Dongjun Liu

Early apoptosis is associated with improved developmental potential in bovine oocytes.

The poor quality of oocytes may be the main reason for the low efficiency of the current in vitro embryo production. However, efforts are required to understand the mechanisms of oocyte development, which is believed to be largely regulated by apoptosis in vivo. The aim of this study was to investigate the levels of apoptosis in bovine immature oocytes with different developmental potentials and to determine whether early apoptosis in bovine oocytes is correlated with their subsequent development. Cumulus-oocyte complexes (COCs) were selected and classified into four groups according to oocyte cytoplasm and cumulus status. Early and late stages of apoptosis were detected by Annexin-V and TUNEL staining, respectively. Developmental competence was evaluated by nuclear maturation (MII) after in vitro maturation and development rates in different stages following in vitro fertilization. Meanwhile, the transcripts of Bcl-2 and Bax genes were carried out in immature oocytes by real-time RT-PCR. Results indicated that Annexin-V-positive oocytes were detected in various groups at different percentages, and Group III showed the highest positive ratio. No TUNEL-positive oocytes were found in any immature COCs. Group III oocytes demonstrated the highest nuclear maturation, cleavage, blastocyst, and hatching blastocyst rates. Meanwhile, Group III oocytes exhibited the highest Bax (initiating apoptosis) transcriptional level and the lowest Bcl-2 (preventing apoptosis) transcriptional level. Taken together, Annexin-V and quantitative PCR results indicated that early apoptosis was beneficial for developmental competence, while TUNEL staining showed that none of the immature oocytes were undergoing late-stage apoptosis. This is the first time that Bax and Bcl-2 transcripts were characterized in the immature bovine oocyte, and results indicated that the genes are good markers of early apoptosis and embryo development. This research overthrows the traditional view that oocytes undergoing apoptosis have poor developmental competence, and the findings will facilitate oocyte selection and improvement of in vitro embryo production.

Isolation, expansion, and differentiation of goat adipose-derived stem cells

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSCs using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures.

Expression of IGF receptors and its ligands in bovine oocytes and preimplantation embryos

The objectives of this study were to assess the mRNA expression and protein location of IGF receptors and its ligands in bovine oocytes and different stages of preimplantation embryos, and then evaluate the effect of different concentrations of IGF-II when added to either the maturation or culture medium on in vitro embryo development. For the assessment of mRNA expression by RT-PCR three replicates each of 100 oocytes, and 60 embryos at each of the 2-cell, 8-cell, morula and blastocyst stages of development were used. Immunocytochemical techniques were used to study the location of IGFs and their receptors for COC, oocytes, and embryos at the same stages of development (n=25). The effect of supplementing maturation medium with IGF-II was examined using groups of 20 oocytes exposed to 0 (control), 10, 20, 50 or 100 ng IGF-II/ml medium. Each treatment was replicated five times. To study the effect of IGF-II added to culture medium, groups of 10 zygotes were cultured in the presence of 0 (control), 50, 100 or 150 ng IGF-II/ml medium and the treatments replicated four times. The results showed that IGF-I mRNA could not be detected but IGF-II, IGF-IR and IGF-IIR mRNA existed in bovine preimplantation embryos. Proteins for IGF-II, IGF-IR and IGF-IIR were detected on the cell plasma membrane of cumulus cells of COC, immature and mature oocytes, and 2-cell stage embryos. They were observed in blastomere cytoplasm of 8-cell and morula stage embryos. In blastocysts, the IGF proteins were distributed in the trophectoderm but not in the inner cell mass. Adding 20 ng/ml IGF-II to maturation medium resulted in higher rates of post-fertilization development than control at 8-cell (58.2% versus 44.5%; p<0.05) and blastocyst (37.0% versus 25.0%; p<0.05) stages of development; and the number of viable cells per blastocyst were significantly higher (126+/-6 versus 103+/-5; p<0.05). When IGF-II was added to the culture medium, no significant treatment differences were observed at 8-cell embryo stage but the development rate of zygotes cultured in the presence of 100 ng IGF-II/ml medium to blastocysts was significantly higher than that of control (30.0% versus 19.2%; p<0.05). It was concluded that supplementation of in vitro maturation or culture media with IGF-II affects the development of bovine embryos and could be used to improve in vitro embryo production.

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization.

AbstractAlthough isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found thatn exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle‐derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2‐step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2‐step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α‐Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARα2 (peroxisome‐proliferator‐activated receptor α2) and clear lipid droplets were present around the cells, with Oil Red‐O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.

FasL-induced apoptosis in bovine oocytes via the Bax signal

The factor associated suicide (Fas) and its ligand (FasL) signaling is an important regulatory pathway of apoptosis in mammalian follicles. However, whether apoptosis in bovine oocytes is regulated by the Fas-FasL signaling pathway remains unknown. In this study, localization of Fas and FasL in immature oocytes and FasL in cumulus cells were examined using immunofluorescence staining. In addition, exogenous FasL was added to an in vitro culture system to investigate apoptotic changes in bovine oocytes, using annexin-V and terminal uridine nick-end labeling staining, and real-time quantitative polymerase chain reaction. In this study, Fas was expressed in immature oocytes, whereas FasL was expressed in cumulus cells, but not in immature oocytes; annexin-V- and terminal uridine nick-end labeling-positive rates of oocytes treated with 2, 10, or 50 ng/mL FasL were higher than those of control oocytes (P < 0.05); and oocytes from the three treatment groups had higher expression levels of Fas and B cell lymphoma/leukemia-2 associated X than those in the control group (P < 0.05). Taken together, we concluded that the Fas-FasL signaling pathway was involved in regulation of bovine oocyte apoptosis, perhaps related to B cell lymphoma/leukemia-2 associated X upregulation.

Isolation and morphological characterization of ovine amniotic fluid mesenchymal stem cells

Mesenchymal stem cells (MSCs) are one of the most promising cell populations for tissue engineering and regenerative medicine. Of utmost importance to MSC research is identification of MSC sources that are easily obtainable and stable. Several studies have shown that MSCs can be isolated from amniotic fluid. The sheep is one of the main types of farm animal, and it has many biophysical and biochemical similarities to humans. Here, we obtained MSCs from ovine amniotic fluid and determined the expansion capacity, surface and intracellular marker expression, karyotype, and multilineage differentiation ability of these ovine amniotic fluid mesenchymal stem cells (oAF-MSCs). Moreover, expression levels of differentiation markers were measured using reverse transcription-qPCR (RT-qPCR). Our phenotypic analysis shows that the isolated oAF-MSCs are indeed MSCs.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Knockdown of IGF-IR by siRNA injection during bovine preimplantation embryonic development

This study aimed to assess the efficiency and effects of insulin-like growth factor receptor-1 (IGF-IR) siRNA knockdown during bovine preimplantation embryonic development. In oocytes injected with IGF-IR siRNA, the relative IGF-IR mRNA levels compared to controls were 28% and 46% at 6 and 24xa0h after injection, respectively. With respect to the injection of IGF-IR siRNA in zygotes, 24xa0h after injection the relative levels of IGF-IR mRNA and protein in the two-cell embryos were 74% and 78% of those in the controls, respectively. IGF-IR siRNA reduced blastocyst formation (23.2%) compared to siRNA controls (33.0%) and uninjected oocytes (35.4%; Pxa0<xa00.05) and the number of viable cells per IGF-IR siRNA-treated blastocyst (64xa0±xa03) was significantly reduced, compared to control siRNA and uninjected blastocysts (81xa0±xa03 and 116xa0±xa04; Pxa0<xa00.01). In conclusion, IGF-IR siRNA knockdown reduces the development of bovine embryos, and microinjection in zygotes can decrease blastocyst cell number.

Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

In this study, highly purified hair follicle stem cells from Arbas Cashmere goat (gHFSCs) were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of the gHFSCs were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The gHFSCs were in small cell size with typical cobblestone morphology, good adhesion and high refractive index. Immunocytochemistry staining showed the cells were expressing Krt15, Krt19, CD34, Itgβ1 and Krt14. Cell growth curve indicated that cultured gHFSCs had strong proliferation ability. Krt14 and CD34 were high expressed at the mRNA level, respectively, 39.68 and 24.37 times of the Cashmere goat keratinocytes, and krt15 expression was 5.62 times and itgβ1 expression was 1.81 times higher (pxa0<xa00.01). Western blot detected the expression of all the above markers. After osteogenic induction, the cells were positive for Von Kossa staining and expressed Osteocalcin. Sulfated proteoglycans in cartilaginous matrices were positively stained by Alcian blue after chondrogenic induction and COL2A1 was expressed. In myogenic induction, Hoechst 33342 staining evidenced cytoplasm fusion and positive expression of MyoG was detected by immunocytochemistry.