Abstract
Glutathione (GSH) is the most abundant intracellular non-protein thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate limiting enzyme in GSH synthesis, have decreased tissue levels of GSH. Our and others previous work supports roles for GSH in inhibiting apoptosis in ovarian follicles and in preimplantation embryonic development. We hypothesized that Gclm-/- mice have accelerated age-related declines in ovarian follicles and increased preimplantation embryonic demise. Ovarian total GSH concentrations in Gclm-/- mice were 27% of those in Gclm+/+ littermates. The ratio of reduced to oxidized glutathione was decreased in Gclm-/- ovaries, consistent with a state of relative ovarian oxidative stress. At 2 and 21 days of age, there were no differences in the numbers of oocytes and follicles in the ovaries of Gclm-/- and Gclm+/+ mice. However, by 3 months of age, there were significantly fewer follicles in Gclm-/- ovaries, and this difference became more pronounced with increasing age. A more rapid decline in primordial follicle numbers appeared to be driving the accelerated age-related decline in ovarian follicles in Gclm-/- mice. In a continuous breeding assay beginning at 2 months of age and lasting 20 weeks, Gclm-/- females produced similar numbers of litters as Gclm+/+ littermates, but the litters were significantly smaller. The numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. A timed breeding study revealed fewer uterine implantation sites in the Gclm-/- females at gestational day 18 and no evidence for increased postimplantation mortality. We next investigated the effects of maternal lack of Gclm on early embryonic development in vivo and in vitro. Prepubertal Gclm-/- and Gclm+/+ females were superovulated with 5 IU eCG followed 46 h later by 5 IU hCG, then mated overnight with a Gclm+/+ male. The following morning, at 0.5 days postcoitum (dpc), Gclm-/- females had significantly lower percentages of zygotes with 2 pronuclei and significantly higher percentages of zygotes with one pronucleus and of unfertilized oocytes than Gclm+/+ females. At 3.5 dpc, a significantly lower percentage of blastocyst stage embryos and higher percentages of 3-cell and 8-cell embryos were recovered from the uteri of Gclm-/- females than of Gclm+/+ females. Total GSH concentrations in oocytes from superovulated Gclm-/- females were less than 20% those of wild type females. To test the effects of low oocyte GSH on in vitro embryo development, superovulated oocytes were harvested from Gclm-/- and Gclm+/+ females and were fertilized in vitro with sperm from wild type males; embryos were cultured for 102 h. There was no effect of Gclm genotype on progression to the 2-3 cell stage by 30 h, but significantly fewer embryos of Gclm-/- females progressed to the blastocyst stage by 102 h after gamete mixing. These results demonstrate critical roles for GSH in regulating the ovarian reserve of follicles during reproductive aging and in supporting normal preimplantation development. Supported by NIH AG032087. (platform)
Published Version
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