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Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group (P0.05). This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group (P

Mouse somatic cells have been successfully reprogrammed into embryonic stem cell-like cells by enforced expression of defined transcription factors, and the reprogrammed cells are termed as induced pluripotent stem cells. Such cell reprogramming strategy is involved in gene transfection with retroviral, lentiviral, adenoviral and plasmid vectors. In addition, proteins and small molecules, synthetic modified mRNA and microRNA also have been used to efficiently generate mouse and human iPS. As a high efficient transposon, piggyBac has been used to generate induced pluripotent stem cells. In this study, piggyBac carrying four transcription factors called Oct4 , Sox2 , Klf4 and c-Myc has been employed to generate mouse induced pluripotent stem cells from murine embryonic fibroblasts, and the differentiation potential of the cells was assessed in vivo and in vitro . The reprogrammed cells showed positive staining for alkaline phosphatase and positive for OCT4, SSEA-1 and NANOG by immunofluorescent analyses. RT-PCR results showed that Oct4 , Sox2 , Klf4 and c-Myc was expressed in the reprogrammed cells. Immunofluorescent staining of AFP, BRACHYURY and NCAM1 indicated that the cells have the potential to differentiate spontaneously into all three germ layers in vitro . Teratomas formed after the cells subcutaneously injected into NOD-SCID mice. Chimeric blastocysts were developed when the cells were aggregated with mouse 8-cell embryos. These results suggested that mouse induced pluripotent stem cells can be generated from fibroblast cells by piggyBac transfection system. Such induced pluripotent stem cells are similar to mouse ES cells and have potential to differentiate into three germ layers in vitro and in vivo .

We recently reported that electrical activation followed by secondary chemical activation greatly enhanced the developmental competence ofin vitromatured porcine oocytes fertilized by intracytoplasmic sperm injection (ICSI). We hypothesized that sperm treatment with disulfide bond reducing agents will enhance the development competence of porcine embryos produced by this ICSI procedure. We examined the effects of glutathione (GSH), dithiothreitol (DTT), GSH or DTT in combination with heparin on sperm DNA structure, paternal chromosomal integrity, pronuclear formation, and developmental competence ofin vitromatured porcine oocytes after ICSI. Acridine orange staining and flow cytometry based sperm chromatin structure assay were used to determine sperm DNA integrity by calculating the cells outside the main population (COMP αT). No differences were observed in COMP αT values among GSH-treated and control groups. COMP αT values in GSH-treated groups were significantly lower than that in DTT-treated groups. Following ICSI, GSH treatments did not significantly alter paternal chromosomal integrity. Paternal chromosomal integrity in sperm treated with DTT plus or minus heparin was also the lowest among all groups. GSH-treated sperm yielded the highest rates of normal fertilization and blastocyst formation, which were significantly higher than that of control and DTT-treated groups. The majority of blastocysts derived from control and GSH-treated spermatozoa were diploid, whereas blastocysts derived from DTT-treated spermatozoa were haploid. In conclusion, sperm treatment with GSH enhanced the developmental capacity of porcine embryos produced by our optimized ICSI procedure.

In total, three different tissues from the rough-legged buzzard were obtained by culture and successfully cryopreserved and then recovered. During the subculture process, biological characteristics including as cell morphology, growth curve, cell adhesion rate, and karyotype were analyzed and compared, and overall all three kinds of tissue cells exhibited fibroblast-like growth. Oviduct-derived cells had the strongest adherent ability, followed by lung-derived cells and trachea-derived cells. The doubling times of lung-derived cells, trachea-derived cells, and oviduct-derived cells were 29.91±0.39 h, 33.18±0.21 h, and 30.67±0.28 h, respectively, with population doubling times 3.54±0.01, 4.52±0.02, and 4.38±0.03, respectively. Likewise, we noted the chromosome number of the rough-legged buzzard was 68, within the typical type of ZW. These results may potentially provide material and a basis for further research in the field, with the successful preservation of genetic information of rough-legged buzzard.用组织块培养法对毛脚鵟不同组织进行原代培养,获得了3种不同组织来源的细胞,并成功对细胞进行了冷冻保存和复苏。在传代培养过程中,对比分析了3种组织来源细胞的形态学、生长曲线、贴壁率、核型等生物学特性。形态学方面,3种来源细胞均为成纤维样细胞。对于3种组织来源细胞的贴壁能力分析显示,输卵管源细胞最强,肺源细胞和气管源细胞次之。3种不同组织来源细胞的倍增时间分别为(29.91±0.39)、(33.18±0.21)和(30.67±0.28) h,群体倍增次数分别为 3.54±0.01、4.52±0.02和4.38±0.03。毛脚鵟细胞的染色体数目为2

To study the effects of cryptotanshinone on androgen synthesis for the prenatally androgenized male rats.On days 16-18 of pregnancy, rats were injected s. c. with testosterone propionas continuously for 3 days; male offspring were studied as subject. Serum concentrations of testosterone (T), 17a-hydroxy progesterone (17-OHP), blood glucose, and insulin were measured by radioimmunoassay. Then, the rats were treated with cryptotanshinone by gavage for 14 days, and the levels of serum T, 17-OHP and insulin were detected and the 17a-hydroxylase protein expression in interstitial cell was measured using the method of immunohistochemistry.There was no difference between the male groups who were prenatally androgenized in serum levels of T, but the 17-OHP, fasting insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) elevated significantly (P0.05). Cryptotanshinone could lower the levels of 17-OHP (P0.05) but had no effect on 17a-hydroxylase.Prenatally androgenized male rats exhibit elevated 17-OHP and diminished insulin sensitivity. Cryptotanshinone could decrease 17-OHP, but has no effect on insulin, indicating it may reduce androgen synthesis.

The aim of this study was to investigate the effect of electrical pulse, ethanol, and ionomycin combined with cycloheximide (CHX), cytochalasin B (CB), and 6-dimethylaminopurine (6-DMAP) on parthenogenetic developmental competence of in vitro matured porcine oocytes. In experiment 1, oocytes were treated with direct current electrical pulse (DC pulse) and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX, and CB + 6-DMAP for 6 h, respectively. The rate of blastocyst development in DC pulse + CB + 6-DMAP group was significantly higher than those in other groups (42.4% vs 23.9% approximately 35.8%; P < 0.05); however, there were no differences in both of the cleavage rate and the cell number of blastocysts among four groups. In experiment 2, oocytes were treated with NCSU-23 medium containing 20 muM ionomycin for 40 min and then incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, CB + CHX and CB + 6-DMAP for 6 h, respectively. The rates of cleavage and blastocyst development in ionomycin + 6-DMAP group were higher than those obtained in other groups (66.2% vs 46.3% approximately 57.3%; 22.3% vs 7.4% approximately 16.1%; P < 0.05). In experiment 3, the activation effects of ethanol combined with 6-DMAP, CHX, CB + 6-DMAP and CB + CHX were investigated. The rates of cleavage and blastocyst development in ethanol + CB + 6-DMAP group were significantly higher than those in other groups (55.5% vs 42% approximately 46.2%; 18.0% vs 7.1% approximately 11.9%; P < 0.05). In experiment 4, the optimal activation protocols in each group plus DC pulse + ionomycin + 6-DMAP were compared. The results showed the rates of cleavage in DC pulse + CB + 6-DMAP group and ionomycin + 6-DMAP were higher than those in ethanol + CB + 6-DMAP and DC pulse + ionomycin + 6-DMAP (73.8-74.4% vs 56.5-57.5%; P < 0.05), but the blastocyst development only in DC pulse + CB + 6-DMAP group was significantly higher than that in other groups (34.1% vs 13.4% approximately 22.3%; P < 0.05). Total cell number of blastocysts in the group of DC pulse + ionomycin + 6-DMAP was higher than that in other groups (34.1 vs 25.3-27.2; P < 0.05). In conclusion, DC pulse, ethanol, CB, and 6-DMAP all affected the parthenogenesis of porcine oocytes matured in vitro, but their combination of DC pulse + CB + 6-DMAP showed the best result in both of cleavage and blastocyst development.