Fig 1.
(A) Cumulus-oocyte complexes (COCs) were aspirated from bovine ovaries collected at the slaughterhouse and were in-vitro matured (IVM) and in-vitro fertilized (IVF). (B) Putative zygotes were individually cultured for 190 h in an incubator equipped with a time-lapse system to allow non-invasive, continuous monitoring of morphokinetic patterns throughout embryonic development. (C) Kinetics evaluation considered: (1) the timing of the 1st, 2nd, 3rd, and 4th cleavages, morula formation, blastulation, blastocyst and expanded blastocyst formation and (2) the characterization of the cleavage pattern into synchronous (sync.; corresponding to cleavages to 4-, 8-, and 16-cell stage embryos) and asynchronous (async.; corresponding to cleavages to 3-, 5-, 6-, 7-, 10-, and 12-cell stage embryos). (D) Kinetics and morphological evaluation of embryos from the 1st cleavage; characterization of normal or abnormal (direct, unequal, and reverse) cleavages and their morphological distribution into good, fair, and poor. (E) Morphology and transcriptomic evaluation of the developed blastocysts; blastocysts that developed from synchronously, asynchronously, and directly cleaved embryos were morphologically evaluated as good, fair, or poor. No blastocyst development was recorded for the unequally or reverse-cleaved embryos. Samples of 4 blastocysts from each of the three cleavage categories (synchronous, asynchronous, and direct) were subjected to RNA extraction, followed by microarray analysis, and samples of 3 blastocysts from each group were validated by RT-qPCR (4 replicates). Fig 1 was created by Shira Yaacobi-Artzi with Microsoft PowerPoint software.
Fig 2.
Developmental kinetics of cleaved bovine embryos.
Cumulus-oocyte complexes (COCs) were in-vitro-matured, fertilized, and individually cultured for 190 h in CultureCoin dishes in an incubator equipped with a time-lapse system. Embryonic development was continuously monitored with image acquisition every 5 min. (A) Representative images of the morphological categories of embryos at the first cleavage: normally cleaved embryos are characterized by two equal-sized blastomeres (putative zygote after fertilization) (a), after normal cleavage (a’); directly cleaved embryos are characterized by direct cleavage from 1 cell (putative zygote after fertilization) to 3 blastomeres (b), after cleavage into a 3-cell-stage embryo (b’); or direct cleavage from 1 cell to 4 blastomeres (c and c’); an unequally cleaved embryo is characterized by two blastomeres of unequal size (d”); a reverse-cleaved embryo is characterized by reduced number of blastomeres through the first division (e and e’), and after merging to 1 cell (e”). The precise time (h:min) post-fertilization is presented in the lower-left corner of the pictures. Scale bar = 100 μm. (B) For each embryo in the synchronously cleaved embryos, the cleavage timing post-fertilization of the 1st, 2nd, and 3rd cleavages, and the time of morula, early blastocyst, and blastocyst formation, as well as the blastocyst expansion were recorded. Data are presented in whisker plots. Boxes indicate the 25th and 75th percentiles, and the middle horizontal line indicates the median. Whiskers indicate the maximum and minimum values within the acceptable range defined by the two quartiles, n = 431 embryos. (C) Presented is the cleavage timing post-fertilization (1st, 2nd, and 3rd cleavages) of synchronously cleaved embryos that further developed or did not develop to the blastocyst stage (the white or gray column, respectively). The line inside the box is the median; the lower hinge of the box indicates the 25th percentile, and the upper hinge indicates the 75th percentile. The upper whisker indicates the 95th percentile values and the lower whisker indicates the 10th percentile values for the cleaved embryos that further developed or did not develop to the blastocyst stage. The Kruskal Wallis test, followed by the Wilcoxon test pairwise comparisons, was used to compare the median value in the first, second, and third cleavages. *P < 0.05. (D) The pattern of asynchronous embryonic division was divided into six distinct classes, according to the number of asynchronous events expressed (i.e., one to six events). The proportion of embryos that either developed or did not develop to blastocysts was analyzed by the Chi-squared test was followed by Pearson’s test. *P < 0.05; n = 271 cleaved embryos. (E) Presented is the time of the first cleavage for all the cleavage patterns. The timing of the 1st divisions in synchronously (Sync), asynchronously (Async), and abnormally cleaved embryos was recorded. The line inside the box is the median; the lower hinge of the box indicates the 25th percentile and the upper hinge indicates the 75th percentile. The upper whisker indicates the 95th percentile values and the lower whisker indicates the 10th percentile values. n = 656 embryos. The Kruskal Wallis test, followed by the Wilcoxon test, was used to compare the median value in the first cleavage between groups.
Table 1.
Primers used for qPCR analysis.
Table 2.
Embryonic development and blastocyst formation in normally and abnormally cleaving groups (2A) and subgroups (2B and 2C).
Fig 3.
Morphology of bovine cleaved embryos.
(A) Representative images of a 2-cell stage embryo with equal-sized blastomeres (1, good morphology); a 2-cell stage embryo with equal sized blastomeres and up to 20% fragmentation (2, fair morphology); a 2-cell stage embryo with unequal sized blastomeres and >50% fragmentation (3, poor morphology); a 4-cell stage embryo with four equal sized blastomeres (4, good morphology); a 4-cell stage embryo with fragmentation, equal sized blastomeres (5, fair morphology); a 4-cell stage embryo with unequal sized blastomeres and >50% fragmentation (6, poor morphology); an 8-cell stage embryo with equal sized blastomeres (7, good morphology); an 8-cell stage embryo with unequal sized blastomeres and minor (<20%) fragmentation (8, fair morphology); and an 8-cell stage embryo with unequal sized blastomeres and severe (>50%) fragmentation (9, poor morphology). (B) Distribution of normal synchronously cleaved embryos that developed (+) or did not (-) to the blastocyst stage into the different morphological categories (good, fair, and poor). A Chi-squared test, followed by Fisher’s exact test, was used for pair comparison within each cleavage. *P < 0.05; n = 431 embryos. (C) Distribution of 2-cell stage embryos with different cleavage patterns (synchronous; sync, unequal, and reverse) in different morphological categories. A Chi-squared test was followed by Pearson’s test; *P < 0.05; n = 403 embryos. Distribution of 3-cell stage embryos (from asynchronous and direct cleavage) in different morphological categories; n = 188 embryos. Distribution of 4-cell stage embryos (from synchronous and direct cleavage) in different morphological categories; n = 319 embryos. The distribution in morphological categories was calculated from cleaved embryos. A Chi-squared test, followed by Fisher’s exact test, was used. (D) Representative images of blastocysts with evident inner cell mass (ICM) with many tightly packed cells and many trophoblast (TE) cells forming a cohesive epithelium (1 and 2, good morphology); a blastocyst with a few TE cells forming a loose epithelium (3 and 4, fair morphology); a blastocyst with ICM presenting loosely grouped cells and TE composed of very few cells (5, fair morphology); a blastocyst with ICM presenting loosely grouped cells and TE composed of a few large cells (6, fair morphology), bar = 100 μm. (E) The distribution of synchronously (Sync), asynchronously (Async), and directly (Direct) cleaved embryos that developed to the blastocyst stage in the different morphological categories. A Chi-squared test, followed by Pearson’s test, was used, n = 170 blastocysts.
Fig 4.
Differential gene expression in blastocysts that developed from synchronously, asynchronously, and directly cleaved embryos.
(A) PCA plots. Purple, blastocysts from synchronously cleaved embryos (Sync); blue, from asynchronously cleaved embryos (Async); red, from directly cleaved embryos (Direct). Each symbol represents one replicate consisting of 4 blastocysts. (B) Hierarchical clustering (unsupervised) analysis of differentially expressed genes in blastocysts from synchronously, asynchronously, and directly cleaved embryos. Blastocysts from synchronously cleaved embryos are in 3 replicates (n = 4 blastocysts each) corresponding to samples Sync_1, Sync_3, and Sync_2; blastocysts from asynchronously cleaved embryos are in 5 replicates (n = 4 blastocysts each) corresponding to samples Async_1, Async_2, Async_3, Async_4, and Async_5; blastocysts from directly cleaved embryos are in 3 replicates (n = 4 blastocysts each) corresponding to samples Direct_1, Direct_3, and Direct_2. Colors indicate the expression levels of the detected genes: red, higher expressed; blue, lower expressed. Each horizontal line represents a single gene, and each column represents a single sample from each group. Left branches indicate the relationships among different genes and the upper branches indicate differences among samples.
Fig 5.
Differentially expressed genes between groups.
Volcano plots are presented to visualize the differential gene expression patterns between groups. The values plotted on the x- and y-axes represent the average normalized signal values of each group (log2-scaled). For each gene, the P-value was plotted against the fold change. Vertical bars represent statistical significance and dots represent the genes that were higher expressed (red) or lower expressed (green). (A) Volcano plots present the differential gene expression between synchronously (sync) and asynchronously (async) cleaved embryos (n = 180 differentially expressed genes); (A’) Distribution of lower (green) and higher (red) differentially expressed genes within the most associated biological processes between synchronously vs. asynchronously cleaved embryos (B) A volcano plot presents the differential gene expression between synchronously and directly cleaved embryos (n = 895; differentially expressed genes); (B’) Distribution of lower (green) and higher (red) differentially expressed genes within the most associated biological processes and between synchronously vs. directly cleaved embryos. (C) A volcano plot presents the differential gene expression between asynchronously and directly cleaved embryos (n = 643; differentially expressed genes). (C’) The distribution of lower (green) and higher (red) differentially expressed genes within the most associated biological processes between synchronously vs. asynchronously cleaved embryos.
Table 3.
Enrichment analysis of differentially expressed genes in blastocysts that developed from synchronously vs. asynchronously cleaved embryos (DAVID).
Fig 6.
A Venn diagram of the differentially expressed genes (n = 1078 genes) in blastocysts derived from synchronously vs. directly cleaved embryos (pink, n = 435 genes); asynchronously vs. directly cleaved embryos (blue, n = 183 genes); and overlapping genes in blastocysts from synchronously and asynchronously vs. directly cleaved embryos (purple, n = 460 genes).
Fig 7.
RT-qPCR validation of microarray analysis.
Cumulus-oocyte complexes were in-vitro matured for 22 h and then fertilized. Putative zygotes were then in-vitro cultured for an additional 190 h to allow for blastocyst formation. Samples of the developed blastocysts (n = 3 for each sample; 4 replicates) from synchronously, asynchronously, and directly cleaved embryos were collected and their RNA was extracted for RT-qPCR validation based on four representative selected genes (HSPA1A, ZP3, DDX3Y, and GDF9). Presented is the fold change calculated by the 2−ΔΔCT method, normalized against the reference genes YWHAZ and SDHA and relative to the control (synchronous or asynchronous, expressed as 1). Data are presented as the means ± SEM, *P < 0.05.
Fig 8.
Possible protein–protein connections of overlapping differentially expressed genes.
STRING analysis of the overlapping genes expressed differentially in blastocysts from synchronously and asynchronously vs. directly cleaved embryos. Only genes with nodes in the network association are presented. For a better view of the figure, it is highly recommended to use the magnifier tool.
Table 4.
Enrichment analysis of overlapping genes in blastocysts that developed from synchronously and asynchronously cleaved embryos that are differentially expressed in directly cleaved embryos (DAVID).
Fig 9.
Possible protein–protein connections of non-overlapping differentially expressed genes.
STRING analysis of non-overlapping genes expressed differentially in blastocysts from synchronously vs. directly cleaved embryos. Only genes with nodes in the network association are presented. For better view of the figure, it is highly recommended to use the magnifier tool.
Fig 10.
Possible protein–protein connections of non-overlapping differentially expressed genes.
STRING analysis of non-overlapping genes expressed differentially in blastocysts derived from asynchronously vs. directly cleaved embryos. Only genes with nodes in the network association are presented. For a better view of the figure, it is highly recommended to use the magnifier tool.
Table 5.
Enrichment analysis of non-overlapping genes in blastocysts that developed from synchronously or asynchronously cleaved embryos that are differentially expressed in directly cleaved embryos (David).