REPRODUCTIONRESEARCH

Human and mouse embryonic development, metabolism and gene expression are altered by an ammonium gradient in vitro

D K Gardner, R Hamilton1, B McCallie1, W B Schoolcraft2 and M G Katz-Jaffe1 Department of Zoology, University of Melbourne, Parkville, Victoria 3101, Australia, 1National Foundation for Fertility Research, Lone Tree, Colorado 80124, USA and 2Colorado Center for Reproductive Medicine, Lone Tree, Colorado 80124, USA Correspondence should be addressed to D K Gardner, Department of Zoology, University of Melbourne, Parkville, Victoria 3010, Australia; Email: [email protected]

Abstract

Ammonium is generated in culture media by the spontaneous deamination of amino acids at 37 8C and through the metabolism of amino acids by human embryos. The appearance of ammonium is a time-dependent phenomenon and can compromise embryo physiology, development and viability. In this study, the effects of a gradient of ammonium on the development, metabolism and transcriptome of human and mouse embryos were investigated. Pronucleate oocytes were cultured in the presence of an ammonium gradient that mimicked the spontaneous deamination of Eagle’s amino acids together with 1 mM glutamine. All embryos were cultured in sequential

media G1/G2 at 5% O2,6%CO2 and 89% N2. Human embryo metabolism was assessed through a non-invasive fluorometric analysis of pyruvate consumption. Transcriptome analysis was performed on the resultant blastocysts from both species using a microarray technology. Embryo development prior to compaction was negatively affected by the presence of low levels of ammonium in both species. Human embryo metabolism was significantly inhibited after just 24 and 48 h of culture. Transcriptome analysis of blastocysts from both species revealed significantly altered gene expression profiles, both decreased and increased. Functional annotation of the altered genes revealed the following over represented biological processes: metabolism, cell growth and/or maintenance, transcription, cell communication, transport, development and transcription regulation. These data emphasize the enhanced sensitivity of the cleavage-stage embryo to its environment and highlight the requirement to renew culture media at frequent intervals in order to alleviate the in vitro induced effects of ammonium build-up in the environment surrounding the embryo. Reproduction (2013) 146 49–61

Introduction Over the past 15 years, amino acids have subsequently become integral components of embryo culture media The environment within the female reproductive tract is and their presence can be linked to the increases in the characterized by relatively high levels of specific amino success rates of human IVF observed over the past acids, such as alanine, glycine, proline, serine and decade (Bavister 1995, Gardner 2008). The means by taurine, while the developing embryo possesses carrier systems to transport amino acids and maintain a which amino acids confer benefit to the embryo appear specific intracellular pool (Schultz et al. 1981, Miller to differ according to the stage of development. Prior to & Schultz 1987, Gardner & Leese 1990, Van Winkle compaction, several amino acids including alanine, 2001, Harris et al. 2005). The beneficial effects of aspartate, glycine, glutamate, glutamine, proline, serine amino acids on embryo development have been well and taurine help to maintain homoeostasis within the documented in several mammalian species (Spindle & blastomeres by acting as buffers of pH (Edwards et al. Pedersen 1973, Bavister & McKiernan 1993, Gardner & 1998), osmolytes (Baltz 2001), antioxidants (Liu & Foote Lane 1993, Lane & Gardner 1997, Steeves & Gardner 1995) and chelators (Gardner 2008). After compaction, 1999, Devreker et al. 2001). At the appropriate the embryo utilizes a wider array of amino acids as concentrations and temporal exposure to the embryo, it differentiates (Lane & Gardner 1997, Martin & amino acids not only increase embryo development Sutherland 2001) and implants (Martin et al. 2003). and differentiation in vitro, but also increase subsequent At all stages of development, amino acids serve as both viability after transfer, leading to increases in both energy sources and regulators of carbohydrate metab- implantation and pregnancy rates (Lane & Gardner olism (Lane & Gardner 2005a). Furthermore, the 1994, Gardner & Lane 2003). utilization of amino acids by the human preimplantation

q 2013 Society for Reproduction and Fertility DOI: 10.1530/REP-12-0348 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/25/2021 04:24:50PM via free access 50 D K Gardner and others embryo has been linked to both developmental capacity pathologies induced by the addition of exogenous in culture and subsequent viability after transfer ammonium are strikingly similar to those caused by the (Houghton et al. 2002, Brison et al. 2004). spontaneous deamination of amino acids in the medium Unfortunately, amino acids in any culture medium are (Lane & Gardner 1994), indicating that this is a suitable labile at 37 8C and spontaneously break down to release model to investigate the actions of ammonium on the ammonium in a time-dependent manner (Gardner & preimplantation embryo. Typically, a concentration of Lane 1993, Heeneman et al. 1993, Virant-Klun et al. 300 mM exogenous ammonium is employed as the 2006). Of the amino acids commonly used in a culture concentration of ammonium added to a culture medium medium, glutamine is the most labile, which breaks (Lane & Gardner 2003, Zander et al. 2006), representing down to ammonium and pyrrolidone carboxylic acid the amount present after 72 h of culture (Gardner & Lane (Tritsch & Moore 1962). However, even in the absence of 1993), and has been demonstrated to significantly glutamine, there are significant levels of ammonium compromise the viability of cultured human cell lines produced by the spontaneous deamination of other (Heeneman et al. 1993). However, such studies on the amino acids (Gardner & Lane 1993). It has been mouse have been criticized for employing too high an documented that the in situ build-up of ammonium ammonium exposure during early embryo development from amino acids in embryo culture media has negative (Biggers et al. 2004, Biggers & Summers 2008). During effects on mouse embryo physiology, viability and fetal cleavage-stage development, embryos would typically development (Lane & Gardner 1994, 2003, Zander et al. be exposed to between 75 and 225 mM ammonium 2006) and a negative effect on human embryo develop- (Gardner & Lane 1993). ment (Virant-Klun et al. 2006). A concentration of Therefore, it was the aim of this study to determine ammonium as low as 20 mM can affect cell allocation the effects of an increasing ammonium gradient on the in the blastocyst (Lane & Gardner 2003). Of interest, development and metabolism of human and mouse Biggers et al. (2004) observed much lower levels of embryos during the preimplantation period, as well as on exencephaly when exposing mouse embryos to reduced gene expression at the blastocyst stage. levels of amino acids over extended culture without medium renewal, raising questions about the appro- priate concentration of amino acids in a culture medium. Materials and methods The human embryo has a considerable capacity to Mouse embryo culture affect the concentration of ammonium in its immediate vicinity, with it being able to metabolize amino acids Pronucleate oocytes were isolated from CF1xCF1 mice and subsequently release ammonium into the medium at and randomly allocated to one of the two groups: control a rate of up to 30 pmol/embryo per h for the human (Group 1) and exposure to increasing ammonium blastocyst (Gardner et al. 2001). Subsequently, four (Group 2). Group 1 (control) embryos were cultured in human embryos in a 50 ml drop of medium containing groups of ten in 20 ml of medium G1 (Vitrolife, Engle- amino acids have the capacity to increase the concentra- wood, CO, USA) for 48 h and then in medium G2 tion of ammonium by w60 mM above the baseline levels. (Vitrolife) (48 h) under 3.5 ml paraffin oil (Ovoil, Indeed, Gardner (2008) discussed the concept that Vitrolife) in 35-mm Primaria dishes (BD Bioscience, embryo culture should not be considered a static San Jose, CA, USA). The media were renewed every 24 h Z phenomenon due to the fact that the embryo is (n 180). Group 2 embryos were cultured in the same continuously changing the composition of the surround- way as the control but with increasing concentrations of Z ing medium through its use of nutrients and by the ammonium (n 190). Group 2 embryos were exposed to release of metabolic products. Consequently, this has 75, 150, 225 and 300 mM on successive days of culture significant implications for how human embryos should in order to mirror the levels of ammonium generated be cultured in a specific volume and how frequently when Eagle’s amino acids, with 1 mM glutamine, media should be renewed. The latter point is of deaminate over time (Fig. 1). All embryos were cultured significance, given the recent reports of uninterrupted in 5% O2,6%CO2, and 89% N2. Embryo development human embryo culture for 5 days (Reed et al. 2009, was scored on successive days of development, and a Sepulveda et al. 2009, Wirleitner et al. 2010). transcriptome analysis was performed on the resultant Subsequently, there have been several studies on the blastocysts on day 5. Embryos were collected over four effects of exogenous ammonium on the development of replicate experiments. the mouse embryo in order to understand the aetiology of its adverse effects (Lane & Gardner 2003, Zander et al. Human embryo culture 2006). Perturbations to embryos induced by exogenous ammonium include retarded development in culture, Pronucleate oocytes, cryopreserved and thawed using reduced mitosis, decreased inner cell mass develop- the technique of Lassalle et al. (1985) (nZ130) were ment, altered metabolism and gene expression, and randomly allocated to either Group 1 or Group 2 (as subsequently retarded fetal development. Of note, the described above) and subsequently cultured individually

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2100 Bioanalyzer and RNA 6000 Nano Assay Kit

M) 400 (Agilent Technologies, Santa Clara, CA, USA). Samples µ were then fragmented and hybridized to either the 300 Codelink Whole Genome Bioarrays for human (nZ6) or 200 those for mouse (nZ6) (Applied Microarrays, Tempe, Ammonium 100 AZ, USA) according to the manufacturer’s instructions. concentration ( Each Codelink Bioarray was scanned using the GenePix 0 Day 1Day 2Day 3Day 4Day 5 4000B Scanner (Axon Instruments, Sunnyvale, CA, USA).

Figure 1 Ammonium gradient used to culture mouse and human embryos to the blastocyst stage. The ammonium concentration Quantitative real-time PCR (Q RT-PCR) represents the gradual accumulation by the spontaneous deamination of Eagle’s amino acids with 1 mM glutamine over a 5-day Microarray results were validated using the same preimplantation culture period (Gardner & Lane 1993). samples by quantitative real-time PCR on the Roche LightCycler (Roche Diagnostics) using the LightCycler in 7 ml medium for 24 h in 5% O2,6%CO2 and 89% N2. FastStart DNA Master SYBR Green I with 2.5 mlof After 24 h, embryos were washed and moved individu- amplified RNA template. After 10-min incubation at ally to a new 7 ml drop, and the spent medium was 95 8C, the following thermal cycling profile was used for snap–frozen for metabolic analysis. On days 1 and 2, 45 cycles of amplification: denaturation at 95 8C for 10 s, medium G1 was used. From day 3 onwards, medium G2 annealing for 5 s at primer-dependent temperatures and was used. Embryo development was scored and extension at 72 8C for varying times depending on the metabolic activity was quantified on successive days of size of the amplicon. The following are the genes that development, while global transcriptome analysis was were chosen for validation: human APC, DLD and used to study the resultant blastocysts. All pronucleate ERCC3 as well as mouse Phgdh and Ruvbl1.The oocytes were donated with consent and the analysis quantification of each gene was done relative to the received the approval of IRB. transcription in every sample of an internal constant housekeeping gene, ACTB (human) or Gapdh (mouse). Validation of non-amplified cDNA in mouse blastocysts Pyruvate consumption was done with results that were the same as those for Pyruvate uptake by individual human embryos was amplified mouse blastocyst cDNA. determined through ultramicrofluorimetry, whereby the concentration of pyruvate in nanolitre samples of Statistical analysis medium was quantified in coupled enzymatic reactions (Leese & Barton 1984, Gardner 2007). Assays were Embryo development was analysed using Fisher’s exact performed in 20 nl drops on siliconized microscope test, and differences in pyruvate uptake were slides under a layer of heavy mineral oil and determined using Student’s t-test. Transcriptome micro- the pyruvate content of the medium was quantified array data were analysed using the GeneSpring through a fluorescence microscope equipped with software (Agilent Technologies) with their optimized a photometer. analysis algorithms including data normalized per chip to the 50th percentile and per gene to the mean. Unsupervised hierarchical clustering was performed RNA isolation and transcriptome analysis with Pearson’s correlation creating a condition tree Total RNA was isolated from groups of five blastocysts, grouping the samples according to a relationship in three replicates per group, using a PicoPure RNA their overall gene expression profiles. One-way Isolation Kit (Molecular Devices, Sunnyvale, CA, USA) ANOVA test revealed transcripts that were differentially with modifications. Briefly, samples were lysed and expressed across the groups using Benjamini and bound to a silica-based filter where they were treated Hochberg False Discovery correction and Student– with RNase-free DNase I (Qiagen) and washed several Newman–Keuls post hoc test, with significance at times prior to elution in 20 ml. Following isolation, two P!0.05. Annotation of differentially expressed genes rounds of linear RNA amplification incorporating a T7 was performed with EASE bioinformatics (www.ease. polymerase promoter were performed using the Messa- com) to identify the overrepresented GO biological geAmp II RNA Amplification Kit (Ambion, Carlsbad, CA, processes (www.geneontology.org)andKEGG USA) and iExpress Assay Reagent Kit (GE Healthcare, pathways (www.genome.jp/kegg). Statistical analysis Little Chalfont, Buckinghamshire, UK). RNA for quantitative real-time PCR was performed with the concentration was evaluated using the NanoDrop REST-2005q software using the pair-wise fixed reallo- spectrophotometer (Thermo Scientific, Waltham, MA, cation randomization test. Gene expression fold USA) and RNA integrity was evaluated with the Agilent differences with P!0.05 were considered significant. www.reproduction-online.org Reproduction (2013) 146 49–61

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90 twofold increase in expression. Hierarchical clustering analysis separated the control and ammonium gradient 80 samples into distinct branches, confirming differential 70 transcriptome profiles, as displayed in the microarray heat map in Fig. 5. 60 ** ** Functional annotations of the transcripts identified as 50 being differentially expressed greater than twofold across ** the groups were classified according to their Gene 40 Ontology (GO) and possible involvement in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. 30 GO terms and KEGG pathways represented biological Embryo development (%) 20 processes common across the two species, including metabolism, cell growth and/or maintenance, transcrip- 10 tion, cell communication, transport, development and 0 transcription regulation (Tables 1 and 2). ≥8-cell Blastocyst Degeneration Table 1 presents a list of genes corresponding to on day 3 on day 5 the altered biological processes in mouse blastocysts, Figure 2 Effect of ammonium on mouse embryo development. Control while Table 2 presents a list of genes corresponding to (solid bars) and ammonium gradient (open bars). Significantly different the altered biological processes in human blastocysts. ! from the control; **P 0.01. The lists of both decreased and increased transcripts were grouped as altered processes including metabolism, cell growth and/or maintenance, transcription and cell com- Results munication. Following exposure to ammonium gradients Mouse embryo development in the presence of an in both species, genes involved in transport mechanisms increasing ammonium concentration was significantly were only identified as decreased, whereas genes slower than that of the control group after 48 h, with just involved in development and regulation of transcription 54% of embryos at the eight-cell stage, compared with were identified as increased (Tables 1 and 2). 73% in the control group (P!0.01). By day 5, only 43% Validation was performed for both the mouse and of the embryos exposed to ammonium formed blasto- human blastocyst microarray data. Differential cysts, compared with 60% in the control group (P!0.01). expression was confirmed by Q RT-PCR for the randomly Furthermore, embryo degeneration was higher in the selected mouse genes: Phgdh and Ruvbl1. For each ammonium group (54 vs 34%; P!0.01) (Fig. 2). sample, the quantification of both these target genes was Human embryos showed retarded development on done relative to the transcription of a housekeeping day 3 when cultured in the presence of an increasing gene, Gapdh (P!0.05) (Fig. 6). For the human blastocyst ammonium concentration compared with the control microarray data, differential expression was confirmed group, with fewer embryos having R7 cells (44 vs 70% ! respectively; P 0.05). In the presence of an ammonium 80 gradient, 42% of the embryos reached the blastocyst stage compared with 53% in the control group (Fig. 3). 70 The analysis of pyruvate uptake, as a measure of oxidative potential, revealed that the metabolism of 60 embryos during the cleavage stages was significantly 50 affected after 24 and 48 h of culture (P!0.05). Pyruvate * uptake at subsequent stages up to the blastocyst 40 stage was not affected by ammonium from day 3 onwards (Fig. 4). 30 In the mouse, transcriptome analysis of the resulting blastocysts revealed alterations in gene expression, with (%) Embryo development 20 66 genes displaying greater than twofold decrease in expression and 85 genes showing greater than twofold 10 increase in expression following ammonium gradient 0 exposure compared with the control blastocysts. Day 3 Total Likewise, in the human, the blastocyst transcriptome ≥ 7-cell blastocyst was altered following ammonium gradient exposure, Figure 3 Effect of ammonium on human embryo development. Control with 205 genes displaying greater than twofold decrease (solid bars) and ammonium gradient (open bars); significantly different in expression and 185 genes showing greater than from the control; *P!0.05.

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50 In this study, the presence of 75–150 mM ammonium was sufficient to impair the development of the cleavage- 40 stage embryo of both species. The strain of mouse, CF1xCF1, was chosen over an F1 hybrid strain as it exhibits greater sensitivity to its environment in vitro.On 30 day 5, significantly fewer mouse embryos had formed blastocysts. A previous analysis of this strain of mice over 20 a range of fixed ammonium concentrations also revealed a trend to fewer blastocysts developing and a significant * decrease in cell number as ammonium concentration 10 * increased (Lane & Gardner 2003). In support of that

Pyruvate uptake (pmol/embryo per h) study, a significant increase in mouse embryo degener- 0 24 48 72 96 117 ation and a decrease in the rate of human embryo development past the seven-cell stage on day 3 in the Hour post thaw presence of an increasing ammonium concentration Figure 4 Effect of an increasing ammonium gradient on human embryo were recorded in this study, suggesting that ammonium metabolism. Control (solid bars) and ammonium gradient (open bars); is detrimental to embryo development and the effects are significantly different from the control; nZ20 embryos in each not species specific. treatment and at each developmental stage. Data are meanGS.E.M.; *P!0.05. The analysis of pyruvate uptake by the human embryo revealed a significant decrease in embryos cultured in by Q RT-PCR for the following randomly selected human the presence of ammonium at 24 and 48 h, reflecting the genes: APC, DLD and ERCC3. Similarly, the quantifi- increased sensitivity of the cleavage-stage embryo to cation of each gene was done relative to the transcription in vitro induced stressors (Lane & Gardner 2005b). From of a housekeeping gene, ACTB (P!0.05) (Fig. 7). 72 h onwards, there were marginal, but not significant differences in pyruvate uptake between the two groups. Discussion This pattern of nutrient utilization can perhaps be explained by two factors. First, after 72 h, the post- These data confirm that the accumulation of ammonium compaction human embryo becomes less reliant on in culture media affects the development of the pyruvate and switches to glucose as its primary energy preimplantation mouse (Gardner & Lane 1993, Lane & Gardner 1994) and human (Virant-Klun et al. 2006) embryos and that even at low concentrations, 75 and 150 mM, ammonium alters human embryo metabolism at the cleavage stages. Furthermore, chronic exposure to an increasing concentration of ammonium results in altered gene expression in the resultant blastocysts of both species. This study provides further evidence for the need to avoid the build-up of ammonium in the culture environment during early embryonic development, reinforcing the imperative to renew media used for human embryo culture at least every 48 h, irrespective of whether one or two formulations of media are used for culture to the blastocyst stage. The lability of amino acids at 37 8C, in particular, glutamine, and the subsequent accumulation of toxic levels of ammonium in tissue culture have been well characterized for a number of cell types, where growth in vitro had been compromised (Newland et al. 1990, Schneider et al. 1996). Indeed, viable somatic cell lines can be significantly compromised in the presence of just 300 mM ammonium (Heeneman et al. 1993), a concen- tration that has been documented to adversely affect mouse embryo development and metabolism (Lane & Figure 5 Heat map representation with hierarchical clustering of altered Gardner 2003). In all tissue culture media containing genes in human blastocysts following ammonium exposure and separation of control (green lines) and three ammonium (red lines) amino acids, ammonium can be detected and its rate of samples into distinct branches. Gene expression is related to colour, appearance is both time- and temperature-dependent with red representing the highest levels of gene up-regulation and blue (Heeneman et al. 1993). down-regulation. www.reproduction-online.org Reproduction (2013) 146 49–61

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Table 1 Gene list including the most common biological processes affected by an increasing gradient of ammonium during mouse embryo culture. Biological process Common name GenBank Description Decreased expression in ammonium Metabolism C330022B21Rik AK021218 RIKEN cDNA C330022B21 gene Alppl2 (Akp5) NM_007433 Alkaline phosphatase, placental-like 2 Elf3 NM_007921 E74-like factor 3 Fkbp2 NM_008020 FK506-binding protein 2 Tbx15 NM_009323 T-box 15 Cryab NM_009964 Crystallin, aB Got1 NM_010324 Glutamate oxaloacetate transaminase 1, soluble Lamp2 NM_010685 Lysosomal-associated membrane protein 2 Hprt NM_013556 Hypoxanthine guanine phosphoribosyl transferase Cdk2ap1 NM_013812 Cyclin-dependent kinase 2 (CDK2)-associated protein 1 Phgdh NM_016966 3-Phosphoglycerate dehydrogenase Ostf1 NM_017375 Osteoclast stimulating factor 1 Sertad1 NM_018820 SERTA domain containing 1 Ruvbl1 NM_019685 RuvB-like protein 1 Usp14 NM_021522 Ubiquitin specific peptidase 14 Pex13 NM_023651 Peroxisomal biogenesis factor 13 Mrpl15 NM_025300 Mitochondrial ribosomal protein L15 Ssr2 NM_025448 Signal sequence receptor, b Mrpl51 NM_025595 Mitochondrial ribosomal protein L51 Mad2l1bp NM_025649 MAD2L1 binding protein Vdrip NM_026119 Mediator of RNA polymerase II transcription, subunit 4 homolog (yeast) Ppa1 (Pyp) NM_026438 Pyrophosphatase (inorganic) 1 2510010F15Rik NM_026454 Ubiquitin-conjugating enzyme E2F Txndc17 (Txnl5) NM_026559 Thioredoxin domain containing 17 Gtf2e2 NM_026584 General transcription factor II E, polypeptide 2 (b-subunit) Denr NM_026603 Density-regulated protein Cpsf5 NM_026623 Nudix (nucleoside diphosphate linked moiety X)-type motif 21 1110012L19Rik NM_026787 RIKEN cDNA 1110012L19 gene Mrpl32 NM_029271 Mitochondrial ribosomal protein L32 Cpn1 NM_030703 Carboxypeptidase N, polypeptide 1 Mrps21 NM_078479 Mitochondrial ribosomal protein S21 Gm9735 NM_183121 Predicted gene 9735 Cell growth and/or Ssr4 NM_009279 Signal sequence receptor, d maintenance Lgals9 NM_010708 Lectin, galactose binding, soluble 9 Cdk2ap1 NM_013812 Cyclin-dependent kinase 2 (CDK2)-associated protein 1 Plp2 NM_019755 Proteolipid protein 2 Arl6ip5 NM_022992 ADP-ribosylation factor-like 6 interacting protein 5 Pex13 NM_023651 Peroxisomal biogenesis factor 13 Crip2 NM_024223 Cysteine-rich protein 2 Mrpl15 NM_025300 Mitochondrial ribosomal protein L15 Cks2 NM_025415 CDC28 protein kinase regulatory subunit 2 Mad2l1bp NM_025649 MAD2L1 binding protein Denr NM_026603 Density-regulated protein Transcription C330022B21Rik AK021218 RIKEN cDNA C330022B21 gene Elf3 NM_007921 E74-like factor 3 Tbx15 NM_009323 T-box 15 Ostf1 NM_017375 Osteoclast stimulating factor 1 Sertad1 NM_018820 SERTA domain containing 1 Gtf2e2 NM_026584 General transcription factor II E, polypeptide 2 (b-subunit) Gm9735 NM_183121 Predicted gene 9735 Cell communication Rohn NM_009708 Rho family GTPase 2 Lgals9 NM_010708 Lectin, galactose binding, soluble 9 Hax1 (Hs1bp1) NM_011826 HCLS1 associated X-1 Ostf1 NM_017375 Osteoclast stimulating factor 1 Transport Alppl2 NM_007433 Alkaline phosphatase, placental-like 2 Ssr4 NM_009279 Signal sequence receptor, d Lgals9 NM_010708 Lectin, galactose binding, soluble 9 Plp2 NM_019755 Proteolipid protein 2 Arl6ip5 NM_022992 ADP-ribosylation factor-like 6 interacting protein 5 Pex13 NM_023651 Peroxisomal biogenesis factor 13 Ssr2 NM_025448 Signal sequence receptor, b Increased expression in ammonium Metabolism Ptgis NM_008968 Prostaglandin I2 (prostacyclin) synthase Ptprn NM_008985 Protein tyrosine phosphatase, receptor type, N St6galnac3 (Siat7c) NM_011372 ST6 ((a-N-acetylneuraminyl 2,3-b-galactosyl-1,3)-N-acetyl galactosaminide-a-2,6-sialyltransferase) C Vezf1 NM_016686 Vascular endothelial zinc finger 1 Rce1 NM_023131 RCE1 homolog, prenyl protein peptidase (S. cerevisiae)

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Table 1 Continued.

Biological process Common name GenBank Description Pcyt2 NM_024229 Phosphate cytidylyltransferase 2, ethanolamine 1110030J09Rik NM_025397 Mediator of RNA polymerase II transcription, subunit 11 homolog (S. cerevisiae) Mina NM_025910 Myc-induced nuclear antigen Dnase1l1 NM_027109 Deoxyribonuclease 1-like 1 Ndufs7 NM_029272 NADH dehydrogenase (ubiquinone) Fe–S protein 7 Zmym2 (Zfp198) NM_029498 Zinc finger, MYM-type 2 Rspo1 (Rspondin) NM_138683 R-spondin homolog (Xenopus laevis) Hnrpll NM_144802 Heterogeneous nuclear ribonucleoprotein L-like (2810036L13Rik) Cell growth and/or Cap1 NM_007598 CAP, adenylate cyclase-associated protein 1 (yeast) maintenance Bmp15 NM_009757 Bone morphogenetic protein 15 Nuf2 (Cdca1) NM_023284 NUF2, NDC80 kinetochore complex component, homolog (S. cerevisiae) Mina NM_025910 Myc-induced nuclear antigen Slc25a46 NM_026165 Solute carrier family 25, member 46 (1200007B05Rik) Cell communication Drd5 AK032054 Dopamine receptor D5 Ptprn NM_008985 Protein tyrosine phosphatase, receptor type, N Development Mbnl3 AK040522 Muscleblind-like 3 (Drosophila) Sca1 NM_009124 Ataxin 1 Dpysl5 NM_023047 Dihydropyrimidinase-like 5 Regulation of Vezf1 NM_016686 Vascular endothelial zinc finger 1 transcription/ Med11 NM_025397 Mediator of RNA polymerase II transcription, subunit 11 homolog (S. cerevisiae) transcription Zmym2 NM_029498 Zinc finger, MYM-type 2 source (Hardy et al. 1989); therefore, pyruvate would be be changed when both statistical significance and fold less indicative of metabolic impacts. Second, the change were accounted for. pre-compaction embryo is far more susceptible to Of general interest was the observation that more environmental stress, particularly, that affecting REDOX genes were affected (both increased and decreased) processes (Gardner & Lane 2005). Indeed, Zander et al. in the human blastocyst than in the mouse blastocyst. (2006) have previously shown that the cleavage stage of One possible explanation for this could be the fact that mouse embryo is the period of development that is most the human embryos were created through IVF and were sensitive to ammonium. The decrease in pyruvate uptake subsequently frozen and thawed, whereas the mouse by the human embryo after 24 and 48 h of culture is embryos were obtained from naturally mated mice. consistent with the documented decrease in pyruvate Of the genes affected in the human blastocyst, several oxidation measured in two-cell mouse embryos in the were linked to carbohydrate and amino acid meta- presence of ammonium (Lane & Gardner 2005b). The bolism, such as pyruvate dehydrogenase phosphatase observed negative impact of ammonium on human isoenzyme 2, dihydrolipoamide dehydrogenase, embryo metabolism is also likely in view of its 6-phosphofructokinase/fructose-2,6-bisphosphatase and documented ability to dramatically alter amino acid glutamine–phenylpyruvate aminotransferase, which utilization in the cow and mouse models (Orsi & Leese were all decreased in expression. It is, therefore, 2004, Wale & Gardner 2013). tempting to speculate that such a decrease in gene In both mouse and human, culture of the embryo expression is a causative factor for the altered metab- in the presence of an increasing concentration of olism in the blastocyst. Both pyruvate dehydrogenase ammonium resulted in the aberrant expression of phosphatase isoenzyme 2 and 6-phosphofructokinase/ numerous genes. There were 34 967 unique transcripts fructose-2,6-bisphosphatase are key enzymes regulating on the mouse array and 45 674 on the human array. It is pyruvate metabolism and glycolysis (Roche et al. 2001, estimated that there are w20 000 protein-coding genes Rider et al. 2004) and their potential down-regulation in the human and mouse genomes. The higher number of could conceivably impact metabolic flux. However, the transcripts on the arrays represents at least one or more enzyme dihydrolipoamide dehydrogenase is present transcripts per protein-coding gene reflecting known throughout the preimplantation period from maternal splicing variants. In the human and mouse blastocysts sources synthesized during oocyte maturation, and examined in this study, only 50% of the transcripts were consequently changes in gene expression at the expressed (i.e. w22 000 in the human blastocysts). In blastocyst stage may well only have effects after total, 151 displayed significant altered expression in the implantation (Johnson et al. 2009). Furthermore, in the mouse blastocyst and 390 in the human blastocyst presence of ammonium, there was decreased expression (greater than twofold; P!0.05). This represents a finding of the glucose transporter SLC2A1 (GLUT1). This similar to those of other studies such as that of Giritharan transporter was first shown to be present in the human et al. (2007), who observed only a minority of genes to blastocyst by Dan-Goor et al. (1997), and glucose uptake www.reproduction-online.org Reproduction (2013) 146 49–61

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Table 2 Gene list including the most common biological processes affected by an increasing gradient of ammonium during human embryo culture. Biological process Common name GenBank Description Decreased expression in ammonium Metabolism PDP2 AB037769 Pyruvate dehydrogenase phosphatase catalytic subunit 2 FLJ38705 AK024438 ZFP41 zinc finger protein ZNF419 AK026886 Zinc finger protein 419 LYZ BC004147 Lysozyme DLD NM_000108 Dihydrolipoamide dehydrogenase ERCC3 NM_000122 Excision repair cross-complementing rodent repair deficiency, complementation group 3 LEP NM_000230 Leptin UROS NM_000375 Uroporphyrinogen III synthase STK11 NM_000455 Serine/threonine kinase 11 CSTF2 NM_001325 Cleavage stimulation factor, 3’ pre-RNA, subunit 2, 64 kDa POLR3D NM_001722 Polymerase (RNA) III (DNA-directed) polypeptide D, 44 kDa EEF1A2 NM_001958 Eukaryotic translation elongation factor 1 a2 FNTA NM_002027 Farnesyltransferase, CAAX box, a FUT4 NM_002033 Fucosyltransferase 4 (a-(1,3) fucosyltransferase, myeloid-specific) HOXB5 NM_002147 Homeo box B5 INPP1 NM_002194 Inositol polyphosphate-1-phosphatase LIG3 NM_002311 Ligase III, DNA, ATP-dependent PI4KB NM_002651 Phosphatidylinositol 4-kinase, catalytic, b ST3GAL1 (SIAT4A) NM_003033 ST3 b-galactoside-a-2,3-sialyltransferase 1 TCEB3 NM_003198 Transcription elongation factor B (SIII), polypeptide 3 (110 kDa, elongin A) USP4 NM_003363 Ubiquitin-specific protease 4 (proto-oncogene) CCBL1 NM_004059 Cysteine conjugate-beta lyase, cytoplasmic PFKFB4 NM_004567 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 SIAH2 NM_005067 Siah E3 ubiquitin protein ligase 2 HDLBP NM_005336 High-density lipoprotein-binding protein RBPJ (RBPSUH) NM_005349 Recombination signal binding protein for immunoglobulin kappa J region CTDSPL NM_005808 Carboxy-terminal domain (CTD, RNA polymerase II, polypeptide A) small phosphatase-like PPP1R2 NM_006241 Protein phosphatase 1, regulatory (inhibitor) subunit 2 RAD54B NM_006550 RAD54 homologue B (S. cerevisiae) APOBEC2 NM_006789 Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 2 POLI NM_007195 Polymerase (DNA directed) i ESPL1 NM_012291 Extra spindle pole bodies homolog 1 (S. cerevisiae) ACIN1 NM_014977 Apoptotic chromatin condensation inducer 1 ANO10 (FLJ10375) NM_018075 Anoctamin 10 CYP3A43 NM_022820 Cytochrome P450, family 3, subfamily A, polypeptide 43 GRHL2 (TFCP2L3) NM_024915 Grainyhead-like 2 (Drosophila) TNKS1BP1 NM_033396 Tankyrase 1-binding protein 1, 182 kDa Cell growth and/or PEX1 NM_000466 Peroxisome biogenesis factor 1 maintenance POLR3D NM_001722 Polymerase (RNA) III (DNA-directed) polypeptide D, 44 kDa CCND3 NM_001760 Cyclin D3 LIG3 NM_002311 Ligase III, DNA, ATP-dependent PI4KB NM_002651 Phosphatidylinositol 4-kinase, catalytic, b SH3GL1 NM_003025 SH3-domain GRB2-like 1 USP4 NM_003363 Ubiquitin-specific peptidase 4 (proto-oncogene) SLC6A5 NM_004211 Solute carrier family 6 (neurotransmitter transporter, glycine), member 5 HDLBP NM_005336 High-density lipoprotein-binding protein SLC2A1 NM_006516 Solute carrier family 2 (facilitated glucose transporter), member 1 SLC20A2 NM_006749 Solute carrier family 20 (phosphate transporter), member 2 APOBEC2 NM_006789 Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 2 ESPL1 NM_012291 Extra spindle poles bodies homolog 1 (S. cerevisiae) ACIN1 NM_014977 Apoptotic chromatin condensation 1 COG4 NM_015386 Component of oligomeric golgi complex 4 SLC50A1 NM_018845 Solute carrier family 50 (sugar transporter), member 1 LEFTY1 (LEFTB) NM_020997 Left–right determination, factor B SLC30A1 NM_021194 Solute carrier family 30 (zinc transporter), member 1 TNKS1BP1 NM_033396 Tankyrase 1-binding protein 1, 182 kDa Transcription ZFP41 (FLJ38705) AK024438 ZFP41 zinc finger protein ZNF419 AK026886 Zinc finger protein 419 ERCC3 NM_000122 Excision repair cross-complementing rodent repair deficiency, complementation group 3 POLR3D NM_001722 Polymerase (RNA) III (DNA-directed) polypeptide D, 44 kDa HOXB5 NM_002147 Homeobox B5 TCEB3 NM_003198 Transcription elongation factor B (SIII), polypeptide 3 (110 kDa, elongin A) RBPJ NM_005349 Recombination signal binding protein for immunoglobulin kappa J region CTDSPL NM_005808 Carboxy-terminal domain (CTD, RNA polymerase II, polypeptide A) small phosphatase-like

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Table 2 Continued.

Biological process Common name GenBank Description SMAD2 (MADH2) NM_005901 SMAD family member 2 RAD54B NM_006550 RAD54 homolog B (S. cerevisiae) ESPL1 NM_012291 Extra spindle pole bodies homolog (S. cerevisiae) ACIN1 NM_014977 Apoptotic chromatin condensation inducer 1 GRHL2 NM_024915 Grainyhead-like 2 (Drosophila) Cell communication EFNB3 (EFL6); EPLG8; AK027329 Ephrin-B3 LERK8 SSTR1 BC035618 Somatostatin receptor 1 APC NM_000038 Adenomatosis polyposis coli KAL1 NM_000216 Kallmann syndrome 1 sequence LEP NM_000230 Leptin FNTA NM_002027 Farnesyltransferase, CAAX box, a INPP1 NM_002194 Inositol polyphosphate-1-phosphatase ITGA5 NM_002205 Integrin, a5 (fibronectin receptor, a-polypeptide) PI4KB NM_002651 Phosphatidylinositol 4-kinase, catalytic, b SH3GL1 NM_003025 SH3-domain GRB2-like 1 SIAH2 NM_005067 Siah E3 ubiquitin protein ligase 2 SOS2 NM_006939 Son of sevenless homologue 2 (Drosophila) TOCA1 NM_017737 Formin binding protein 1-like LEFTY1 NM_020997 Left–right determination, factor 1 DEPTOR (DEPDC6) NM_022783 DEP domain containing MTOR-interacting protein PIK3R1 NM_181504 Phosphoinositide-3-kinase, regulatory subunit a VAC14 (FLJ10305) U25801 Vac14 homolog (S. cerevisiae) Transport PI4KB NM_002651 Phosphatidylinositol 4-kinase, catalytic, b SLC6A5 NM_004211 Solute carrier family 6 (neurotransmitter transporter, glycine), member 5 HDLBP NM_005336 High-density lipoprotein-binding protein SLC2A1 NM_006516 Solute carrier family 2 (facilitated glucose transporter), member 1 SLC20A2 NM_006749 Solute carrier family 20 (phosphate transporter), member 2 APOBEC2 NM_006789 Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 2 COG4 NM_015386 Component of oligomeric golgi complex 4 SLC50A1 NM_018845 Solute carrier family 50 (sugar transporter), member 1 SLC30A1 NM_021194 Solute carrier family 30 (zinc transporter), member 1 Increased expression in ammonium Metabolism ERV3-2 AB040899 Endogenous retrovirus group 3, member 2 HES2 AK091122 Hairy and enhancer of split 2 (Drosophila) ARSE NM_000047 Arylsulphatase E (chondrodysplasia punctata 1) MSX1 NM_002448 Msh homeobox 1 HS3ST1 NM_005114 Heparan sulphate (glucosamine) 3-O-sulphotransferase 1 CSF1R NM_005211 Colony-stimulating factor 1 receptor HAS3 NM_005329 Hyaluronan synthase 3 NNMT NM_006169 Nicotinamide N-methyltransferase ZNF22 NM_006963 Zinc finger protein 22 ELL2 NM_012081 Elongation factor, RNA polymerase II, 2 KIAA0141 NM_014773 KIAA0141 KIAA0663 NM_014827 Zinc finger CCCH-type containing 11A KLF3 NM_016531 Kruppel-like factor 3 (basic) N4BP2 NM_018177 NEDD4-binding protein 2 MNS1 NM_018365 Meiosis-specific nuclear structural DNAJA4 NM_018602 DnaJ (Hsp40) homolog, subfamily A, member 4 LARS NM_020117 Leucyl-tRNA synthetase S100A14 NM_020672 S100 calcium-binding protein A14 TNS3 (TENS1) NM_022748 Tensin 3 DHDDS NM_024887 Dehydrodolichyl diphosphate synthase MARK4 NM_031417 MAP/microtubule affinity-regulating kinase 4 TTC25 NM_031421 Tetratricopeptide repeat domain 25 (DKFZP434H0115) POLR3GL (MGC3200) NM_032305 Polymerase (RNA) III (DNA directed) polypeptide G (32kD)-like ZC3H8 NM_032494 Zinc finger CCCH-type containing 8 PTPDC1 (PTP9Q22) NM_152422 Protein tyrosine phosphatase domain containing 1 Cell growth and/or HAP1 NM_003949 Huntingtin-associated protein 1 (neuroan 1) maintenance KIF23 NM_004856 Kinesin family member 23 CSF1R NM_005211 Colony-stimulating factor 1 receptor S1PR3 (EDG3) NM_005226 Sphingosine-1-phosphate receptor 3 LMO2 NM_005574 LIM domain only 2 (rhombotin-like 1) RBL2 NM_005611 Retinoblastoma-like 2 (p130) RAMP1 NM_005855 Receptor (G protein-coupled) activity-modifying protein 1 RAMP3 NM_005856 Receptor (G protein-coupled) activity-modifying protein 3 KDELR3 NM_006855 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 3 INTS6 (DDX26) NM_012141 Integrator complex subunit 6 www.reproduction-online.org Reproduction (2013) 146 49–61

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Table 2 Continued.

Biological process Common name GenBank Description TMEFF2 NM_016192 Transmembrane protein with EGF-like and two follistatin-like domains 2 FLJ10188 NM_018017 Chromosome 10 open reading frame 118 FLJ10420 NM_018090 NECAP endocytosis associated 2 N4BP2 NM_018177 NEDD4-binding protein 2 S100A14 NM_020672 S100 calcium-binding protein A14 FLJ22557 NM_024713 Katanin p80 subunit B-like 1 Cell communication GRM2 NM_000839 Glutamate receptor, metabotropic 2 CDH11 NM_001797 Cadherin 11, type 2, OB-cadherin (osteoblast) RGS12 NM_002926 Regulator of G-protein signalling 12 HAP1 NM_003949 Huntingtin-associated protein 1 S1PR3 NM_005226 Sphingosine-1-phosphate receptor 3 RAMP1 NM_005855 Receptor (G protein-coupled) activity-modifying protein 1 RAMP3 NM_005856 Receptor (G protein-coupled) activity-modifying protein 3 CLEC1 NM_016511 C-type lectin domain family 1, member A TREM1 NM_018643 Triggering receptor expressed on myeloid cells 1 TNS3 NM_022748 Tensin 3 ARRDC4 NM_183376 Arrestin domain containing 4 Development ARSE NM_000047 Arylsulphatase E (chondrodysplasia punctata 1) CDH11 NM_001797 Cadherin 11, type 2, OB-cadherin (osteoblast) MSX1 NM_002448 Msh homeobox 1 HAP1 NM_003949 Huntingtin-associated protein 1 S1PR3 NM_005226 Sphingosine-1-phosphate receptor 3 LMO2 NM_005574 LIM domain only 2 (rhombotin-like 1) KLF3 NM_016531 Kruppel-like factor 3 (basic) MTPAP NM_018109 Mitochondrial poly(A) polymerase SPECC1 NM_152904 Sperm antigen with calponin homology and coiled-coil domains 1 Regulation of HES2 AK091122 Hairy and enhancer of split 2 (Drosophila) transcription/ MSX1 NM_002448 Msh homeobox 1 transcription RBL2 NM_005611 Retinoblastoma-like 2 (p130) ZNF22 NM_006963 Zinc finger protein 22 ELL2 NM_012081 Elongation factor, RNA polymerase II, 2 KLF3 NM_016531 Kruppel-like factor 3 (basic) POLR3GL NM_032305 Polymerase (RNA) III (DNA directed) polypeptide G (32kD)-like ZC3H8 NM_032494 Zinc finger CCCH-type containing 8 ZSCAN4 (ZNF494) NM_152677 Zinc finger and SCAN domain containing 4 has been shown to be directly related to the viability of expression of Got1 observed in the presence of the human embryo at this stage of development (Gardner ammonium could, therefore, be associated with com- et al. 2011). Consequently, a decrease in the levels of promised metabolic activity and regulation and may SLC2A1 could realistically impact blastocyst metabolism well be a causative factor for the reduced viability of and viability. Of note, the expression of the enzyme glutamate 1.50 oxaloacetate transaminase 1 (GOT1), also know as aspartate aminotransferase (cytoplasmic), was decreased 1.00 in mouse blastocysts that developed in the presence of an ammonium gradient. The activity of this enzyme actually increases with development (Moore & Brinster 0.50 1973) and its substrate, aspartate, is the highest utilized amino acid by the mouse blastocyst (Lamb & Leese 0.00 1994, Wale & Gardner 2012). Furthermore, the GOT1 enzyme plays a key role in the malate–aspartate shuttle, –0.50 which transfers NADH from the cytoplasm into the * mitochondria via a series of reactions catalysed in the –1.00 * cytoplasm by malate dehydrogenase 1 (MDH1) and

GOT1 and in the mitochondria by mitochondrial MDH2 Gene expression levels relative to the control –1.50 and GOT2. The activity of this shuttle is essential for the Gapdh Phgdh Ruvbl1 regulation of metabolism during the preimplantation Figure 6 Validation of mouse blastocyst microarray data by Q RT-PCR period (Lane & Gardner 2005a), and if its activity is for the Phgdh and Ruvbl1 genes. Quantification was done relative to compromised in the blastocyst, then subsequent implan- the transcription of an internal constant housekeeping gene, Gapdh tation and fetal development are significantly reduced (*P!0.05), in every sample. Control (solid bars) and ammonium (Mitchell et al.2009). The alterations in the gene gradient (open bars).

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1.50 In this study, by pooling samples, it was possible to look for a group effect from ammonium concentration 1.00 instead of an individual biology. However, it is possible 0.50 that individual embryos did differ in their response to ammonium. Furthermore, gene expression is not always 0.00 a predictor (w50%) of protein abundance (Gygi et al. 1999). This is due to several mechanisms, including –0.50 * targeted mRNA and protein degradation that allows a * –1.00 cell to respond to external stimuli. In conclusion, these data reveal the sensitivity of the –1.50 human embryo to ammonium in the culture environ- ment, which is manifested by reduced development and –2.00 ** impaired oxidative capacity at early cleavage stages, Gene expression levels relative to the control –2.50 culminating in altered gene expression profiles. The ACTB APC DLD ERCC3 alterations in gene expression are consistent with Figure 7 Validation of human blastocyst microarray data by Q RT-PCR ammonium having effects on metabolism and neural for the following genes: APC, DLD and ERCC3. Quantification was development, which have both been reported to be done relative to the transcription of an internal constant housekeeping directly affected by the exposure of embryos to ! ! gene, ACTB (*P 0.05; **P 0.01), in every sample. Control (solid ammonium in culture using animal models. Conse- bars) and ammonium gradient (open bars). quently, these data have significant implications for how human embryos should be cultured in terms of specific mouse embryos exposed to ammonium (Lane & Gardner volume and frequency of medium renewal. Lane & 1994, Sinawat et al. 2003). Gardner (1995) developed an enzymatic method for the Across the two species studied, both revealed a removal of ammonium from embryo culture media, decrease in the expression of ubiquitin-specific pro- through its transamination to glutamate (Lane & Gardner teases (USPs). These proteases are deubiquitinating 1995). However, even though this approach was suitable enzymes associated with the ubiquitin–proteasome as an experimental procedure, it did not translate into system. The USPs have a significant role to play in the clinical use. Although the most labile of the amino acids, maintenance of cellular homoeostasis and are especially glutamine, can be substituted for more stable dipeptide important for neuronal function (van Tijn et al. 2008). In forms, such as alanyl- and glycly-glutamine, the the case of the human and mouse embryos, those remainder of the amino acids in a culture medium, affected were USP4 and USP14 respectively. Among present at the levels of standard tissue culture media, i.e. other pathways, USP4 is involved in the regulation of the Eagle’ s and Ham’s, still break down to release activity of the signalling pathways mediated by Wnt considerable amounts of free ammonium (Gardner & (Zhao et al. 2009) and p53 (Zhang et al. 2011), both of Lane 1993). Furthermore, the human embryo has the which have been shown to be involved in embryo capacity to generate considerable amounts of development (Ganeshan et al. 2010) and blastocyst ammonium through amino acid transamination activation (Xie et al. 2008). Furthermore, USP4 regulates (Gardner et al. 2001), sufficient to change the concen- the ubiquitination status of phosphoinositide-dependent tration present in a culture drop (Gardner 2008). A kinase 1, which is part of the signalling cascade for many practical means to alleviate concerns over ammonium growth factors (Uras et al. 2012). USP14 deficiency in toxicity would be to renew media every 48 h. mice has been linked to neuronal dysfunction, and a reduction in levels of the protein following gene knockout in transgenic mice subsequently leads to male sterility (Crimmins et al. 2009). Given that one of Declaration of interest the gross manifestations of ammonium toxicity after The authors declare that there is no conflict of interest that transfer in the mouse is the development of exencephaly could be perceived as prejudicing the impartiality of the (Lane & Gardner 1994, Sinawat et al. 2003), the altered research reported. gene expression profile reported herein may indicate a possible aetiology of this neural tube defect. In the human blastocyst, increased expression of nicotinamide N-methyltransferase (NNMT), which catalyses the Funding N-methylation of nicotinamide and structurally related D K Gardner and W B Schoolcraft received funding from pyridines, was also observed. An increase in NNMT Vitrolife A B for the development of culture media. This study activity has also been associated with increased was supported by the Colorado Centre for Reproductive neuronal cell death in a mouse model (Mori et al. 2012). Medicine. www.reproduction-online.org Reproduction (2013) 146 49–61

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