Nuclear Reprogramming: Kinetics of Cell Cycle and Metabolic Progression as Determinants of Success

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development

Time-lapse cinematography of ICSI and NT mouse embryos.

<p>A) Until 48 hours post activation (hpa), bright-field images were captured every 20 minutes. From 48 until 96 hpa, confocal optical sections of <i>H2b-GFP</i> expressing embryos were captured every 20 minutes. Time-lapse movies were evaluated to obtain the timing of cleavages. B) Cell division aberrancies such as failing cytokinesis (top panel; filled arrows) or cell fusions (bottom panel; empty arrows) were detected in both NT and ICSI embryos. Dotted lines indicate cell membranes. Scale bar, 20 µm.</p

Cleavage pace predicts blastocyst formation of cloned embryos.

<p>Embryos were scored <i>fast</i> or <i>slow</i> according to the time spent until the three-cell stage was reached. Then blastocyst formation was recorded, and ESCs were derived (data pooled from 4 experiments), or fetal rates were determined at E10.5 after transfer in utero 12 hours after scoring (data pooled from 2 experiments). The <i>p</i>-value of Fisher's exact test shows that the difference in blastocyst formation of <i>fast</i> and <i>slow</i> is significant, difference in ESC formation efficiency is of borderline significance, and difference in fetal formation is not significant. Data were pooled from five independent NT experiments. Development data from fertilized control embryos (ICSI) are shown in bottom row. Note that fertilized embryos were not sorted into fast and slow, and therefore frequencies relate to the two-cell stage. Derived ESCs were pluripotent regardless of their origin (fast, slow) as demonstrated by in vitro differentiation into derivatives of the three germ layers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035322#pone.0035322.s013" target="_blank">figure S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035322#pone.0035322.s006" target="_blank">movies S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035322#pone.0035322.s007" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035322#pone.0035322.s008" target="_blank">S8</a>) and by teratoma formation (data not shown).</p

Scatter plot of gene expression levels.

<p>A) NT <i>fast</i> and NT <i>slow</i> largely overlap (5 genes differently expressed, red/blue dots), B) ICSI <i>fast</i> and ICSI <i>slow</i> also largely overlap (34 genes differently expressed, red/blue dots), C) NT and ICSI show great differences in gene expression (218 genes differently expressed, red/blue dots). D) Fold-change of gene expression in ICSI <i>fast</i> versus ICSI <i>slow</i> and NT <i>fast</i> versus NT <i>slow</i>. Red dots, oocyte/1-cell-specific (<i>maternal</i>) transcripts; blue dots, 4-cell/blastocyst-specific transcripts (<i>embryonic</i>); black dots, transcripts not different between maternal and embryonic stages; grey dots, transcripts not represented in downloaded data sets. Embryonic transcripts are over-represented in <i>fast</i> ICSI embryos.</p

Accuracy of predicting developmental success from cleavage timings.

<p>A,B) Duration of three-cell stage plotted against the average length of cell cycle 2 of single embryos for ICSI and NT, respectively. Empty dots, embryos that did not develop to blastocyst; filled dots, embryos that developed to blastocyst. The window of highest prediction accuracy is highlighted, and F-score, , a combined measure of accuracy, sensitivity (<i>sn</i>) and precision (<i>pr</i>) of the predictions are indicated. The variability of cleavage timings is much higher between NT embryos than between ICSI embryos, therefore the time window for prediction are larger in NT. C) Accuracy of predicting blastocyst formation by particular measures of cell cycles in cloned (NT) and fertilized (ICSI) embryos up to the 16-cell stage (F-score). cc2, cc3, cc4, average length of cell cycles 2, 3, 4, respectively; ck1, duration of 1^st cytokinesis; diff2, diff3, diff4, length of inter-stages, i.e. 3-cell, 5- to 7-cell, 9- to 15-cell stages, respectively; div1, div2.1, div2.2, div3.1, div3.4, div4.1, div4.8, time of division to 2-, 3-, 4-, 5-, 8-, 9-, 16-cell stages, respectively. Until 4-cell stage, there are no measured variables predicting blastocyst formation of NT embryos with <i>F</i>>0.45.</p

Cell cycle duration of ICSI and NT mouse embryos in hours, starting from activation.

<p>Green, cell cycle 1 (one-cell stage); blue, cell cycle 2 (two-cell stage); red, cell cycle 3 (four-cell stage); orange, cell cycle 4 (eight-cell stage). Boxed, inter-stages (three-cell stage, five- to seven-cell stage, nine- to fifteen-cell stage). Centred black numbers indicate median length of the entire cleavage stage; white numbers indicate the inter-stage length only. Error bars, median average deviation of entire cleavage stage length; <i>n</i>, number of embryos at respective stage. *, significantly different from same cell cycle in ICSI (Wilcoxon rank sum test, <i>p</i><0.01).</p

Proposed model of reprogramming mode, adapted from Hanna et al. [<b>8</b>].

<p>After NT, reprogramming proceeds with directed and stochastic components (A) with variable latency to yield embryos of different developmental stages (B). Due to the stochastic component of the reprogramming process, some embryos have been reprogrammed more than others at a certain point in time (C). If a critical reprogramming threshold of genes essential for the respective chronologically timed embryonic stage is not reached, the embryo halts development (C). It cannot be excluded that certain oocytes reprogram better than others (elite oocytes, D), for example due to higher levels of certain factors of the “reprogrammome” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035322#pone.0035322-Pfeiffer1" target="_blank">[61]</a>.</p