Effects of Alcohol and Liquid Paraffin on Development of Early Mouse Embryos in vitro

In culturing early mouse embryos in vitro,liquid paraffin and alcohol exert deleterious influence on the development of embryos. Some of light liquid paraffin produced by Chinese factories have proved harmful for early mouse embryos. As shown by our experiments, the nitronaphthalene contained and the specific gravity of liquid paraffin were not involved in the injurious effects.However,alcohol mingled in medium had harmful effects on the development of embryos. At the 0.1% concentration of alcohol in medium the proportion of embryos developing to blastocysts decreased to 73.9%. When the concentration of alcohol was increased to 0.8%, all embryos ceased developing. In our experiments, CO_2 which contained 0.13% alcohol had no visible effects on the development of embrvos in vitro.

Glucose Metabolism During Kunming Mouse Preimplantation Development:Analysis of Gene Transcription in Embryos in Vivo

In order to investigate glucose metabolism pathways and their changes in Kunming mouse preimplantation 1-,2-,4-,8-cell,and morula embryos,the mRNA level for the genes involved in glucose metabolism was tested by nested RT-PCR on embryos at different development stages in vivo.These genes were glucose 6-phosphate dehydrogenase(G6PDH),phospho-fructokinase(PFK),and phosphoglucomutase(PGM),representing pentose phosphate pathway(PPP),glycolysis,and glycogensis and glycogenolysis respectively.Three sets of inner and outer primers were designed and synthesized based on cDNA sequences of G6PDH,PFK and PGM.RT-PCR results revealed that G6PDH gene transcription was found in Kunming mouse 1-8 cell embryos,and not in morula embryos;it indicated that 1-8 cell embryos may metabolize glucose by pentose phosphate pathway,but morula embryos can not do so.PFK gene transcription was found in 1-8 cell and morula embryos;it is probable that there exists glycolysis in those embryos.PGM gene transcription was not found in 1-8 cell and morula embryos,so glycogenesis and glycogenolysis in these embryos were not present.

Effects of Colchicine on Distribution of Mitochondria in 2-cell and Compacted 8-cell Mouse Embryos

The distribution of mitochondria during early development of mouse embryos was visualized bymitochondria-specific vital fluorescent dye, rhodamine 123(Rh 123). Mitochondrial clusters wasmarkedly conceotrated to perinuclear area in blastomere of normal 2-ccll embryos. In blastomere ofuncompacted 8-cell embryos, mitochondria were randomly distributed throughout the cytoplasm, butthey were reorganizcd to the cytocortices beneath the apposed surfaces of blastomere duringcompaction. As demonstrated in our study, colchicine (10 μg/ml) produced marked effect onmitochondrial distribution in blastomcre of 2-cell and compacted 8-cell embryos: mitochondriabecame scattered throughout the cytoplasm ofblastomere. It is suggested that the spatial distributionof mitochondria in early mouse embryo are maintained by microtubule.

Effect of Different High CO_2 Concentrations on the Development of 2-cell Mouse Embryos in vitro

Objective To investigate effects of different high CO_2 concentrations on the development of 2-cell mouse embryos in vitro Methods At levels of 5% CO_2 (control group), 5.7% CO_2, 6.0% CO_2 and 15% CO_2, embryos were incubated in drops with CZB medium, respectively, and the drops were covered by paraffin oil which was treated with three-distilled water. In addition, at the level of 15% CO_2, there were another two groups, in which paraffin oil was treated with phosphate-buffered saline (PBS) solution or the drops were uncovered. The development of embryos in all stages was noted. Results The developmental rates of blastocysts in five experimental groups were significantly lower than that of the control group (P<0.01). At the level of 5.7% CO_2, the developmental rate of blastocysts was 4.3%, and those of other experimental groups were 0. At the levels of 5.7% and 6.0% CO_2, embryos were blocked in the 2-cell or the 4-cell stage, and no significant difference was showed between the two groups (P>0.05). At the level of 15% CO_2, 15% embryos developed in the 4-cell stage with irregular blastomere and degenerated quickly in the group which paraffin oil was treated with distilled water; 2.2% embryos developed in the 4-cell stage in the group which paraffin oil was treated with PBS and the rest stagnated in the 2-cell stage. Conclusions High CO_2 concentrations had toxic effect on the in vitro development of 2-cell mouse embryos, and was responsible for the inhibition of the embryos. It is important for the development of embryos in vitro to detect strictly CO_2 concentration.

Effect of an Improved Mechanical Method for Assisted Hatching on the in vitro Development of Mouse Embryos

Objective To evaluate the safety and efficiency of an improved shape of opening for mechanical assisted hatching （AH） on the in virto development of mouse embryos. Methods A total of 622 KM BAI mouse embryos in 2-cell-4-cell stage were randomly divided into group A, group B and control group. A new mechanical AH method by improving the shape of opening in the ZP was used in group A, and a ＂-/ ＂-shaped opening was created. A ＂＋ ＂ -shaped opening was made in group B, while no opening was made in control group. Comparisons have been made among the three groups with regard to the duration of AH, the blastocyst formation and complete hatching rate, etc. Results The duration of AH in group A （43.25 ±3.46 s） was significantly shorter than that in group B （52.81 ±4.32 s, P 〈0.05）. The blastocyst formation rate on d 5 was not significantly different among the three groups （92.27%, 93.66% and 94.92% respectively, P 〉0.05）. The complete hatching rate of blastocysts on d 6 between group A and group B was no statistical difference （94.09% vs 92.71%, P 〉0.05）, but significantly higher than that in control group （43.32%, P 〈0.001）. No significant difference in the percentage of grade 1 blastocysts was found among the three groups on d 5 （85.22%, 82.81% and 86.63% respectively, P 〉0.05）. Conclusion R could enhance the process of embryo hatching and facilitate the hatching rate of blastocysts by using the improved mechanical AH method, which is of safety and efficiency to mouse embryo in the in vitro development.

小鼠整胚石蜡切片制备方法的改进

Objective:The morphology analysis of whole-mount mouse embryos was observed using an improved paraffin section technique.Methods:Mouse embryos of varying embryonic ages were collected and whole-mount embryo paraffin sections were prepared using PFA-intravenously injected fixation,prolonged dehydration,and paraffin embedding.Hematoxylin and eosin(H&E)staining and immunohistochemical staining were employed to evaluate the quality of sections,with different tissues being observed labeled by CD34.Results:Following a series of tissue processing and staining procedures,the structure of the whole-mount mouse embryo was well-preserved,and the staining was clear and easily distinguishable.Embryos of different embryonic ages were treated differently,yet the quality of tissue processing remained highly consistent.Conclusion:Tissue processing and staining have been significantly improved,allowing for the easy acquisition of whole-mount mouse embryos of different ages through simplified methods of tissue fixation and dehydration duration.The staining results are clear and stable,providing technical support for the study of mouse embryo development.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornifiedenvelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then wedetermined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. Wefound a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cellsfrom E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed inthe granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin develop-ment provided several morphological evidences for the epidermal differentiation. The above findings suggest that theexpression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

To investigate the effect of clenbuterol hydrochloride on the in vitro development of both 1 cell and 2 cell mouse embryos. Methods The cultural systems of both 1 cell and 2 cell mouse embryo were used to determine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and 10 ng/mL on developmental rates of mouse embryos. Results When 1 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, developmental rates from the 4 cell stage to blastocyst stage were significantly lower than those in the control group (P<0.05), but on dosages of 3 ng/mL and 10 ng/mL, the inhibiting effects on embryo development were significantly increased (P<0.01). When 2 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious differences in developmental rates were not found between the 2 cell embryo group and the control (P>0.05). However, at levels of 3 ng/mL and 10 ng/mL, significant decrease of developmental rates in 2 cell embryos was observed from the 4 cell and from the 8 cell stage, respectively (P<0.05). Embryos cultured with clenbuterol hydrochloride appeared to have more granules, fragments and degeneration than those in the control. Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and the effect is in a dose dependent. 1 cell mouse embryos cultured with clenbuterol hydrochloride could be easily inhibited at 2 cell stage, but the effect of clenbuterol hydrochloride on development of the late 2 cell embryos would be reduced.

LIVE IMAGING OF EARLY DEVELOPMENTAL PROCESSES IN MAMMALIAN EMBRYOS WITH OPTICAL COHERENCE TOMOGRAPHY

Early embryonic imaging of cardiovascular development in mammalian models requires a methodthat can penetrate through and distinguish the many tissue layers with high spatial and temporalresolution. In this paper we evaluate the capability of Optical Coherence Tomography (OCT)technique for structural 3D embryonic imaging in mouse embryos at different stages of thedevelopmental process ranging from 7.5 dpc up to 10.5 dpc. Obtained results suggest that thecollected data is suitable for quantitative and qualitative measurements to assess cardiovascularfunction in mouse models, which is likely to expand our knowledge of the complexity of theembryonic heart, and its development into an adult heart.

Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.

Sensitivity of sperm motility assay for detecting endotoxin effect on human sperm in vitro

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endotoxin, there was a significant reduction in the development rate at all developmental stages with 1-cell and 2-cell mouse embryo assays and at the level of 10 ng/mL, the embryo development was completely arrested. Conclusion: The sperm motility assay could detect high levels of endotoxin effect in vitro, but its sensitivity is low as compared with the 1-cell or 2-cell mouse embryo bioassay.

Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B （AurkB） is ubiquitously expressed while Aurora kinase C （AurkC） is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 （H3S10P） and regulate metaphase timing. Using an Oct4-photoactivat- able GFP fusion protein （Oct4-paGFP） and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

Chemical compositions,cytotoxicity and antioxidant activity of the endophytic fungus Fusarium napiforme isolated from Psidium guajava

The bioactive secondary metabolites from the endophytic fungus Fusarium napiforme was evaluated for the cytotoxic effect and antioxidant activity.The total antioxidant capacity(TAC)of the extract was determined by 2,2-diphenyl-1-picrylhydrazyl(DPPH),phosphomolybdate,and reducing power assay methods.The cytotoxicity of the extract was evaluated against lung adenocarcinoma(A549)cells and mouse embryo fibroblast(NIH3T3)cells by methyl thiazolyl tetrazolium(MTT)method.The major composition of the crude extract was identified by the Gas Chromatography-Mass Spectrometry(GC-MS)analysis.Estimation of the endophyte crude extract revealed a high amount of the total flavonoid content(TFC)and total phenolic content(TPC).The extract showed high cytotoxic activity against the A549 cell line with the mean cytotoxicity of 69.74±0.49%.The extract did not show any cytotoxic effect against the NIH3T3 cell line.The extract exhibited high antioxidant activity as a function of the concentrations.At a test concentration of 1 mg ml-1,the extract showed the highest inhibition against DPPH radical at 75.4607%±0.47688,ferric ion reducing power at 0.882±0.0120,and 255.434±21.404 AAE/g of the extract by phosphomolybdenum assay(PMA).There is a significant correlation between TPC and antioxidant activity at p<0.05.The correlation between reducing power and DPPH is significant at p<0.01.The major types of bioactive compounds identified by the GC-MS have shown the presence of nine major compounds.This result strongly exhibits that the endophyte F.napiforme can be a potential source for the formulation of natural anticancer drugs and protecting the body from oxidative damages.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

The crystal structure of Ac-AChBP in complex with α-conotoxin LvlA reveals the mechanism of its selectivity towards different nAChR subtypes

The a3＊ nAChRs, which are considered to be promising drug targets for problems such as pain, addiction, cardiovascular function, cognitive disorders etc., are found throughout the central and peripheral nervous system. The α-conotoxin （α-CTx） LvlA has been identified as the most selective inhibitor of α3β2 nAChRs known to date, and it can distinguish the α3132 nAChR subtype from the α6/α3β2β3 and α3β4 nAChR subtypes. However, the mechanism of its selectivity towards α3132, α6/α3β2β3, and α3β4 nAChRs remains elusive. Here we report the co-crystal structure of LvlA in complex with Aplysia californica acetylcholine binding protein （Ac-AChBP） at a resolution of 3.4 A. Based on the structure of this complex, together with homology modeling based on other nAChR subtypes and binding affinity assays, we conclude that Asp-11 of LvlA plays an important role in the selectivity of LvlA towards α3132 and α31o6132133 nAChRs by making a salt bridge with Lys-155 of the rat α3 subunit. Asn-9 lies within a hydrophobic pocket that is formed by Met-36, Thr-59, and Phe-119 of the rat β2 subunit in the α3β2 nAChR model, revealing the reason for its more potent selectivity towards the a3β2 nAChR subtype. These results provide molecular insights that can be used to design ligands that selectively target α3β2 nAChRs, with significant implications for the design of new therapeutic a-CTxs.

MR-seq:measuring a single cell's transcriptome repeatedly by RNA-seq

We described a novel single-cell RNA-seq technique called MR-seq （measure a single-cell transcriptome repeatedly）, which permits statistically assessing the technical variation and identifying the differentially expressed genes between just two single ceils by measuring each single cell twice. We demonstrated that MR-seq gave sensitivity and reproducibility similar to the standard single-cell RNA-seq and increased the positive predicate value, Application of MR-seq to early mouse embryos identified hundreds of candidate intra-embryonic heterogeneous genes among mouse 2-, 4- and 8-cell stage embryos. MR-seq should be useful for detecting differentially exnre^ed ~enes ~rnnn~ ~ ~m^ll nHmhpr nf c^ll~