Prdx6 is required to protect human corneal epithelial cells against ultraviolet B injury.

BACKGROUND: The protective role of Prdx6 on rat corneal tissue against ultraviolet B injury in vivo has been confirmed previously. We further investigated the function and molecular mechanism of Prdx6 in human corneal epithelial cells under ultraviolet B radiation. METHODS: The experimental groups were designed as follows: (1) Prdx6 RNAi, (2) Prdx6 RNAi + ultraviolet B radiation, (3) normal human corneal epithelial cells, (4) normal human corneal epithelial cells + ultraviolet B radiation, (5) wild-type Prdx6 overexpression, (6) wild-type Prdx6 overexpression + ultraviolet B radiation, (7) mutant-type Prdx6 overexpression, and (8) mutant-type Prdx6 overexpression + ultraviolet B radiation. The cell survival rate was detected by a Thiazolyl Blue Tetrazolium Bromide assay. Apoptosis, reactive oxygen species, and malondialdehyde were detected with a commercial kit. Gene expression was detected by real-time polymerase chain reaction. RESULTS: We found the following results. (1) Compared to normal cells, the survival rates were 32%, 87%, and 58% under ultraviolet B radiation in the Prdx6 interference, wild-type overexpression, and mutant-type overexpression groups, respectively. The survival rates were decreased to 50% at 24 h and 31% at 48 h when the phospholipase A2 activity of Prdx6 was inhibited after ultraviolet B radiation. (2) Apoptosis, reactive oxygen species content, and malondialdehyde levels were increased when Prdx6 was downregulated. This phenomenon became more severe under ultraviolet B radiation. (3) The expression levels of apoptosis-related and antioxidant genes all changed along with the changes in expression of Prdx6. CONCLUSION: (1) Both peroxidase and phospholipase A2 activities of Prdx6 are crucial for its protective role in corneal tissue. (2) Downregulated expression of Prdx6 resulted in high endoplasmic reticulum stress. (3) Apoptosis in human corneal epithelial cells with downregulated Prdx6 coupled with ultraviolet B radiation was related to the pathways of DNA damage and the death receptor. (4) Low levels of antioxidants are sufficient for maintaining homeostasis in human corneal epithelial cells without external stimuli. Under the condition that Prdx6 was downregulated, human corneal epithelial cells were more sensitive to ultraviolet B radiation.

Combined inhibitory effects of low temperature and N-acetyl-l-cysteine on the postovulatory aging of mouse oocytes.

The postovulatory aging of oocytes eventually affects the development of oocytes and embryos. Oxidative stress is known to accelerate the onset of apoptosis in oocytes and influence their capacity for fertilisation. This study aimed to reveal the roles of temperature and the antioxidant N-acetyl-l-cysteine in preventing the aging of postovulatory mouse oocytes. First, newly ovulated mouse oocytes were cultured at various temperature and time combinations in HCZB medium with varying concentrations of N-acetyl-l-cysteine to assess signs of aging and developmental potential. When cultured in HCZB with 300 µM N-acetyl-l-cysteine at different temperature and incubation time combinations (namely 25°C for 12 h, 15°C for 24 h and 5°C for 12 h), the increase in the susceptibility of oocytes to activating stimuli was efficiently prevented, and the developmental potential was maintained following Sr2+ activation or in vitro fertilisation. After incubation at either 15°C for 36 h or 5°C for 24 h, oocytes that had decreased blastocyst rates displayed unrecoverable abnormal cortical granule distribution together with decreased BCL2 levels, total glutathione concentrations and glutathione/glutathione disulphide (GSH/GSSG) ratios. In conclusion, postovulatory oocyte aging could be effectively inhibited by appropriate N-acetyl-l-cysteine addition at low temperatures. In addition, a simple method for the temporary culture of mature oocytes was established.

The Kunming mouse: as a model for age-related decline in female fertility in human.

To ascertain whether the Kunming (KM) mouse is an available model for age-related decline in female fertility in human or not, oocytes from young (6-8 weeks), middle-aged (9 months) and aged (12 months) female mice were compared with respect to number of oocytes, frequency of in-vitro maturation (IVM) and in-vitro fertilization (IVF), and meiotic chromosome segregation and alignment. The mean number of pups born per mouse decreased significantly from the young to the middle-aged and the aged mice. The mean number of ovarian follicles, ovarian germinal vesicle oocytes and ovulated MII oocytes decreased significantly with maternal age. The rate of IVM in oocytes from young mice (73.9%) was less significantly than that in oocytes from middle-aged and aged mice (86.1% and 84.4%, respectively). Immunocytochemical analysis showed that ageing caused a significantly higher rate (49.3%) of chromosome misalignment than that (15.7%) of the young mice. The presence of premature chromatids was also significantly higher in MII oocytes of aged mice as compared with young mice (37.8 versus 8.3%). Pronuclear formation was delayed in oocytes of middle-aged and aged females (35.5 and 42.3% respectively in 5 h of IVF) as compared with young mice (88.1%). The study suggests that KM mouse exhibits an age-related decline in female fertility. Significant reduction of germinal vesicle (GV) and MII oocytes and significant increase of metaphase chromosome misalignment and premature chromatid segregation after meiotic maturation of oocytes, similar to human, presumably contribute to the decline in aged KM mice.

Effect of age, GV transfer and modified nucleocytoplasmic ratio on PKCα in mouse oocytes and early embryos.

Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.

Nucleocytoplasmic ratio of fully grown germinal vesicle oocytes is essential for mouse meiotic chromosome segregation and alignment, spindle shape and early embryonic development.

BACKGROUND: This study examined the effect of nucleocytoplasmic ratio of fully grown germinal vesicle (GV) oocytes on meiotic chromosome segregation and alignment, spindle shape, Ca(2+) oscillations and capacity of early embryonic development in mouse. METHODS: GV oocytes with reduced volume (equal to 1/5 to 4/5 of an intact oocyte) were made by micromanipulation to remove different amounts of cytoplasm, and then matured and fertilized in vitro. RESULTS: When >1/2 of GV oocyte cytoplasm was removed, the time-course of GV breakdown (GVBD) was delayed and oocyte maturation rate decreased significantly. Abnormal chromosome segregation rate increased if >1/2 of the cytoplasm was removed from the oocyte. Length and structure of meiotic spindle and chromosome alignment were also impaired by the reduction of cytoplasmic volume. Once matured in vitro, the oocytes could undergo Sr(2+)-induced Ca(2+) oscillations and form pronuclei in a manner independent of nucleocytoplasmic ratio, but their ability to develop to 2-cell embryos was affected if >1/2 of their cytoplasm was removed from the GV oocytes. CONCLUSIONS: These results suggest that nucleocytoplasmic ratio is essential for normal meiotic chromosome segregation, spindle formation and chromosome alignment over the metaphase spindle, and development to 2-cell stage, for which 1/2 of the volume of the GV oocyte appears to be a threshold.

Transfer of germinal vesicle to ooplasm of young mice could not rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice.

BACKGROUND: [corrected] Transferring a germinal vesicle (GV) from an aged woman's oocyte into ooplasm from a younger woman has been proposed as a possible way to overcome the problem of age-related decline in female fertility. Here we assessed this possibility by determining whether ooplasts derived from young mice could rescue ageing-associated chromosome misalignment in meiosis of oocytes from aged mice. METHODS: Three groups of reconstructed oocytes, young GV-young cytoplast (group YY), aged GV-young cytoplast (group AY), and young GV-aged cytoplast (group YA), were created by micromanipulation and electrofusion. RESULTS: Nuclear transplantation was successful in 89.8-94.4% of GV-ooplast complexes, and maturation rate of the reconstructed oocytes was 93.5-97.9%. Confocal microscopy analysis showed a significantly higher rate (49.2%) of chromosome misalignment in ageing mice than in young mice (16.9%), and 57.1% of oocytes in group AY exhibited chromosome misalignment, while the abnormality rate in groups YY and YA was 16.3 and 16.7% respectively. Calcium imaging showed that the three groups of reconstructed oocytes exhibited a similar pattern of calcium oscillations upon stimulation with bovine sperm extracts. Fertilization rate and developmental capacity to 2-cell embryos were also similar among the three groups of oocytes. CONCLUSIONS: Our findings suggest that: (i) the ooplasm from young mice could not rescue ageing-associated chromosome misalignment in meiosis of GV from aged mice; and (ii) behaviour of chromosome alignment over metaphase spindle is predominantly determined by GV material.