Regulation of gamma-glutamate-cysteine ligase expression by oxidative stress in the mouse preimplantation embryo.

The present study examined expression of gamma-glutamate-cysteine ligase (GLCL; also known as gamma-glutamylcysteine synthetase), the rate-limiting enzyme for de novo synthesis of glutathione, in the preimplantation mouse embryo. Previous studies indicated that the cleavage stage embryo is unable to synthesize glutathione de novo. It is hypothesized that GLCL mRNA and protein are not normally expressed in the cleavage stage embryo, but either glutathione depletion or oxidation may induce their expression. In untreated embryos, RT-PCR and Western blotting revealed GLCL heavy subunit (GLCL-H) mRNA and protein only at the blastocyst stage of development. Furthermore, while diethyl maleate (DEM) exposure to deplete cellular glutathione did not induce expression of GLCL-H, exposure to tertiary-butyl hydroperoxide (tBH), an oxidizing agent, resulted in significant upregulation of GLCL-H expression in two-cell embryos. Neither treatment affected expression in blastocysts. Finally, HPLC analysis confirmed that tBH-treated embryos experienced oxidative stress, as indicated by an increase in the ratio of oxidized to reduced glutathione. This oxidative stress induced de novo glutathione synthesis in the cleavage stage embryo, as demonstrated by the subsequent recovery of reduced glutathione levels following DEM-induced depletion. In the absence of tBH treatment, however, cleavage stage embryos could not recover GSH after DEM-mediated depletion. This study demonstrates that the preimplantation embryo has the capacity to upregulate glutathione synthesis in response to oxidative stress but not GSH depletion. These results suggest that, while the preimplantation embryo is well adapted to dealing with oxidative stress, it may be poorly protected from GSH-depleting toxicants.

Effects of nutrient intake and number of oestrous cycles on in vitro development of preimplantation pig embryos.

The effects of nutrient intake and insemination of gilts at first versus third oestrus on the in vitro development of preimplantation pig embryos were investigated. Standard swine management involves ad libitum feeding of gilts at first oestrus and restricted feeding of gilts at third oestrus. According to previous research, gilts inseminated at first oestrus demonstrate greater embryonic mortality than gilts inseminated at third oestrus, and it is possible that differences in nutrient intake between gilts inseminated at first versus third oestrus affect the viability of eggs or embryos. In the present study, experimental gilts were assigned to three treatments: animals designated 1A were inseminated at first oestrus and fed ad libitum; animals designated 3R were inseminated at third oestrus and were fed a restricted diet; and 3A animals were inseminated at third oestrus and fed ad libitum. Embryos collected from each treatment group were cultured in vitro, and data were evaluated according to cell stage at collection. Comparison of treatments 1A and 3R supported the contention of increased embryo mortality in gilts inseminated at first oestrus under normal management conditions. When cultures were initiated at the one- to two-cell or two- to four-cell stages, the percentage of 1A embryos developing to the morula stage (50.9%, 68.0%) was significantly lower than that of 3R embryos (88.9%, 90.9%; P < 0.05). Comparison of treatments 1A and 3A addressed effects due to the number of oestrous cycles. Significantly more two- to four-cell embryos from gilts inseminated at third oestrus and fed ad libitum reached the morula and expanded blastocyst stages of development (87.0%, 41.3%) compared with embryos from gilts inseminated at first oestrus and fed ad libitum (68.0%, 20.3%; P < 0.05). Finally, the effects of ad libitum feeding were determined by comparing treatments 3A and 3R. These data were inconclusive, as both positive and negative effects were observed. More one- to two-cell embryos from treatment 3R developed to the morula stage (88.9%) compared with 3A embryos collected at the same stage (64.7%), whereas a greater number of 3A embryos in the two- to four-cell category reached the expanded blastocyst stage (41.3%) than 3R embryos (21.2%; P < 0.05). These results support the hypothesis of lower in vitro developmental capacity for embryos collected from gilts inseminated at first oestrus. Furthermore, the findings indicate that differences in embryo viability between gilts inseminated at first versus third oestrus are related to the number of oestrous cycles and possibly to differential nutrition.

Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion.

We investigated the hypothesis that reduced glutathione (GSH) is present in secretions of the female reproductive tract and that this extracellular GSH may protect preimplantation mouse embryos after intracellular GSH depletion. The cleavage-stage mouse embryo cannot synthesize GSH de novo and is unable to recover from glutathione depletion in vitro. Analysis of GSH and total protein of oviduct flushings, quantified by HPLC and the Bradford method, respectively, revealed 51 nmol GSH per mg total protein. Embryos were treated with 60 microM diethyl maleate (DEM) to deplete cellular GSH. When cultured with 1 mM GSH, these embryos exhibited improved development compared to those cultured in control medium (96% vs. 87% morula [p < 0.05], 78% vs. 75% blastocyst, 58% vs. 54% expanded blastocyst, 21% vs. 17% initiating hatching blastocyst). However, intracellular GSH content of embryos was not significantly increased by the culture of DEM-treated embryos in medium containing GSH for 16, 40, or 64 h of incubation, suggesting that the embryo is not capable of taking up intact GSH. Furthermore, addition of buthionine sulfoximine (which inhibits synthesis of GSH) or acivicin (which inhibits breakdown of GSH at the membrane) to culture medium blocked the improvement in development. These data suggest that GSH in reproductive tract fluid may help protect preimplantation embryos from the adverse effects of toxicant-induced and endogenous depletion of embryonic GSH.