Molecular cloning, expression, and functional features of IGF1 splice variants in sheep.

Insulin-like growth factor 1 (IGF1), also known as somatomedin C, is essential for the regulation of animal growth and development. In many species, the IGF1 gene can be alternatively spliced into multiple transcripts, encoding different pre-pro-IGF1 proteins. However, the exact alternative splicing patterns of IGF1 and the sequence information of different splice variants in sheep are still unclear. In this study, four splice variants (class 1-Ea, class 1-Eb, class 2-Ea, and class 2-Eb) were obtained, but no IGF1 Ec, similar to that found in other species, was discovered. Bioinformatics analysis showed that the four splice variants shared the same mature peptide (70 amino acids) and possessed distinct signal peptides and E peptides. Tissue expression analysis indicated that the four splice variants were broadly expressed in all tested tissues and were most abundantly expressed in the liver. In most tissues and stages, the expression of class 1-Ea was highest, and the expression of other splice variants was low. Overall, levels of the four IGF1 splice variants at the fetal and lamb stages were higher than those at the adult stage. Overexpression of the four splice variants significantly increased fibroblast proliferation and inhibited apoptosis (P < 0.05). In contrast, silencing IGF1 Ea or IGF1 Eb with siRNA significantly inhibited proliferation and promoted apoptosis (P < 0.05). Among the four splice variants, class 1-Ea had a more evident effect on cell proliferation and apoptosis. In summary, the four ovine IGF1 splice variants have different structures and expression patterns and might have different biological functions.

Growth performance, reproductive traits and offspring survivability of genetically modified rams overexpressing toll-like receptor 4.

Genetic modification provides a means to enhancing disease resistance in animals. Toll-like receptor 4 (TLR4), a member of the TLR family, is critical for the recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by host immune cells, which initiates cell activation and subsequently triggers a proinflammatory response to the invading pathogens. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. Compared with wild-type (WT) rams, the GM rams have similar growth performance, basic semen quality and spermatozoon ultrastructure. The offspring birth rates after cervical artificial insemination were also similar between GM (90.32%) and WT (92.38%) rams. Overall, the presence and expression of the TLR4 transgene in the genome did not appear to interfere with normal semen production, reproductive traits and the ability of transgene transmission to offspring. The expression levels of TLR4, tumor necrosis factor and interferon gamma genes in monocyte/macrophages from GM sheep were significantly higher than that from WT sheep at early stages after LPS stimulation. The GM offspring born from the founder transgenic ram inseminated ewes had similar survival rate with WT offspring (88.89% vs 84.86%) at weaning. The TLR4 transgene showed no deleterious effects on growth performance, reproductive traits and offspring survivability of GM rams. Therefore, the GM sheep overexpressing TLR4 provide a powerful experimental model for analyzing function of TLR4 in vivo during infection and inflammation.

Long-term cryopreservation had no adverse effect on viability of embryos and their offspring in sheep.

Cryopreservation has been widely utilized in livestock and human embryos, which allows for storage of worthy embryos for a long period of time, although it is still uncertain as how long embryos can be cryopreserved in liquid nitrogen. The aims of this study were to evaluate the effects of long-term cryopreservation on birth rate of transferred sheep embryos at morula or blastocyst stage, and to investigate growth performance and viability of their offspring. A total of 373 sheep embryos from the same batch, which had been cryopreserved by conventional procedure for 0.5 yr (n=259) or 7.5 yr (n=114), respectively, were transferred to 373 recipient ewes. In parallel, artificial inseminations, acting as controls, were conducted in the same month in both years (n=81 and n=110) that embryo transfers were performed. Results showed that there were no significant differences in birth rate between short-term cryopreservation group (cryopreserved for 0.5 yr in 2003) and long-term cryopreservation group (cryopreserved for 7.5 yr in 2010) either at the morula or blastocyst stage (p>0.05). No specific differences in birth weight were observed among short-term cryopreservation, artificial insemination 1 (performed in 2003), long-term cryopreservation and artificial insemination 2 (performed in 2010) group (p>0.05). The weaning weights were similar between the short-term cryopreservation and long-term cryopreservation group (p>0.05). The mortality rates of the offspring were similar in both groups as well (p>0.05). We concluded that the long-term cryopreservation did not appear to adversely affect birth rate of the embryos, growth performance and viability of their offspring. Our results indicated that the cryopreserved sheep embryos should be stable in liquid nitrogen for at least 7.5 years.

Melatonin promotes embryonic development and reduces reactive oxygen species in vitrified mouse 2-cell embryos.

Two-cell embryos of mouse were vitrified by the open-pulled straw (OPS) method. The vitrified embryos were warmed and introduced into M16 medium for culture that contains melatonin at different concentrations (10(-3), 10(-5), 10(-7), 10(-9), 10(-11) m). This process caused reactive oxygen species (ROS) formation and jeopardized the development of the embryos. Melatonin, at different concentrations, significantly suppresses ROS production and promotes embryonic development in vitrified embryos compared with untreated ones. The mechanistic studies indicated that the beneficial effects of melatonin on vitrified 2-cell embryos of mouse were melatonin receptor (MT1 and MT2) independent. The direct free radical scavenging activity, the enhancement of endogenous glutathione levels, and the anti-apoptotic capacity of melatonin may account for its protective effects on vitrified embryonic development.