Depletion of glutathione causes dna damage and increase of hydrogen peroxide levels in bovine embryos

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Theriogenology

DEPLETION OF GLUTATHIONE CAUSES DNA DAMAGE AND INCREASE OF HYDROGEN PEROXIDE LEVELS IN BOVINE EMBRYOS M. Takahashi, N. Saka, Y. Kanai and A. Okano Department of Animal Reproduction, Glutathione(GSH)

National Institute of Animal Industry, Ibaraki 30.5, Japan

is a tripeptide that has a role for keeping a reducing environment in the

cells and the level is highly correlated to the early development embryos after freezing.

and viability

of bovine

One of the most important roles of GSH is to protect the cells against

the toxic effect caused by reactive oxygen species(RGS).

Reduction of redox state causes

harmful effect to cells such as breaking DNA strands or increase of oxidative stress.

We have

previously reported the detection of DNA damage in individual mammalian embryos by using a comet assay techniques. DNA.damage

In the present study, we investigated the effect of GSH depletion on

and the levels of H,O, in bovine embryos.

Bovine 6-8 cell stage embryos

obtained by in vitro fertilization were used for the experiment. Embryos were cultured for 24 h in 1) TCM-199 with 5 % FCS (control), 2) TCM-199 with FCS plus 50 PM 8-mercaptoethanol @-ME :a thiol that promotes intracellular GSH synthesis) PM @ME plus Buthionine

sulfoximine

and 3) TCM-199 with FCS and 50

(BSO: a specific inhibitor of GSH synthesis).

In

experiment 1, DNA damage of individual embryos in each treatment was analyzed by comet assay technique.

Comet assay analysis was achieved as follows.

Embryos were mixed with

small volume of 1% low melting agarose at 37 “C and solidificated on a slideglass.

Then the

embryos were lysed by detergents and high salt for 3 h. After lysing, the slides were set on a horizontal

gel electrophoresis

unit and equilibrated

Electrophoresis was conducted for 20 min at 25 V.

for 20 min in electrophoretic

buffer.

Most procedures were achieved in the

dark to prevent additional DNA damage caused by fluorescence light. After electrophoresis, the slides were stained by ethidium bromide. observed by using fluorescence microscope.

DNA damage of individual

embryos was

Photographs of single embryo were taken and the

length of migrated DNA of each embryo was measured.

In experiment 2, the levels of H,O,

in embryos in each treatment were measured by staining with 2’,7’-dichlorodihydrofluorescein diacetate (DCHFDA).

Fluorescence caused by oxidation of DCI-IFDA in the embryos were

detected with fluorescent microscope and analyzed by image analysis. achieved by using student’s t-test.

Statistical analysis was

The length of damaged DNA in embryos significantly

increased when GSH synthesis was inhibited by BSO treatment (236.1r22.4 (159.6215.1 pm).

m)

than control

Intensity of fluorescence by oxidation of DCHFDA also increased when

GSH synthesis was inhibited by BSO treatment.

These results indicate

that the reduction of

intracellular redox state causes damage to embryos by increasing of hydrogen peroxide level and damaging of DNA.