Crosstalk between Endoplasmic Reticulum Stress and Oxidative Stress in Heat Exposure-Induced Apoptosis Is Dependent on the ATF4-CHOP-CHAC1 Signal Pathway in IPEC-J2 Cells.

The intestinal epithelium is susceptible to heat stress (HS), which leads to gut leakage and inflammation. However, the mechanisms underlying HS-induced intestine dysfunction have yet to be elucidated. We established an in vitro chronic heat exposure-induced intestinal injury of intestinal porcine epithelial cells (IPEC-J2) exposed to high temperatures (43 °C) for 12 h. The results revealed that HS increased reactive oxygen species (ROS) generation and decreased superoxide dismutase 2 (SOD2) expression, leading to oxidative stress. Western blotting analysis demonstrated that HS induced apoptosis as evidenced by increased cytochrome c (Cyt c) release in the cytoplasm and caspase 3 activation. Transcriptome sequencing analysis revealed that HS activated the endoplasmic reticulum stress (ERS) response/unfolded protein response (UPR) but inhibited glutathione metabolism. Specifically, HS triggered the pro-apoptotic activating transcription factor 4 (ATF4)/CEBP-homologous protein (CHOP) branch of the UPR. Interestingly, glutathione-specific gamma-glutamylcyclotransferase1 (CHAC1) involved in glutathione degradation was upregulated due to heat exposure and was proved to be downstream of the ATF4-CHOP signal pathway. Knockdown of CHAC1 attenuated the HS-induced decrease in glutathione level and cell apoptosis. These studies suggest that crosstalk between ERS and oxidative stress in HS-induced apoptosis might be dependent on the ATF4-CHOP-CHAC1 signal pathway in IPEC-J2 cells.

Expression profiling and functional characterization of miR-192 throughout sheep skeletal muscle development.

MicroRNAs (miRNAs) are evolutionarily conserved, small, non-coding RNAs that have emerged as key regulators of myogenesis. Here, we examined the miRNA expression profiles of developing sheep skeletal muscle using a deep sequencing approach. We detected 2,396 miRNAs in the sheep skeletal muscle tissues. Of these, miR-192 was found to be up-regulated in prenatal skeletal muscle, but was down-regulated postnatally. MiR-192 expression also decreased during the myogenic differentiation of sheep satellite cells (SCs). MiR-192 overexpression significantly attenuated SCs myogenic differentiation but promoted SCs proliferation, whereas miR-192 inhibition enhanced SCs differentiation but suppressed SCs proliferation. We found that miR-192 targeted retinoblastoma 1 (RB1), a known regulator of myogenesis. Furthermore, knockdown of RB1 in cultured cells significantly inhibited SCs myogenic differentiation but accelerated SCs proliferation, confirming the role of RB1 in myogenesis. Taken together, our findings enrich the ovine miRNA database, and outline the miRNA transcriptome of sheep during skeletal muscle development. Moreover, we show that miR-192 affects SCs proliferation and myogenic differentiation via down-regulation of RB1.

Cultivation and Biological Characterization of Chicken Primordial Germ Cells

The purpose of this work was to investigate the isolation, culture process of chicken gonadal primordial germ cells (PGCs) and study their biological characterization. PGCs were harvested from 5.5-day-old chicken embryonic genital ridges and explanted onto chicken embryonic fibroblasts (CEFs). The results showed that the primary cultivation of chicken PGCs on their own gonadal stroma cells were better than CEFs at first two days for reproduction. The conditioned media supported the growth and colony formation of PGCs for a prolonged time in vitro and maintained a normal diploid karyotype, which were positively stained by alkaline phosphatase (AKP), periodic acid Schiff (PAS) and reacted with anti-SSEA-1, SSEA-3, Oct4, Blimp1 and Sox2. Real-time PCR showed that they expressed the stage specific genes CVH, Blimp1 and Dazl, the stem cell specific genes Sox2, Pouv and Nanog. They also formed the embryoid bodies (EBs). These results suggested that the chicken PGCs cultured in vitro not only had strong self-renewal ability, but also had the potential capability of multi-lineage differentiation.

[Isolation and biological characterization of mesenchymal stem cells from Beijing fatty chicken fetal liver].

Mesenchymal stem cells (MSCS) from chicken fetal liver are multipotent stem cells that can differentiate in vitro into various terminally differentiated cells. The majority of studies have focused on rats and mice now. Reports from other animals are less and analyses on domestic animals are few. In this study, chicken liver-derived MSCs were isolated from 7-day-old embryo of Beijing fatty chickens. Primary liver-derived MSCs were subcultured to passage 15. The surface markers of liver-derived MSCs, CD29, and CD44 were detected by immunofluorescence and the surface markers CD34 and CK19 of hematopoietic progenitor cells/hepatic oval cells were not detected. RT-PCR analysis detected positive expression of CD29, CD44, CD71, and CD73. The growth curves were typically sigmoidal. Liver-derived MSCs of different passages were successfully induced and differentiated into neuronal and osteoblast cells. The results suggest that the MSCs isolated from chicken fetal liver possess similar biological characteristics with those derived from mice, and their multilineage differentiation provides many potential applications.

Dynamic analysis of Ca²+ level during bovine oocytes maturation and early embryonic development.

Mammalian oocyte maturation and early embryo development processes are Ca(2+)-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca(2+) and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca(2+) was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca(2+) was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca(2+) was present throughout the blastomere. In PA embryos, Ca(2+) was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca(2+) in the SCNT embryos. However, Ca(2+) was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca(2+) showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca(2+) location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate.

[Effect of different choices and treatments with donor cells on reprogramming].

The donor nucleus must experienced the epigenetic modification of the process reprogramming and went back to the initial state after the donor cell was injected into the oocytes. If the reprogramming is not completed, the efficiency of cloning will be reduced. However, reprogramming of nucleus muct was not only embodied in its ability after it was transferred into the oocytes. It was different in the potential if the cell type was not identical. In addition, different treatment to the donor cells resulted in different ability and the level of reprogramming. This paper described different effects of the type, algebra, cycles, age, and species of the donor cells after nuclear transplantation on the reprogramming. An overview of the exposition and analysis through the donor cell cryopreservation, serum starvation, and different reagent treatments were discussed.

Effects of granulosa cell mitochondria transfer on the early development of bovine embryos in vitro.

The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.

[The application of co-culture system on the in vitro development of bovine somatic nuclear transferred embryos].

To establish a co-culture system of nuclear transferred embryos in bovine, effects of co-culture cell types, passages and cryopreservation as well as addition of BFF or FBS were investigated. The results showed that embryos co-cultured with oviductal epithelial cell and granulosa cell achieved significantly higher blastocyst rate compared with the control group (P < 0.05) and co-cultured with oviductal epithelial cell had more embryo cell number than those with granulosa cell. Passages of co-culture cells significantly affected the blastocyst rate and embryo cell number (P < 0.05), and cryopreservation decreased the blastocyst rate and embryo cell number remarkably. Supplemention of BFF increased blastocyste rate significantly (P < 0.05). In conclusion, co-cultured with fresh primary oviductal epithelial cell along with addition of 10% BFF in SOFaa could improve development of nuclear transferred bovine embryo in vitro.