Uptake and Salvage of Hypoxanthine Mediates Developmental Arrest in Preimplantation Mouse Embryos

BIOLOGY OF REPRODUCTION 56, 1-13 (1997) Uptake and Salvage of Hypoxanthine Mediates Developmental Arrest in Preimplantation Mouse Embryos' Mary K. Dienhart, 3 Marilyn J. O'Brien, 4 and Stephen M. Downs 23, Marquette University,3 Department of Biology, Milwaukee, Wisconsin 53210-1881 Jackson Laboratory, 4 Bar Harbor, Maine 04609 ABSTRACT Preimplantation mouse embryos become arrested after first or second cleavage when cultured in hypoxanthine-supplemented Whitten's medium. We present evidence that the hypoxanthine-induced arrest isdependent on uptake and salvage of hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of ['14C]hypoxanthine metabolism revealed that hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme hypoxanthine-guanine phosphoribosyltransferase, hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by hypoxanthine. We conclude that metabolism of hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in hypoxanthine or glucose uptake, or by changes in the relative metabolism of hypoxanthine. INTRODUCTION Rapidly growing and dividing cells have an absolute requirement for purine and pyrimidine nucleotides essential for the synthesis of DNA and RNA as well as for many of the coenzymes involved in bioenergetic processes. Although most mammalian cells in culture are capable of synthesizing these compounds de novo, media used for cell culture are often supplemented with precursors for nucleic acid synthesis, which has resulted in improved growth rate and survival of cells [1]. Preimplantation mouse embryos do not require an exogenous source of nucleic acid components, having either an adequate endogenous supply or the capability of synthesizing them as needed [2]. If the culture medium contains nucleosides or nucleoside bases, they can be incorporated into embryonic DNA and RNA [3-6], but these compounds have also been found to inhibit embryo development [7-10]. Preimplantation embryos from some strains of mice are extremely sensitive to micromolar concentrations of the purine base hypoxanthine, exhibiting developmental arrest during G2 of the second or third cell cycle [8, 9, 11-13]. Accepted August 13, 1996. Received May 9, 1996. 'This work was supported by a grant from the NIH (HD 25291). Marilyn J. O'Brien was supported by a grant to John Eppig from the NIH (HD21 970). 2 Correspondence: Stephen M. Downs, Marquette University, Department of Biology, P.O. Box 1881, Milwaukee, WI 53210-1881. FAX: (414) 288-7357; e-mail: 1 This response to hypoxanthine is not limited to the mouse: the in vitro development of human [14, 15] and rabbit [16] embryos is also compromised. The hypoxanthine-induced block in mouse embryos, which is dependent on the culture medium used and the presence of glucose [11, 13], can be reversed by transfer of embryos to purine-free conditions [9]. Compounds that elevate cAMP levels have also been reported to reverse the inhibitory effect [17], although in Whitten's medium the beneficial effect of these agents is dependent on the presence of EDTA [13]. The means by which hypoxanthine acts in embryos is not understood; however, results from the reversal experiments suggest that the mechanism is different from that found in oocytes [9, 13], where maintenance of elevated cAMP levels is thought to mediate the meiosis-suppressing influence of hypoxanthine [18]. Purine bases are utilized via the salvage pathway. In mammalian cells, the X-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of guanine and hypoxanthine to their respective monophosphate nucleotides (guanosine monophosphate [GMP] and inosine monophosphate [IMP], respectively). Activity of HPRT in preimplantation mouse embryos before the 4-cell stage has been found to be low and is maternally controlled but increases rapidly after the 8-cell stage up to the blastocyst stage [19-23]. A required cosubstrate for enzyme activity is phosphoribosylpyrophosphate (PRPP), an activated form of ribose phosphate that can be derived from glucose via the pentose phosphate pathway. The hypoxanthine-induced block in mouse embryos is dependent on the presence of glucose [11, 13], but the reason for this dependence is unknown. Mouse preimplantation embryos require pyruvate for first cleavage [24], and exposure to glucose before the 4-cell stage may be detrimental to embryo development [25-27]. Although glucose alone is unable to support development until the late 4- to early 8-cell stage [25, 28], it has been reported that exposure to this hexose is critical for completion of the later stages of preimplantation development [26, 29-32]. Barbehenn et al. [33, 34] proposed that the inability of early embryos to utilize glucose resides at the phosphofructokinase step of glycolysis, although both glucose transport and hexokinase activity have been reported in mouse embryos as early as the 2-cell stage [35-38]. The pentose phosphate pathway, on the other hand, is active in embryos as early as the 2-cell stage [39], and utilization of glucose by this route could result in the synthesis of ribose-5-phosphate (ribose-5-P), the immediate precursor for PRPP. Phosphoribosylation of the purine base by PRPP is the rate-limiting step controlling incorporation of hypoxanthine into cellular material [40]. The principal goals of this study were to determine whether hypoxanthine uptake and salvage are required for the hypoxanthine-induced block to development in preimplantation mouse embryos and to ascertain the means by 2 DIENHART ET AL. which glucose, EDTA, and cAMP-elevating agents may alter embryo response to this purine base. We measured hypoxanthine and glucose uptake, assayed for ATP and PRPP, and analyzed hypoxanthine metabolism via HPLC. We also used HPRT-deficient mice (Hprt-) to confirm a role for hypoxanthine salvage in the purine-induced developmental block. MATERIALS AND METHODS Chemicals Oocyte and Embryo Collection and Culture Female mice, 20-22 days old, from the following strains were used: 57BL/6 X SJL/J (BL6SJL) F l , 129/SvJ (Hprt+), and 129/SvEv-Hprt b-m 2 (Hprt-) (Jackson Laboratory, Bar Harbor, ME). The Hprt null mutation was produced by microinjecting Hprt- embryonic stem cells into blastocysts and breeding the resultant chimeric (...truncated)