MiR-34a affects hepatocyte proliferation during hepatocyte regeneration through regulating Notch/HIF-1α signaling pathway.

OBJECTIVE: To explore the influences of micro ribonucleic acid (miR)-34a on liver function and hepatocyte proliferation during hepatocyte regeneration in rats and its mechanism. MATERIALS AND METHODS: A total of 80 Sprague-Dawley rats were randomly divided into 4 groups: Sham-2 d group (2 days after hepatectomy), Sham-10 d group (10 days after hepatectomy), miR-34a siRNA-2d group (miR-34a knockdown + 2 days after hepatectomy) and miR-34a siRNA-10 d group (miR-34a knockdown + 10 days after hepatectomy), with 20 rats in each group. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were detected at 2 d and 10 d after the operation. The rat liver was harvested for calculating the liver/body weight ratio. In addition, the deoxyribonucleic acid (DNA) content in rat hepatocytes was detected via Feulgen staining. The pathological changes in rat liver were detected via hematoxylin-eosin (H&E) staining. Moreover, the hepatocyte apoptosis in each group was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Expression levels of proliferating cell nuclear antigen (PCNA), Notch1 intracellular domain (NICD), and hypoxia-inducible factor-1α (HIF-1α) in liver tissues of each group were detected via immunohistochemistry and Western blotting. RESULTS: No significant differences in the liver/body weight ratio, serum levels of ALT, AST, LDH, pathological structure of the liver, hepatocyte apoptosis level, and PCNA expression in hepatocytes were found between miR-34a siRNA-2 d group and Sham-2 d group. However, the expression levels of NICD and HIF-1α in the liver significantly increased in miR-34a siRNA-2 d group compared with those in Sham-2 d group (p<0.05). On the contrary, compared with those in Sham-10 d group, the liver function and hepatocyte regeneration level significantly increased in miR-34a siRNA-10 d group. Increased liver/body weight ratio, remarkable decline in serum levels of ALT, AST, and LDH, significant alleviation of pathological injury of liver tissues, decreased the apoptosis level and upregulated PCNA protein were observed in miR-34a siRNA-10 d group than those of Sham-10 d group. The Notch/HIF-1α signaling pathway was also significantly activated. CONCLUSIONS: MiR-34a knockdown can significantly enhance the liver function and hepatocyte regeneration ability in rats at 10 d after hepatectomy through activating the Notch/HIF-1α signaling pathway.

Preliminary studies on the vitrification of red sea bream (Pagrus major) embryos.

The objective was to identify an appropriate cryoprotectant and protocol for vitrification of red sea bream (Pagrus major) embryos. The toxicity of five single-agent cryoprotectants, dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), glycerol (GLY), and methyl alcohol (MeOH), as well as nine cryoprotectant mixtures, were investigated by comparing post-thaw hatching rates. Two vitrifying protocols, a straw method and a solid surface vitrification method (copper floating over liquid nitrogen), were evaluated on the basis of post-thaw embryo morphology. Exposure to single-agent cryoprotectants (10% concentration for 15 min) was not toxic to embryos, whereas for higher concentrations (20 and 30%) and a longer duration of exposure (30 min), DMSO and PG were better tolerated than the other cryoprotectants. Among nine cryoprotectant mixtures, the combination of 20% DMSO+10% PG+10% MeOH had the lowest toxicity after exposure for 10 min or 15 min. High percentages of morphologically intact embryos, 50.6+/-16.7% (mean+/-S.D.) and 77.8+/-15.5%, were achieved by the straw vitrifying method (20.5% DMSO+15.5% acetamide+10% PG, thawing at 43 degrees C and washing in 0.5M sucrose solution for 5 min) and by the solid surface vitrification method (40% GLY, thawing at 22 degrees C and washing in 0.5M sucrose solution for 5 min). After thawing, morphological changes in the degenerated embryos included shrunken yolks and ruptured chorions. Furthermore, thawed embryos that were morphologically intact did not consistently survive incubation.

Flow cytometry and ultrastructure of cryopreserved red seabream (Pagrus major) sperm.

The objectives were to assess motility, fertilizing capacity, structural integrity, and mitochondrial function in fresh versus frozen-thawed (15% DMSO was used as a cryoprotectant) sperm from red seabream (Pagrus major). Mean (+/-S.D.) rates of motility, fertilization and hatching of frozen-thawed sperm were 81.0+/-5.4, 92.8+/-1.9, and 91.8+/-5.2%, respectively; for fresh sperm, they were 87.5+/-7.7, 95.8+/-2.4, and 93.8+/-4.2%. Although motility was lower in frozen-thawed versus fresh sperm (P<0.05), there was no effect (P>0.05) of cryopreservation on fertilization or hatching. Based on scanning and transmission electron microscopy, 77.8+/-5.6% of fresh sperm had normal morphology, whereas for frozen-thawed sperm, 63.0+/-7.2% had normal morphology, 20.6+/-3.1% were slightly damaged (e.g. swelling or rupture of head, mid-piece and tail region as well as mitochondria), and 16.4+/-4.2% were severely damaged. Sperm were stained with propidium iodide and Rhodamine 123 to assess plasma membrane integrity and mitochondrial function, respectively, and examined with flow cytometry. For fresh sperm, 83.9% had an intact membrane and functional mitochondria, whereas for frozen-thawed sperm, 74.8% had an intact membrane and functional mitochondria, 12.7% had a damaged membrane, 9.9% had nonfunctional mitochondria, and 2.6% had both a damaged membrane and nonfunctional mitochondria. In conclusion, ultrastructure and flow cytometry were valuable for assessment of frozen-thawed sperm quality; cryopreservation damaged the sperm but fertilizing ability was not significantly decreased.