Expression pattern and signification of Cx43,beta-catenin and Smo in the second heart field / 中国组织工程研究

BACKGROUND:The second heart field is crucial for the development of the embryonic heart.Abnormal development of the second heart field can result in multiple cardiac malformations.After Cx43 gene knockout,reduced formation and proliferation of cells of the second heart field can be observed,but the specific reason remains unclear. OBJECTIVE:(1)To determine whether β-catenin,Smo and Cx43 were co-expressed in the second heart field and the endoderm,we observed the expression patterns of these proteins.(2)To explore whether Cx43 interacts with the Wnt/β-catenin pathway or the Shh pathway in the development of the second heart field. METHODS:Serial paraffin sections of the mouse embryos at embryonic days 10-12 were selected for immunohistochemical staining,hematoxylin-eosin staining and immunofluorescence staining.The primitive gut of mouse embryos at embryonic day 11 was separated for western blot assay and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)Cx43 and Isl1 were co-expressed in some mesenchymal cells on the ventral side of the foregut and dorsal wall of the pericardial cavity of mouse embryos at embryonic days 10-12;Isl1 positive cells increased while Cx43 positive cells increased.(2)Cx43 and β-catenin were co-expressed in the ventral part of the endoderm at embryonic days 10-12.(3)Cx43 and Smo were co-expressed in the endoderm at embryonic days 10-12.(4)The co-immunoprecipitation results confirmed that there was an interaction between Cx43 and β-catenin,which suggested that Cx43 interacted with β-catenin to participate in the development of the second heart field.

Embryonic Toxicity Test of Medical Devices for Human Assisted Reproductive Technology / 中国医疗器械杂志

OBJECTIVE@#To introduce the test methods of embryo toxicity applied to medical devices for human assisted reproductive technology (ARTMD), and provide the evaluation reference.@*METHODS@#The embryo toxicity test methods of ARTMD were summarized, and the key procedures and challenges in their safety evaluation were also discussed.@*RESULTS@#Establishing sensitive and stable test system is important to guarantee the safety and efficacy of ARTMD.@*CONCLUSIONS@#It remains development opportunities in improving sample preparation, extending test technology and expending evaluation method.

Advances in the study of the murine hematopoiesis during the early stage of embryogenesis / 解剖学报

The development of the murine embryonic hematopoietic system occurs in spatially and temporally distinct waves, which it is described as three waves so far-primitive hematopoiesis, bipotential erythroid-myeloid progenitors (EMPs) generation and long-lived transplantable hematopoietic stem cells (HSCs) maturation from their precursors and differentiation toward all the adult lineages. The latest point is that HSC-independent hematopoietic lineages are produced in the primitive wave and definitive progenitor wave in the early mammalian embryo, such as primitive erythrocytes or EMPs. The HSC-dependent phase of hematopoietic development produces all the adult lineages derived from HSCs. In this review, the recent studies on the development of hematopoietic cells and HSCs in the yolk sac and aorta-gonad-mesonephron region (AGM) region at cellular and molecular level will be summarized to provide an integrated model of developmental hematopoiesis, although multiple hematopoietic sites are involved in embryonic hematopoiesis. It may offer new insights into the characteristics and its underlying mechanism of hematopoiesis at the early stage of embryogenesis.

Analysis of factors affecting the standardization of in vitro mouse embryo test / 中国比较医学杂志

Infertility has become a global problem affecting human reproductive health.As an important treatment for infertility, assisted reproductive technology has made great progress over the past few decades.Rapid development has also taken place in medical devices for human assisted reproductive technology.It is imperative to establish the risk management and safety evaluation system of these products.In 2016, the industry standard YY/T 1434-2016 Human in vitro Assisted Reproductive Technology With Medical Equipment in vitro Mouse Embryo Test was officially released.In this paper, the key notes and elements of this in vitro mouse embryo test are briefly reviewed.

Investigating the Potential of Nigella Sativa and Thymoquinone in Salvaging the Embryo from Effects of Toxic Paternal Exposure to Cyclophosphamide

Background: Exposure to cyclophosphamide (CPA) for cancer treatment results in over-production of reactive oxygen species and oxidative stress thus affecting the DNA in male germ cell inducing sperm defects. Our goal is to assess the potential effects of Nigella sativa extract (NSE) and thymoquinone (TQ) on sperm and embryo quality following fertlization of sperm produced from germ cells which have been exposed to the damaging alkylating effects of CPA. Methods: Thirty six male ICR mice were divided into six groups; (I) Vehicle-treated control (normal saline), (II) CPA-only, (III) TQ-only, (IV) NSE-only, (V) CPA followed by TQ and (VI) CPA followed by NSE. Treatment with 200mg/kg CPA and 10mg/kg of both NSE and TQ were given by intraperitoneal injection. Animals were sacrificed at 33 days by cervical dislocation and sperm from caudal epidydymis were taken for analysis and in vitro fertilization (IVF) with eggs from untreated female. Fertilization rates and embryo development were monitored for 5 days. The result were analysed by using SPSS 16.Results: TQ and NSE supplementation to CPA-exposed male mice have no significant effect (p>0.05) on the total number of sperm if compared to CPA-only exposed mice. NSE and TQ supplementation have been shown to have significant effect (p<0.05) on the percentage of motile sperm as well as the number of abnormal sperm. Four types of abnormalities of the sperm were found which includes folded sperm, amorphous, banana-like and the head lacking of the usual hook. Finally, the embryo quality shows a significant improvement by the supplementation of TQ and NSE to CPA-exposed male mice (p<0.05). Conclusion: Overall, both NSE and TQ have indicated chemopreventive potential against the cytotoxicity of cyclophosphamide on the reproductive capacity and fertility.

Effects of laser-assisted hatching and exposure time to vitrification solution on mouse embryo development / 대한생식의학회지

OBJECTIVE: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. METHODS: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. RESULTS: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p < 0.05). In the control-8 group (22.1±4.6), the cell number of the inner cell mass was higher than in the LAH groups (p < 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group (92.8±8.9) than in the others (p < 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group (19.5±5.1, p < 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±12.1) than in the other groups, but without statistical significance. CONCLUSION: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

Construction of induced mouse pluripotent stem cells by piggyBac transposon and its identification / 吉林大学学报(医学版)

Objective:To investigate the effect of piggyBac transpon,as a carrier of four defined transcription factors Oct4,Sox2,Klf4 and c-Myc,in the reprogramming of mouse embryonic fibroblasts (MEFs)to induced pluripotent stem cells (iPSCs).Methods:The MEFs were isolated from Oct4-GFP fetal mice and transfected by piggyBac transposon with four factors (Oct4,Sox2,Klf4 and c-Myc).The morphological changes of clones were traced with microscope during the process of induction.The chromosomes were analyzed to evaluate the karyotypic variation of iPSCs.The mRNA expressions of Oct4, Nanog and FGF4 associated with embryonic stem cells (ESCs)in the iPSCs of mice were tested by RT-PCR;the protein expressions of SSEA-1,Nanog and Alkaline phosphatase in the iPSCs of mice were determined by flow cytometry,immunofluorescence and AP staining.The iPSCs were transplanted into the NOD-SCID mouse groin,4 weeks later,the teratomas were removed for HE staining and the differentiation of tissue was observed.Results:The iPSCs were successfully obtained from MEFs by piggyBac carrying Oct4,Sox2,Klf4,and c-Myc.The round or oval iPSCs clones were similar to ESCs with clear boundry and large dense nuleus.The iPSCs showed the normal karyotypic and expressed the marker genes (Oct4,Nanog and FGF4)and proteins (SSEA-1,Nanog and AP)of ESCs.Teratomas containing three germ layers were formed in NOD-SCID mice after tanspalantation of iPSCs.Conclusion:The iPSCs are reprogrammed from MEFs by piggyBac transposon with four transcription factors-Oct4,Sox2,Klf4 and c-Myc,and the iPSCs with normal karyotype possess the characteristics of ESCs.

Effect of cell-penetrating peptide-conjugated estrogen-related receptor beta on the development of mouse embryos cultured in vitro / 대한생식의학회지

OBJECTIVE: Estrogen related receptor beta (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotency-related genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. METHODS: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without 2 microg/mL CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. RESULTS: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. CONCLUSION: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

The effect of various assisted hatching techniques on the mouse early embryo development / 대한생식의학회지

OBJECTIVE: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers. METHODS: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed. RESULTS: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05). CONCLUSION: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture

OBJECTIVE: This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. METHODS: A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula< or =) and 96 hours (blastocyst< or =) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst< or =) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. CONCLUSION: The rate of embryonic development after 96 hours (hatching blastocyst< or =) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.