Glutathione Biosynthesis in the Postimplantation Rat Conceptus in Vitro

Abstract

Glutathione (GSH) biosynthesis in Day 10 rat conceptuses was characterized in whole embryo culture by evaluation of net rates of GSH replenishment in whole conceptuses, embryos. and visceral yolk sacs (VYS); uptake and distribution of [35S]cysteine and [35S]methionine from the culture medium; incorporation of 35S-labeled amino acids into GSH; and efflux of [35S]GSH into the culture medium. Diethyl maleate (DEM, 500 [mu]M) depleted intracellular GSH pools in embryo and VYS to 30% of control values within 45 min. Restoration of GSH pools in VYS began immediately and continued at a rate of 295 pmol/ conceptus/hr until GSH concentrations exceeded initial control levels at 4 hr. GSH pools in the embryo remained depleted for over 2 hr, followed by resynthesis at initial rates of 118 pmol/ conceptus/hr. [35S]Cysteine (0.2 mM) uptake from the culture medium resulted in whole conceptus accumulations that reached 1.6 nmol/conceptus. The portion of intracellular free cysteine obtained through uptake of extraconceptal amino acid was 40-90 pmol/conceptus and represented less than 20% of the total free intracellular cysteine. [35S]Methionine (0.2 mM) accumulation surpassed that of cysteine at all time points by two- to threefold. [35S]Cysteine, added 2 hr after DEM, was incorporated into GSH at rates of 126 pmol/conceptus/hr during the first hour. By 4 hr, rates of incorporation had declined to 22 pmol/conceptusfhr. L-Buthionine-[S,R]-sulfoximine (1 mM) completely eliminated incorporation of [35S]cysteinc into GSH. Net efflux of [35S]GSH into the culture medium accounted for less than 40 pmol of total GSH when measured 5 hr after DEM addition. Although effectively transported into the conceptus and readily utilized in protein synthesis, 35S from methionine was not incorporated into GSH under any conditions tested. After chemical depletion, de novo GSH synthesis occurs exclusively in the VYS. Embryonic recovery begins only after GSH pools in the VYS are replete. Prolonged embryonic GSH depletion and slower recovery rates indicate that the embryo may be selectively susceptible to chemical insult following depletion of GSH.