Embryo-fetal safety evaluation of ondansetron in rats.

BACKGROUND: Ondansetron is a 5HT3 receptor antagonist, used to mitigate the effects of nausea and vomiting after chemotherapy or surgery. Since nausea and vomiting are common experiences during the first trimester of pregnancy, this antiemetic has been the main drug used during this period. METHODS: To evaluate the effects of ondansetron on the embryo-fetal development, which are still very contradictory, pregnant rats were exposed to therapeutic doses of ondansetron (1.7 or 2.5 mg/kg) daily, from gestational day (GD) 6 to 15. RESULTS: No clinical signs of toxicity were observed in dams during the treatment. Although the hemato-biochemical parameters were similar among the groups, histological changes, as well as a reduction in the weight of kidney were found in the treated dams. After fetal examination, no visceral and skeletal abnormalities were observed in treated fetuses. CONCLUSION: In conclusion, therapeutic doses of ondansetron have low teratogenic potential in rats. These data provide important information about the drug safety during pregnancy.

α-Ketoglutarate Improves Meiotic Maturation of Porcine Oocytes and Promotes the Development of PA Embryos, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway.

α-Ketoglutarate (α-KG) is a metabolite in the tricarboxylic acid cycle. It has a strong antioxidant function and can effectively prevent oxidative damage. Previous studies have shown that α-KG exists in porcine follicles, and its content gradually increases as the follicles grow and mature. However, the potential mechanism of supplementation of α-KG on porcine oocytes during in vitro maturation (IVM) has not yet been reported. The purpose of this study was to explore the effect of α-KG on the early embryonic development of pigs and the mechanisms underlying these effects. We found that α-KG can enhance the development of early pig embryos. Adding 20 µM α-KG to the in vitro culture medium significantly increased the rate of blastocyst formation and the total cell number. Compared with to that of the control group, apoptosis in blastocysts of the supplement group was significantly reduced. α-KG reduced the production of reactive oxygen species and glutathione levels in cells. α-KG not only improved the activity of mitochondria but also inhibited the occurrence of apoptosis. After supplementation with α-KG, pig embryo pluripotency-related genes (OCT4, NANOG, and SOX2) and antiapoptotic genes (Bcl2) were upregulated. In terms of mechanism, α-KG activates the Nrf2/ARE signaling pathway to regulate the expression of antioxidant-related targets, thus combating oxidative stress during the in vitro culture of oocytes. Activated Nrf2 promotes the transcription of Bcl2 genes and inhibits cell apoptosis. These results indicate that α-KG supplements have a beneficial effect on IVM by regulating oxidative stress during the IVM of porcine oocytes and can be used as a potential antioxidant for IVM of porcine oocytes.

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro†.

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15 µM AKT-InhIII; 10 µM AKT-InhIV; 10 µM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine.

TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos.

Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.

All-trans-retinoic acid suppresses rat embryo hindlimb bud mesenchymal chondrogenesis by modulating HoxD9 expression.

In vertebrates, 5'-Hoxd genes (Hoxd9), which are expressed in the hindlimb bud mesenchyme, participate in limb growth and patterning in early embryonic development. In the present study, We investigated the mechanisms by which ATRA regulates cultured E12.5 rat embryo hindlimb bud mesenchymal cells (rEHBMCs). Following exposure to ATRA over 24 h, mRNA and protein expression levels of HoxD9 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and western blotting. Flow cytometry was used to detect apoptosis. ATRA inhibited the condensation and proliferation, and promoted the apoptosis rate of the rEHBMCs in a dose-dependent manner. Sox9 and Col2a1 in rEHBMCs were downregulated by ATRA in a dose-dependent manner at both mRNA and protein levels. Similarly, HoxD9 was downregulated by ATRA in a dose-dependent manner, in parallel with the cartilage-specific molecules Sox9 and Col2a1. Both qPCR and western blotting showed that both Shh and Gli3 were downregulated. Overexpression of HoxD9 reversed the effects of ATRA. These results demonstrate that ATRA suppresses chondrogenesis in rEHBMCs by inhibiting the expression of HoxD9 and its downstream protein targets, including Sox9 and Col2a1. This effect may also be correlated with inhibition of the Shh-Gli3 signaling pathway.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Spatiotemporal evaluation of the mouse embryonic redox environment and histiotrophic nutrition following treatment with valproic acid and 1,2-dithiole-3-thione during early organogenesis.

Redox regulation during metazoan development ensures that coordinated metabolic reprogramming and developmental signaling are orchestrated with high fidelity in the hypoxic embryonic environment. Valproic acid (VPA), an anti-seizure medication, is known to increase markers of oxidation and also increase the risk of neural tube defects (NTDs) when taken during pregnancy. It is unknown, however, whether oxidation plays a direct role in failed neural tube closure (NTC). Spatial and temporal fluctuations in total glutathione (GSH) and total cysteine (Cys) redox steady states were seen during a 24 h period of CD-1 mouse organogenesis in untreated conceptuses and following exposure to VPA and the Nrf2 antioxidant pathway inducer, 1,2-dithiole-3-thione (D3T). Glutathione, glutathione disulfide (GSSG), and Cys, cystine (CySS) concentrations, measured in conceptal tissues (embryo/visceral yolk sac) and fluids (yolk sac fluid/amniotic fluid) showed that VPA did not cause extensive and prolonged oxidation during the period of NTC, but instead produced transient periods of oxidation, as assessed by GSH:GSSG redox potentials, which revealed oxidation in all four conceptal compartments at 4, 10, and 14 h, corresponding to the period of heartbeat activation and NTC. Other changes were tissue and time specific. VPA treatment also reduced total FITC-Ab clearance from the medium over 3 h, indicating potential disruption of nutritive amino acid supply. Overall, these results indicated that VPA's ability to affect cellular redox status may be limited to tissue-specific windows of sensitivity during the period of NTC. The safety evaluation of drugs used during pregnancy should consider time and tissue specific redox factors.

Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1.

In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.

Leptin improves the in vitro development of preimplantation rabbit embryos under oxidative stress of cryopreservation.

Vitrification is an economically effective method for embryo cryopreservation in human and livestock animals; however, it carries the risk of damage by the exposure to severe oxidative stress. The present study was conducted to evaluate the effect of leptin at different levels on the in vitro development of fresh and vitrified preimplantation embryos in a rabbit model. Normal embryos at morulae stage were randomly cultured for 2 h with 0, 10, 20 or 100 ng/mL of leptin, then were cultured for further 48 h as freshly or after vitrification. Thereafter, developed blastocysts form the best leptin level in fresh and vitrified embryos along with their controls were allocated to analyze the pro-oxidant (malondialdehyde, MDA; nitric oxide, NO), antioxidant (total antioxidant capacity, TAC; superoxide dismutase, SOD; glutathione peroxidase, GPx), apoptotic (Bcl-2 associated X protein, BAX; heat shock 60kD protein member 1, HSP60; tumor necrosis factor alpha, TNFα) and developmental (sex determining region Y box protein 2, SOX2; Nanog homeobox protein, NANOG; Octamer-binding protein 4, OCT4) biomarkers. Results indicate that expanding and hatching rates of embryos were significantly higher at 20 ng/mL leptin than the other levels, while vitrification had an independent suppression effect on the in vitro development rates. The MDA and NO were significantly higher, while TAC, SOD and GPx were significantly lower in the vitrified than fresh embryos. In contrast, leptin treatment significantly decreased the pro-oxidant biomarkers and increased the antioxidant biomarkers in both fresh and vitrified embryos. Vitrification significantly increased the antiapoptotic biomarkers, and decreased the developmental biomarkers in embryos. In contrast, leptin decreased the BAX and TNFα, increased the HSP60, and moreover, ameliorated the reduction of developmental biomarkers in the vitrified embryos. These results conclude that leptin could be used as antiapoptotic and antioxidant promotor to support the in vitro embryonic development, particularly under oxidative stress emerged from cryopreservation programs.

The effect of lutein and Urtica dioica extract on in vitro production of embryo and oxidative status in polycystic ovary syndrome in a model of mice.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrinopathies in women during the reproductive age. Herbal medicines are used increasingly alone or in supplement with chemical medicines for the treatment of different diseases and dysfunctions. This study was aimed to evaluate the effects of lutein and nettle (Urtica dioica) extract on the biochemical parameters and the reproductive function in the PCOS model of mice. METHODS: Following the induction of PCOS by dehydroepiandrosterone (DHEA), the mice (n = 98) were randomly assigned into seven groups, each consisting of fourteen mice; the groups were included control group (received solvent), PCOS group (received 6 mg/100 g B.W/day IP, DHEA for 21 days), PCOS+ Nettle extract (200 and 400 mg/kg), PCOS+ Lutein (125 and 250 mg/kg), and PCOS+ NL (200 mg/kg nettle extract and 125 mg/kg lutein). The nettle extract and lutein were administrated using gavage for 30 consecutive days after PCOS induction. Malondialdehyde (MDA), total antioxidant capacity (TAC), and estrogen were measured in serum, ovary, and uterus samples by the ELISA method. The total number of oocytes, oocyte quality, fertilization rate, 2-cell blastocyst, and arrested embryos (type I, type II, and type III) were also investigated. RESULTS: A combination treatment of the nettle and lutein produced the lowest concentration of MDA in comparison to other groups which affected by the PCOS. The lowest level of TAC was observed in the PCOS group without treatment. The number of oocytes, oocyte quality, fertilization rate, and 2-cell blastocyst were significantly higher in the control group, but the lowest values were observed in the PCOS group without any treatment. CONCLUSIONS: The most favorable findings include improving antioxidant capacity, oocyte and embryo quality were observed in the PCOS+ 125 L group.

An Embryonic Diapause-like Adaptation with Suppressed Myc Activity Enables Tumor Treatment Persistence.

Treatment-persistent residual tumors impede curative cancer therapy. To understand this cancer cell state we generated models of treatment persistence that simulate the residual tumors. We observe that treatment-persistent tumor cells in organoids, xenografts, and cancer patients adopt a distinct and reversible transcriptional program resembling that of embryonic diapause, a dormant stage of suspended development triggered by stress and associated with suppressed Myc activity and overall biosynthesis. In cancer cells, depleting Myc or inhibiting Brd4, a Myc transcriptional co-activator, attenuates drug cytotoxicity through a dormant diapause-like adaptation with reduced apoptotic priming. Conversely, inducible Myc upregulation enhances acute chemotherapeutic activity. Maintaining residual cells in dormancy after chemotherapy by inhibiting Myc activity or interfering with the diapause-like adaptation by inhibiting cyclin-dependent kinase 9 represent potential therapeutic strategies against chemotherapy-persistent tumor cells. Our study demonstrates that cancer co-opts a mechanism similar to diapause with adaptive inactivation of Myc to persist during treatment.

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

Valproic acid alters nitric oxide status in neurulating mouse embryos.

The molecular bases of the teratogenic effects elicited by valproic acid (VPA) are not fully defined. It was previously shown that inhibition of nitric oxide (NO) synthesis is associated with an enhancement of the teratogenic effects of VPA, while amplification of NO signal by sildenafil prompted a dose-dependent reduction of VPA-induced neural tube defects. In this study, for the first time, the effect of VPA on the NO synthesis was evaluated in mouse embryos during early organogenesis. On gestation day 8, ICR-CD1 mice received 600 mg/kg of VPA. Eight and 24 h later embryos were collected and analyzed for NO synthase (NOS) isoform expression, and for molecular mechanisms involved in their modulation. As main finding, in utero embryonic exposure to VPA determined a time-dependent shift of NOS isoforms expression, with a down regulated expression and activity of constitutive NOS (cNOS) and an increased expression and activity of inducible NOS (iNOS). The teratological relevance of this information remains to be established.

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes.

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.

Daidzein supplementation enhances embryo survival by improving hormones, antioxidant capacity, and metabolic profiles of amniotic fluid in sows.

Daidzein (DAI) is a kind of natural isoflavonic phytoestrogen with estrogenic activity. However, little is known about its influence on early fetal growth in mammalian animals. The current study aimed to explore the characteristics of amniotic fluid exposure to dietary DAI using 1H NMR-based metabolomics and biochemical analysis. Here, we found that DAI supplementation at a dose of 200 mg kg-1 significantly enhanced the number of viable embryos at the early gestation stage (P < 0.05). DAI significantly elevated the concentrations of estrogen (E) and insulin-like growth factor-I (IGF-I) in the amniotic fluid (P < 0.05). Moreover, DAI tended to increase the concentration of progesterone, but decrease the concentration of tumor necrosis factor α (TNF-α) in the amniotic fluid (0.05 < P < 0.10). Interestingly, the activity of glutathione peroxidase (GSH-Px) was higher in the DAI group than in the CON group (P < 0.05). An 1H NMR-based metabolomics analysis identified and quantified more than 30 compounds in the amniotic fluid, and some critical metabolites such as arginine, creatine, and citrate were found to be significantly elevated upon DAI supplementation (P < 0.05). Importantly, the metabolic pathways involved in arginine and proline metabolisms were found to be significantly affected by DAI. Collectively, dietary DAI may improve embryo survival by improving hormones, antioxidant capacity, and metabolic profiles in the maternal amniotic fluid.

Effect of Rhodiola sachalinensis Aqueous Extract on In Vitro Maturation of Porcine Oocytes and Subsequent In Vitro Embryonic Development.

Oxidative stress can impede maturation of the nucleus and cytoplasm of oocytes during in vitro maturation (IVM). Rhodiola sachalinensis, an herb commonly used in traditional Chinese medicine, conveys antioxidative effects to cryopreserved bovine sperm. Therefore, the aims of this study were to evaluate the effects of different concentrations of R. sachalinensis aqueous extract (RSAE) on IVM and subsequent in vitro embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT). The results showed that RSAE supplementation (6 and 60 mg/L) significantly increased intracellular glutathione levels, but had no effect on maturation rates or reactive oxygen species. After in vitro culture, greater blastocyst formation was observed in PA embryos (6 mg/L RSAE), as well as in IVF and SCNT embryos (60 mg/L) matured in RSAE-supplemented IVM media. In conclusion, although there was no significant improvement in the maturation rate, RSAE supplementation conveyed an antioxidative effect during IVM, and improved subsequent embryonic development in vitro. Further studies are needed to explore gene expression pattern in oocytes and embryos treated with RSAE.

Graphene Quantum Dots' Surface Chemistry Modulates the Sensitivity of Glioblastoma Cells to Chemotherapeutics.

Recent evidence has shown that graphene quantum dots (GQDs) are capable of crossing the blood-brain barrier, the barrier that reduces cancer therapy efficacy. Here, we tested three alternative GQDs' surface chemistries on two neural lineages (glioblastoma cells and mouse cortical neurons). We showed that surface chemistry modulates GQDs' biocompatibility. When used in combination with the chemotherapeutic drug doxorubicin, GDQs exerted a synergistic effect on tumor cells, but not on neurons. This appears to be mediated by the modification of membrane permeability induced by the surface of GQDs. Our findings highlight that GQDs can be adopted as a suitable delivery and therapeutic strategy for the treatment of glioblastoma, by both directly destabilizing the cell membrane and indirectly increasing the efficacy of chemotherapeutic drugs.

Suprabasin-null mice retain skin barrier function and show high contact hypersensitivity to nickel upon oral nickel loading.

Suprabasin (SBSN) is expressed not only in epidermis but also in epithelial cells of the upper digestive tract where metals such as nickel are absorbed. We have recently shown that SBSN level is decreased in the stratum corneum and serum of atopic dermatitis (AD) patients, especially in intrinsic AD, which is characterized by metal allergy. By using SBSN-null (Sbsn-/-) mice, this study was conducted to investigate the outcome of SBSN deficiency in relation to AD. Sbsn-/- mice exhibited skin barrier dysfunction on embryonic day 16.5, but after birth, their barrier function was not perturbed despite the presence of ultrastructural changes in stratum corneum and keratohyalin granules. Sbsn-/- mice showed a comparable ovalbumin-specific skin immune response to wild type (WT) mice and rather lower contact hypersensitivity (CHS) responses to haptens than did WT mice. The blood nickel level after oral feeding of nickel was significantly higher in Sbsn-/- mice than in WT mice, and CHS to nickel was elevated in Sbsn-/- mice under nickel-loading condition. Our study suggests that the completely SBSN deficient mice retain normal barrier function, but harbor abnormal upper digestive tract epithelium that promotes nickel absorption and high CHS to nickel, sharing the features of intrinsic AD.

Expression of Fibroblast Growth Factor 4 in a Rat Model of Polydactyly of the Thumb Induced by Cytarabine.

BACKGROUND The aim of this study was to assess the expression and mechanisms of fibroblast growth factor 4 in polydactyly of the thumb induced by cytarabine. MATERIAL AND METHODS Rats were intraperitoneally injected with cytarabine at different gestation periods (12.5 days, 13.5 days, and 14.5 days) to establish a polydactyly of the thumb model. Then, the expression of FGF4 in polydactyly was studied by whole-mount in situ hybridization. We used hematoxylin & eosin stain and cartilage stain to investigate the development of the skeleton and tissues in the embryo. Pictures were taken to determine the general shape of the deformity, then X-rays were taken to detect bone distortion of the rats born with a congenital malformation. RESULTS In the experimental group (11.5 days, 12.5 days, 13.5 days, and 14.5 days), whole-mount in situ hybridization showed that the FGF4 expression at the tip of the embryonic limb bud was significantly increased compared with the control group and FGF4 was distributed in a wider range and lasted longer than in the control group (P<0.01). HE staining and cartilage staining showed that there was an extra metacarpal bone and a phalanx in the rats with polydactyly of the thumb (P<0.01). Images of the deformed limbs showed polydactyly and syndactyly of the thumb in the rats. Further X-ray examination revealed 1 extra metacarpal bone and 1 extra phalanx. CONCLUSIONS Cytarabine can induce polydactyly and syndactyly of the thumb in rats. In this process, cytarabine can induce the expression of FGF4 on the tip of the embryonic limb bud, which further leads to abnormal development of the embryonic limb bud and eventually causes a congenital deformity.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.