage. This indicates that embryo development is improved when a 1‐cell stage embryo is exposed to a glucose‐free medium before and during the 2‐cell stage, and glucose only has an inhibitory effect on embryo development during conversion from the 1‐cell stage to the 2‐cell stage.  相似文献   

17.
The present study was conducted to examine the effects of culture systems and culture media on developmental competence and freezability of bovine embryos obtained by in vitro culture of in vitro matured and fertilized (IVM-IVF) oocytes. No significant difference was observed in the proportions of oocytes developed to blastocysts, the speed at which the oocytes reached the blastocyst stage and the number of cells, when the IVM-IVF oocytes were cultured in CR1aa with or without cumulus cells. Nevertheless, more of the IVM-IVF oocytes cultured either with or without cumulus cells in CR1aa were seen to reach the blastocyst stage much sooner than those cultured with cumulus cells in TCM199 (P<0.05). The proportion of embryos developed to the blastocyst stage by day 7 in CR1aa culture was significantly higher than embryos cultured in TCM199. Viability after frozen-thawed blastocysts were obtained in vitro, was seen in a significantly higher percentage of embryos cultured in TCM199 and developed to the hatched blastocysts than in those cultured in CR1aa (P<0.05). These results indicate that CR1aa was superior to TCM199 for the potential developmental of IVM-IVF oocytes to blastocysts during in vitro culture regardless of co-culture with or without cumulus cells. But the freezability of blastocysts developed in CR1aa was inferior to those developed in TCM199.  相似文献   

18.
生物频谱对家兔胚胎发育影响的初步研究   总被引：4，自引：1，他引：3  
本文首次研究了周林生物频谱照射对家兔胚胎体外发育的影响，初步探讨了周林生物频谱在胚胎工程中应用的可能性。周林生物频谱分别照射家兔２－细胞胚胎３、５、７、１０、１５、２０、２５分钟，结果照射１０、１５、２０、２５分钟，可使７２小时（从注射ＨＣＧ后算起，即照射后４８小时）桑椹胚发育率显著增加，照射２０和２５分钟可提高７２、８４、９６和１０８小时囊胚发育率，照射１０分钟和１５分钟可提高１０８小时囊胚发育率，照射７、１０、１５、２０和２５分钟可提高９６小时扩张囊胚发育率及１０８小时孵化囊胚发育率。周林生物频谱照射家兔１６－细胞胚胎２０分钟，其６０小时（从注射ＨＣＧ后起，即照射后１２小时）致密桑椹胚发育率与对照比较差异极显著（Ｐ＜０．０１），囊胚、扩张囊胚和孵化囊胚发育率与对照比较无显著差异（Ｐ＞０．０５）  相似文献   

19.
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20.
In this study, we aimed at the in vitro effects of anti-fibroblast growth factor-2 (anti-FGF-2 or anti-bFGF) on embryo culture in rats. In vitro effects of anti-bFGF on total embryonic development were investigated in 40 rat embryos (which were divided into four groups) (obtained from five pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), and in WRS+ 2.5, 5, and 10 μg/ml anti-bFGF. After 48 h of culturing, the embryos from each group were harvested to be analysed morphologically according to a morphological scoring system and biochemically to obtain the embryo protein content. The morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in WRS+ anti-bFGF had significant embryonic retardation. Mean morphological scores for the embryos grown in WRS, in the presence of 2.5, 5 and 10 μg anti-FGF-2 were 61.4 ± 1.64, 46.3 ± 8.42, 27 ± 2.58 and13.6 ± 0.96 respectively. These results suggest that bFGF is very important for normal embryonic development and rat anti-bFGF neutralizes bFGF effect.  相似文献   

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