Page 460 - IJB-10-4

International Journal of Bioprinting Biomechanical mimic-based artificial oviduct system

Notably, preimplantation embryos cultured on 10% between the 8% GelMA group and the 10% GelMA and
GelMA hydrogel exhibited the best developmental control groups. Normalized expression levels are illustrated
speed and blastocyst development rate among the in a heatmap (Figure 4F).
experimental groups. GelMA hydrogel culture conditions Differentially expressed genes (DEGs) were thoroughly
positively impacted embryo development, potentially by examined via inter-group comparisons, applying the criteria
recapitulating the biomechanical properties of the oviduct of p < 0.05 and |fold change| > 2, which are visualized in a
environment. Venn diagram (Figure 4G). These analyses revealed that 77

3.3. Transcriptional shifts in morula cultivated on genes were altered in the 8% GelMA group relative to the
the 3D gelatin methacryloyl platform control group (Datasheet 1 [Supplementary File 2]), 157
The 3D-bioprinted GelMA hydrogel culture system genes were altered in the 10% GelMA group relative to the
increased development speed on days 3–4, particularly control group (Datasheet 2 [Supplementary File 2]), and 89
during the morula-to-blastocyst transition. The morula genes were altered in the 8% GelMA group relative to the
stage is pivotal for blastocyst development and activation 10% GelMA group (Datasheet 3 [Supplementary File 2]).
of the entire gene sets of sex chromosomes, playing a key The Hist1h4d gene, which encodes the histone H4 protein,
role in the first differentiation from totipotent embryonic was the only gene differentially expressed in all three
cells into the ICM and trophectoderm (TE). 41 comparisons and was down-regulated in both the 8% and
10% GelMA groups compared with the control group
To assess the effects of GelMA hydrogel culture (Datasheet 4 [Supplementary File 2]).
conditions on gene expression dynamics during
preimplantation development, which is especially crucial Furthermore, 22 DEGs were common in two
for the morula-to-blastocyst transition, we conducted comparisons (Control vs. 8% GelMA and Control vs.
comprehensive RNA-seq analyses of total mRNA 10% GelMA), including seven up-regulated genes (C1ql1,
extracted from morulae derived from zygotes cultivated Chst13, Cpeb1, Depdc5, Gm8300) and 15 down-regulated
on GelMA hydrogels (8% or 10%) or standard hard genes (Arid3a, Ccnk, Edn2, Hist1h4d, Iqcf5). Additionally,
plastic dishes (control group) for 72 h. Intriguingly, the the most significantly changed DEGs (p < 0.05; |fold
principal component (PC) analysis (PCA) scatter plot change| > 1.5) of each comparison are presented in a
revealed 38.88% (PC1) and 19.78% (PC2) variations in volcano plot (Figure 4H).
gene expression between the morulae of the control, 8%, The identified top varied DEGs from PC1 and PC2
and 10% GelMA groups (Figure 4A). Importantly, the PC1 score analyses were linked to the GO terms: transcription,
scores of the top 50 hit genes exhibited distinct expression mRNA processing, endoplasmic reticulum, and protein
patterns of genes associated with the GO categories. transport. GSEA for DEGs related to the GO terms
Noteworthy categories included transcriptional regulation (Control vs. 8% GelMA and Control vs. 10% GelMA)
(GO:0006355), cell differentiation (GO:0030154), were consistent with PCA. In a comparison of the 8%
mRNA processing (GO:006397), cell cycle regulators GelMA group with the control group, many DEGs were
(GO:0051726), lipid metabolism (GO:0006629), and up-regulated (Eif1ad7, Eif1ad8, Eif4a3l1, Cpeb1, Pramef25,
multicellular organism development (GO:0007275) etc.), and were linked to the GO terms: ribosomal large
(Figure 4B and D). The hit genes (Actb, Etv3, Hnrnpa3, subunit binding (GO:0043023), ribonucleoprotein
Barx2, and Cers4) associated with these GO terms were complex binding (GO:0043021), translation regulator
identified as key contributors to most of the differences in activity (GO:0045182), enzyme-substrate adaptor activity
gene expression between the control, 8%, and 10% GelMA (GO:0140767), and ubiquitin-like ligase substrate adaptor
groups. Normalized expression levels of hit genes are activity (GO:1990756) (Figure 4I [left]; Datasheet 5
represented visually in a heatmap (Figure 4E). Likewise, [Supplementary File 2]). Contrastingly, down-regulated
for the PC2 scores, expression of genes associated with GO DEGs in the 10% GelMA group relative to the control
terms related to endoplasmic reticulum (GO:0005783), were Srarpf2, Uhrf2, Sp1, Actb, Med13, etc., and they
transmembrane transport (GO:0055085), protein transport were linked to the GO terms: RNA polymerase II-specific
(GO0015031), membrane (GO:0016020), and nucleotide DNA-binding transcription factor binding (GO:0061629),
binding (GO:0000166) drove the transcriptomic variations, transcription coregulator activity (GO:0003712),
particularly in the 8% GelMA hydrogel group relative to mRNA binding (GO:0003729), mRNA 3´-UTR binding
the 10% GelMA and control groups (Figure 4C and D). (GO:0003730), and mRNA 3´-UTR AU-rich region
The identified hit genes (Apbb2, Abca17, Acsl1, Neo1, and binding (GO:0035925). Cpeb1 and Nxf2 genes were up-
Hap1) associated with these GO terms emerged as key regulated in the 10% GelMA hydrogel group (Figure 4I
contributors to the differences in transcriptional profiles [right]; Datasheet 6 [Supplementary File 2]).

Volume 10 Issue 4 (2024) 452 doi: 10.36922/ijb.3346