To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques

To investigate the expression of phospholipase C-gamma1 (PLC-gamma1) in mouse embryonic tissues, serial tissue sections were prepared routinely for immunocytochemistry for PLC-gamma1. The results showed that PLC-gamma1 was expressed in the cartilage, skeletal muscles, myocardium, the collecting tubule of the kidney, connective tissues and the brain, suggesting the important role PLC-gamma1 and the related signal pathway may play in the development of mouse embryonic tissues.
Animals ; Brain ; embryology ; enzymology ; Cartilage ; embryology ; enzymology ; Embryo, Mammalian ; enzymology ; Female ; Fetal Heart ; enzymology ; Immunohistochemistry ; Kidney ; embryology ; enzymology ; Mice ; Muscle, Skeletal ; embryology ; enzymology ; Phospholipase C gamma ; biosynthesis ; Pregnancy

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.
Animals ; Cloning, Organism/*veterinary ; Culture Media ; Embryo, Mammalian ; Karyotyping ; Nuclear Transfer Techniques/*veterinary ; *Oocytes ; Swine/*embryology ; Whales/*embryology

OBJECTIVETo induce nonparenchymal mesenchymal stem cells (NPMSCs) differentiating into functional hepatocyte-like cells in vitro, and to identify the molecular biology and functional characteristics of those hepatocyte-like cells.

METHODSHuman NPMSCs were isolated and cultured with cell culture technique. NPMSCs were induced (on 1% Matrigel as a matrix and then submitted to 2.5 mmol/L AZA pretreatment for 10-12 h), by adding HGF 10 microg/L + FGF4 10microg/L + HGM into the culture medium. The characteristics of proliferation and growth of human NPMSCs were studied with methyl thiazolyl tetrazolium (MTT). The phenotypes of NPMSCs were identified by flow cytometry, immunohistochemistry and RT-PCR. Albumin (Alb) levels in culture supernatants were determined with ELISA. Staining for glycogen of undifferentiated NPMSCs and NPMSCs derivated hepatocyte-like cells was conducted with periodic acid-Schiff (PAS) test.

RESULTSGrowth and division of adherent cells obtained from fetal livers were good and the amount of NPMSCs resourced from each fetus could be amplified to 109 cells after 10 serial subcultivations. The phenotype of NPMSCs was CD166 positive and CD34 negative. The shape of NPMSCs plated on Matrigel with FGF4 and HGF changed from long fusiform to polygonal or round on days 21-28. The rate of cell rounding was 40% and the ratio of dikaryocytes was 5%. Immunohistochemical and RT-PCR detection showed that undifferentiated NPMSCs expressed few alpha fetoprotein (AFP) and AFP mRNA, and did not express any of the liver-specific transcription factors or cytoplasmic markers. Many cells in early induction expressed GATA4, AFP and CK18 proteins and their mRNAs, and their expressions were reduced at the late induction, but the expressions of Alb, CK18, GST-and hepatocyte transcription factor HNF1increased gradually. The ratio of Alb and CK18 positive cells was 60%. Undifferentiated NPMSCs did not produce Alb. Alb production by induced NPMSCs increased in a time-dependent manner. Glycogen storage was first seen on day 14, and maximum levels were seen after day 28.

CONCLUSIONSThere are MSCs among nonparenchymal cells of fetal livers. A high ratio of hepatocyte-like cells was obtained under our induction condition. NPMSCs differentiate firstly into hepatocyte precursors, and then differentiate into mature hepatocytes and hepatocyte-like cells with positive hepatocyte markers. The induced NPMSCs have hepatocyte specific functional features.

Cell Differentiation ; Cell Separation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fetus ; cytology ; Hepatocytes ; cytology ; Humans ; Liver ; embryology ; Mesenchymal Stromal Cells ; cytology

In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 microg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 microg/mL) for 1 h, and the rate of cytoplast protrusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 microg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 microg/mL colchicine for 0.5 h. 3) A maturation time of 18-23 h did not affect the rates of cytoplast protrusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21-23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.
Animals ; Cloning, Organism ; methods ; Colchicine ; pharmacology ; Embryo, Mammalian ; embryology ; Female ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; drug effects ; Sheep

BACKGROUNDThere have been no detailed reports of the three-dimensional structure and the relationship between the external and internal vascularizations observed successively for a long duration in the rat fetus, although many authors have studied the vascular morphology of the developing brain. This study examined the three-dimensional structure of both the external and internal vascularizations of the prenatal rat telencephalon from embryonic days 12 (E12) to 20 (E20).

METHODA microvascular casting method for scanning electron microscopy (SEM) was used in this study, along with vascular staining using gold-gelatin solution-autometallography (GGS-AMG) after intravascular injection of colloidal gold, as well as hematoxylin-eosin (HE) staining for paraffin embedded specimens.

RESULTSIn GGS-AMG stains, E16 fetuses had a few short perforating cortical blood vessels (SPCVs); E17 fetuses had long perforating cortico-medullary vessels (LPCVs). Older fetuses had specific patterns of vascular networks in the cortex and the deeper subcortical part of the telencephalon. In the cortex, fine longitudinal blood vessels were connected by transverse channels. The deep telencephalon had fine blood vessels running in all directions. Using SEM, the external vascularization was already visible in E12 fetuses as arborizations of arterial branches, forming a mesh of fine vascular networks covering the telencephalon. A coralliform fine venous plexus was observed in the external vascularization of E16 fetuses. There were ring-like anastomoses and bud-like protrusions in the network of small blood vessels, most likely the angiogenesis of fetal vessels. From E12 to E16, an immature and incomplete internal vascularization began to appear. There were short blood vessels with ballooned terminals branching from the external vascularization. They penetrated the brain tissue to form networks in the superficial layer, comparable to SPCVs. In E17 to E20 fetuses, tortuous venous branches, straight arterial blood vessels, and a fine network of small blood vessels formed the external vascularization. There were fewer arterial than venous branches connecting to the fine networks of small blood vessels. LPCVs were noted at E17, at the time the white matter emerged. They branched from the external vascularization, and perpendicularly penetrated the brain surface, traversing the cortical plate, and entering into the deep brain. At E17, arterial and venous blood vessels could be clearly distinguished in the external vascularization. At E20, the cortex and white matter contained specific arrangements of networks of fine blood vessels, as seen by GGS-AMG staining.

CONCLUSIONThese findings show that the development of both the external and internal vascularization follows the development of the telencephalon. In particular, the emergence of the cortical plate and white matter on E16 and E17 influence the development of both the internal and the external vascularization. The laminal arrangement of blood vessels was not observed corresponding to the respective laminal neuronal layers.

Animals ; Blood Vessels ; embryology ; ultrastructure ; Embryo, Mammalian ; Fetus ; Microscopy, Electron, Scanning ; Rats ; Rats, Wistar ; Telencephalon ; blood supply

Animals ; Cdc20 Proteins/metabolism* ; Cell Line ; Embryo, Mammalian/embryology* ; Embryonic Development ; Female ; Infertility, Female/metabolism* ; Mice ; Mutation ; Oocytes/metabolism*

OBJECTIVETo explore the patterns of Cx43 and Pax3 protein expressions in the small intestinal muscular layers of human embryo during early development.

METHODSImmunohistochemistry with SABC method was employed to examine the expression of Cx43 and Pax3 proteins in the muscular layers of the small intestine in early human embryos in the second to fourth months of gestation.

RESULTSIn the second month of gestation, the muscle layer of the small intestine was negative for Cx43 and Pax3 protein expressions. In the third month, Cx43 and Pax3 expressions were negative in the inner circular muscle layer, but some positive cells were found in the longitudinal muscle layer and the myenteric plexus. In the fourth month, positive expression of Cx43 and Pax3 proteins were seen in the entire muscle layer.

CONCLUSIONCx43 and Pax3 proteins are closely related to the growth and development of the cells and tissues in the small intestinal muscle layer in human embryos.

Connexin 43 ; biosynthesis ; Embryo, Mammalian ; metabolism ; Humans ; Immunohistochemistry ; Intestine, Small ; embryology ; metabolism ; Muscle, Smooth ; embryology ; metabolism ; PAX3 Transcription Factor ; Paired Box Transcription Factors ; biosynthesis

The aim of this study was to investigate the roles of chemokine CCL20 in development of CD4(+)CD25(+) thymocytes by means of fetal thymus organ culture. Fetal mouse thymus lobes were removed at the fetus age of 14.5 days and cultured in complete RPMI 1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. The results revealed that from day 14.5 to day 19, the absolute and relative numbers of the CD4(+)CD25(+) thymocytes varied similarly as their development as in vitro culture at 6 days. Data showed that during the 6 days in vitro culture the CD4(+)CD25(+) cell percentage out of CD4(+) cells was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4(+)CD25(+) cell percentage out of CD25(+) cells was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06%. All these data indicated similar characters to their development in vivo. Moreover, at interference with CCL20, the percentage of CD4(+)CD25(+) T cells in thymocytes significantly decreased at the 3 and 6 days from 3.24+/-0.18 and 3.96+/-0.24 to 1.27+/-0.11 (p<0.001) and 1.76+/-0.22 (p<0.001) respectively. It is concluded that the development of CD4(+)CD25(+) thymocytes is similar both in vitro and in vivo, interfering with CCL20 significantly downregulate the expression of CD4(+)CD25(+) T cells. The above data may help to understand the development of naturally arising CD4(+)CD25(+) regulatory T cells.
Animals ; Chemokine CCL20 ; pharmacology ; Embryo, Mammalian ; Embryonic Development ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Organ Culture Techniques ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Thymus Gland ; cytology ; embryology

OBJECTIVETo study the expression and distribution of KDR, VEGF and CD34 in yolk sac and liver of human embryo at different development stage.

METHODSYolk sacs and livers of 15 human embryos were analyzed by the immunohistochemical SP kits for the expression of KDR, VEGF and CD34.

RESULTSKDR, VEGF and CD34 were all expressed in yolk sacs and livers of the embryos. In the intermediate liver group, the grey value of KDR and VEGF were 103.8 +/- 6.1 and 96.4 +/- 6.3, respectively, stronger than that in the late liver group which were 90.4 +/- 6.0 and 87.4 +/- 6.3, respectively (P < 0.05). A positive correlation between the levels of KDR and VEGF was observed (P < 0.05).

CONCLUSIONThe expression of KDR and CD34 in yolk sac and liver of embryo suggests the presence of hemangioblast in these organs. Interaction of KDR and VEGF might relate to survival, proliferation, migration and differentiation of hemangioblasts.

Embryo, Mammalian ; metabolism ; Humans ; Immunohistochemistry ; Liver ; embryology ; metabolism ; Vascular Endothelial Growth Factor A ; biosynthesis ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; Yolk Sac ; metabolism