Ming Cang

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.

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.

Role of thymosin beta 4 in hair growth

Although thymosin beta 4 (Tβ4) is known to play a role in hair growth, its mechanism of action is unclear. We examined the levels of key genes in a Tβ4 epidermal-specific over-expressing mouse model and Tβ4 global knockout mouse model to explore how Tβ4 affects hair growth. By depilation and histological examination of the skin, we confirmed the effect of Tβ4 on hair growth, the number of hair shafts and hair follicle (HF) structure. The mRNA and protein expression of several genes involved in hair growth were detected by real-time PCR and western blotting, respectively. Changes in the expression of β-catenin and Lef-1, the two key molecules in the Wnt signaling pathway, were similar to the changes observed in Tβ4 expression. We also found that compared to the control mice, the mRNA and protein expression of MMP-2 and VEGF were increased in the Tβ4 over-expressing mice, while the level of E-cadherin (E-cad) remained the same. Further, in the Tβ4 global knockout mice, the mRNA and protein levels of MMP-2 and VEGF decreased dramatically and the level of E-cad was stable. Based on the above results, we believe that Tβ4 may regulate the levels of VEGF and MMP-2 via the Wnt/β-catenin/Lef-1 signaling pathway to influence the growth of blood vessels around HFs and to activate cell migration. Tβ4 may have potential for the treatment of hair growth problems in adults, and its effects should be further confirmed in future studies.

CRISPR/Cas9‐mediated specific integration of fat‐1 at the goat MSTN locus

Recent advances in understanding the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, no study has reported simultaneous knockout of endogenous genes and site‐specific knockin of exogenous genes in large animal models. Using the CRISPR/Cas9 system, this study specifically inserted the fat‐1 gene into the goat MSTN locus, thereby achieving simultaneous fat‐1 insertion and MSTN mutation. We introduced the Cas9, MSTN knockout small guide RNA and fat‐1 knockin vectors into goat fetal fibroblasts by electroporation, and obtained a total of 156 positive clonal cell lines. PCR and sequencing were performed for identification. Of the 156 clonal strains, 40 (25.6%) had simultaneous MSTN knockout and fat‐1 insertion at the MSTN locus without drug selection, and 55 (35.25%) and 101 (67.3%) had MSTN mutations and fat‐1 insertions, respectively. We generated a site‐specific knockin Arbas cashmere goat model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer for the first time. For biosafety, we mainly focused on unmarked and non‐resistant gene screening, and point‐specific gene editing. The results showed that simultaneous editing of the two genes (simultaneous knockout and knockin) was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in safe animal breeding.

The role of Sox9 in maintaining the characteristics and pluripotency of Arbas Cashmere goat hair follicle stem cells

In our previous work, we isolated Arbas Cashmere goat hair follicle stem cells (gHFSCs) and explored the pluripotency. In this study, we investigated the expression and putative role of Sox9 in the gHFSCs. Immunofluorescence staining showed that Sox9 is predominantly expressed in the bulge region of the Arbas Cashmere goat hair follicle, and also positively expressed in both nucleus and cytoplasm of the gHFSCs. When the cells were transfected using Sox9-shRNA, cell growth slowed down and the expression of related genes decreased significantly, cell cycle was abnormal, while the expression of terminal differentiation marker loricrin was markedly increased; cells lost the typical morphology of HFSCs; the mRNA and protein expression of gHFSCs markers and stem cell pluripotency associated factors were all significantly decreased; the expression of Wnt signaling pathway genes LEF1, TCF1,c-Myc were significantly changed. These results suggested that Sox9 plays important role in gHFSCs characteristics and pluripotency maintenance.