[Rhesus monkey (Macaca mulatta) muller cells exhibit retinal stem/progenitor cell features in vitro].

Recent evidences indicate that retinal muller cells exhibit retinal progenitor characteristics under certain condition in chick, rat and human. However, there is no report on nonhuman primate, a close relative to human. In this study, we first established a muller cell line of rhesus monkey expressing GS, vimentin, CRALBP, EGFR, barely GFAP, which resemble the expression profile of human muller cells, differ from that of rodent. Expression of pax6, nestin, sox2, otx2, six6, and six3 was detected after one week culture in neural stem cell medium. Further culture with retinoic acid induced some cells differentiate toward neuron. These results suggest that primate muller cell is capable of dedifferentiating to retinal progenitors, which may serve as a potential cell source for cell therapy to treat retinal degenerative diseases.

[Role of PTEN in mouse pre-implantation development].

The PI3K/Akt signal transduction pathway plays an important role in pre-implantation embryonic development. The tumor suppressor gene PTEN negatively regulates the PI3K/Akt pathway. Although PI3K is constitutively activated during pre-implantation embryonic development, currently no evidence shows the presence and possible involvement of PTEN in early embryo development. We investigated the expression of PTEN protein in germinal vesicle (GV) stage oocytes as well as in pre-implantation embryos. The activated form of PTEN was distributed in the peripheral of GV oocytes and compact morula. Treatment of GV oocytes with PTEN inhibitor bpV(pic) did not affect the maturation of the oocyte, but significantly impaired embryonic development. Thus, our study suggests the necessary role of PTEN in pre-implantation embryonic development.

Notch signaling dependent differentiation of cholangiocyte-like cells from rhesus monkey embryonic stem cells.

Rhesus monkey embryonic stem (rES) cells have similar characteristics to human ES cells, and might be useful as a substitute model for preclinical research. Notch signaling is involved in the formation of bile ducts, which are composed of cholangiocytes. However, little is known about the role of Notch signaling in cholangiocytic commitment of ES cells. We analyzed the effect of Notch signaling on the induction of cholangiocyte-like cells from rES cells. About 80% of definitive endoderm (DE) cells were generated from rES cells after treatment with activin A. After treatment with BMP4 and FGF1 on matrigel coated wells in serum-free medium, rES-derived DE gave rise to cholangiocyte-like cells by expression of cholangiocytic specific proteins (CK7, CK18, CK19, CK20, and OV-6) and genes (GSTPi, IB4, and HNF1ß). At the same time, expression of Notch 1 and Notch 2 mRNA were detected during cell differentiation, as well as their downstream target genes such as Hes 1 and Hes 5. Inhibition of the Notch signal pathway by L-685458 resulted in decreased expression of Notch and their downstream genes. In addition, the proportion of cholangiocyte-like cells declined from approximately 90% to approximately 20%. These results suggest that Notch signaling may play a critical role in cholangiocytic development from ES cells.

Effects of various extenders and permeating cryoprotectants on cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa.

The cryoprotective effects of 11 different extenders, TTE, DM, mDM, LG-DM, G-DM, TCG, TEST, TSM, Test-M, Test-H, and LM, on sperm cryopreservation of cynomolgus monkey (Macaca fascicularis) have been compared with glycerol as cryoprotectant. Sperm motility, plasma membrane, and acrosomal integrity were examined to evaluate frozen-thawed sperm function. The results showed that TTE, DM, mDM, LG-DM, G-DM, and TCG exhibited the best and similar protective efficiencies for cynomolgus monkey sperm cryopreservation in terms of sperm motility and plasma membrane integrity (P > .05). The acrosomal integrity for spermatozoa cryopreserved in TCG was statistically lower than that of TTE, DM, mDM, LG-DM, and G-DM (P < .05) but was significantly higher than that of TEST, TSM, Test-M, Test-H, and LM (P < .05). The postthaw sperm motility for 5 other extenders (TEST, TSM, Test-M, Test-H, and LM) did not exceed 30%, and the 3 sperm parameters evaluated for them were significantly lower than that of TTE, DM, mDM, LG-DM, G-DM, and TCG (P < .05). On the basis of these findings, 5 commonly used permeating cryoprotectants, glycerol, ethylene glycol, dimethyl sulfoxide, acetamide and propylene glycol have further been tested for their effectiveness on sperm cryopreservation in extenders of TTE, DM, mDM, LG-DM, G-DM, and TCG. The results showed that the sperm cryoprotective efficiencies of glycerol and ethylene glycol were similar and best among 5 permeating cryoprotectant treatments (P > .05). Dimethyl sulfoxide or acetamide resulted in average cryoprotection for cynomolgus monkey spermatozoa: poorer than glycerol or ethylene glycol but better than that of propylene glycol (P < .05). In addition, the action of permeating cryoprotectant appeared to be independent of extenders. The results in the present study demonstrate that 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation; 2) the mechanism of action of permeating cryoprotectants are not affected by extender composition; 3) ethylene glycol has a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryopreservation in cynomolgus monkeys.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender.

AIM: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. METHODS: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. RESULTS: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. CONCLUSION: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Photodynamic inhibitory effects of three perylenequinones on human colorectal carcinoma cell line and primate embryonic stem cell line.

AIM: To investigate the photodynamic inhibitory effects of Elsinochrome A (EA), Hypocrellin A (HA) and Hypocrellin B (HB) on human colorectal carcinoma Hce-8693 cells and rhesus monkey embryonic stem R366.4 cells, via inducing apoptosis. METHODS: EA, HA and HB were extracted from metabolites of Hypomyces (Fr) Tul.Sp. R366.4 cells or Hce-8693 cells were cultured with different concentrations of EA, HA or HB respectively, irradiated and incubated with fresh medium for 2 h. Cell cycle analysis was performed by flow cytometry (FCM). Data were expressed as means +/-SD and analysis of variance and Student' t-test for individual comparisons. RESULTS: The photodynamic bioactivity of EA was first reported in this study. After irradiation for 5 min, 6 min, 10 min or 20 min, photoactivated EA at lower concentrations, which were 10(-7) Mol/L, 10(-6) Mol/L, 10(-5) Mol/L respectively, had no cytotoxic effects on R366.4 ES cells. Whereas, all of the three perylenequinones could induce apoptosis with a dose-dependent manner when Hce-8693 cells were incubated with photoactivated EA, HA and HB respectively. When Hce-8693 cells were incubated with EA at 10(-6) Mol/L and irradiated 5 min, 6 min, 10 min and 20 min respectively, the rates of EA-induced apoptosis were 0, 0, 13.4 % and 40.5 %. While the rates of HA-induced apoptosis were 29.5 %, 32.0 %, 40.2 % and 22.6 %. And the rates of HB-induced apoptosis were 0, 0, 0 and 13.7 % respectively. Meanwhile, after 10(-5) Mol/L treatment, the rates of EA-induced apoptosis were 32.7 %, 19.3 %, 26.4 % and 52.7 %, the rates of HA-induced apoptosis were 47.2 %, 39.1 %, 45.2 % and 56.6 %, and the rates of HB-induced apoptosis were 0, 0, 20.0 % and 13.9 % respectively. CONCLUSION: EA, HA and HB have significant anti-cancer activity. The order of photodynamic inhibitory effects on tumor cells would be approximately HA>EA>HB. The molecular mechanisms of apoptosis may not be induced by reactive oxygen species and are worth further investigation.