Ammonium Induces Aberrant Blastocyst Differentiation, Metabolism, pH Regulation, Gene Expression and Subsequently Alters Fetal Development in the Mouse

BIOLOGY OF REPRODUCTION 69, 1109–1117 (2003) Published online before print 28 May 2003. DOI 10.1095/biolreprod.103.018093 Ammonium Induces Aberrant Blastocyst Differentiation, Metabolism, pH Regulation, Gene Expression and Subsequently Alters Fetal Development in the Mouse Michelle Lane1 and David K. Gardner Research Department, Colorado Center for Reproductive Medicine, Englewood, Colorado 80110 The presence of ammonium in the culture medium has significant detrimental effects on the regulation of embryo physiology and genetics. Ammonium levels build up linearly over time in the culture medium when media containing amino acids are incubated at 378C. Ammonium in the culture media significantly reduces blastocyst cell number, decreases inner cell mass development, increases apoptosis, perturbs metabolism, impairs the ability of embryos to regulate intracellular pH, and alters the expression of the imprinted gene H19. In contrast, the rate of blastocyst development and blastocyst morphology appear to be normal. The transfer of blastocysts exposed to ammonium results in a significant reduction in the ability to establish a pregnancy. Furthermore, of those embryos that manage to implant, fetal growth is significantly impaired. Embryos exposed to 300 mM ammonium are retarded by 1.5 days developmentally at Day 15 of pregnancy. It is therefore essential that culture conditions for mammalian embryos are designed to minimize the buildup of ammonium to prevent abnormalities in embryo physiology, genetic regulation, pregnancy, and fetal development. assisted reproductive technology, early development, in vitro fertilization INTRODUCTION Formulations for embryo culture media have traditionally consisted of a balanced salt solution, such as Kreb Ringer Salts, supplemented with carbohydrates and a protein source, such as serum albumin [1–3]. However, development and viability of embryos cultured in such media lagged significantly behind in vivo embryo development. During the last several years, significant research has been conducted regarding the physiology and culture of mammalian embryos. Such studies have demonstrated that optimal embryo development requires gradients of carbohydrates, amino acids, and vitamins [1–3]. Clearly, amino acids are an essential component of embryo culture media and are regulators of embryo physiology [4–18]. Amino acids stimulate embryo development by acting as intracellular pH regulators [19], osmolytes [20–23], energy substrates [24], regulators of metabolism [25, 26], and chelators [27]. Development of the cleavage-stage embryo is stimulated by the presence of the amino acids alanine, asparagine, aspartate, glutamate, glycine, proline, and serine [10, 11, 14, 15]. However, after the embryo has Correspondence: Michelle Lane, e-mail: 1 Received: 10 April 2003. First decision: 2 May 2003. Accepted: 21 May 2003. Q 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org compacted, forms the blastocoel, and differentiates into the inner cell mass (ICM) and trophectoderm (TE), development is stimulated by a more complex array of amino acids that have specific effects on the two cell types [14, 15, 28–32]. Perhaps of greatest significance is that the inclusion of specific amino acids increases implantation rates of blastocysts similar to those of in vivo-developed embryos [15]. Amino acids have been shown to be key regulators of embryo development and viability, but they spontaneously break down in culture to produce ammonium [10, 12, 16]. Also, the embryo metabolizes amino acids, resulting in the additional production of ammonium in the medium [10]. Ammonium in the culture medium has been shown to be detrimental to blastocyst development. However, most significantly, when ammonium is present in the medium for embryos from an F1 hybrid mouse, significant fetal retardation and an induction of the birth defect exencephaly occur following embryo transfer [12]. The incidence of this birth defect increases linearly with the ammonium concentration. The mechanism(s) by which ammonium affects embryo development and viability is currently unknown. The aim of the present study was to determine the effects of ammonium in the culture medium on embryo differentiation, apoptosis, physiology, metabolism, and gene expression. MATERIALS AND METHODS Media Composition The medium for embryo collection was a 4-morpholinepropanesulfonic acid (MOPS)-buffered modification of medium G1.2 (MOPS-G1) (Table 1) with a pH of 7.35. Media for embryo culture were G1.2 and G2.2 supplemented with 5 mg/ml of human serum albumin (HSA) (Table 1) [33, 34]. All salts were Analar grade and purchased from BDH (Dorset, U.K.). Pyruvate, lactate, taurine, alanine, asparagine, aspartate, glycine, glutamate, proline, serine, alanyl-glutamine, and MOPS were purchased from Sigma Chemical Co. (St. Louis, MO). Amino acid and vitamin solutions were obtained from ICN (Aurora, OH). Both EDTA and HSA were obtained from Vitrolife AB (Gothenburg, Sweden). All media, media components, and disposables used for embryo culture were screened for ability to support embryo development with a 1-cell mouse embryo bioassay (zygote development to the blastocyst stage in a protein-free medium of .80% and blastocyst cell numbers of .65 on Day 5) before use [2]. Embryo Collection and Culture Embryos were collected from CF1 female mice following superovulation with 5 IU of eCG (Sigma) and 5 IU of hCG 48 h later (Pregnyl; Organon, Inc., West Orange, NJ). Immediately following the second injection, females were placed with males of the same strain to generate CF1 3 CF1 embryos. Mating was indicated by the presence of a vaginal plug the following morning. All experimental protocols were approved by the Institutional Animal Care and Use Committee. Zygotes were collected at 21 h post-hCG in MOPS-G1 and denuded by incubation with hyaluronidase (0.5 mg/ml; bovine testes, type IV; Sig- 1109 ABSTRACT 1110 LANE AND GARDNER TABLE 1. Composition of culture media (mM).a Component G1.2 G2.2 90.08 5.5 0.25 1.0 5.0 1.8 0.5 10.5 0.32 0.01 0.1 0.5 0.1 — 0.1 0.1 — 0.1 0.1 — — — — — — 0.1 0.1 — — — — — — — — — — — — 20.0 90.08 5.5 0.25 1.0 25.0 1.8 0.5 10.5 0.32 0.01 0.1 0.5 0.1 — 0.1 0.1 — 0.1 0.1 — — — — — — 0.1 0.1 — — — — — — — — — — — — — 90.08 5.5 0.25 1.0 25.0 1.8 3.15 5.87 0.1 — — 1.0 0.1 0.6 0.1 0.1 0.1 0.1 0.1 0.2 0.4 0.4 0.4 0.1 0.2 0.1 0.1 0.4 0.5 0.2 0.4 0.0042 0.0072 0.0023 0.010 0.0082 0.0049 0.0003 0.003 — MOPSG2 90.08 5.5 0.25 1.0 5.0 1.8 3.15 5.87 0.1 — — 1.0 0.1 — 0.1 0.1 — 0.1 0.1 — — — — — — 0.1 0.1 — — — — — — — — — — — — 20.0 a All media were supplemented with 0.06 g/L of penicillin and 0.005 g/ L of phenol red. MOPS-G1 and MOPS-G2 were adjusted to a pH of 7.35. Measurement of Intracellular pH Intracellular pH levels were determined by ratiometric fluorescence analysis using the fluorochrome SNARF 1-acetoxymethyl ester (SNARF-1-AM; Molecular Probes, Eug (...truncated)