Impact of Nacr on Bovin Fibroblast Morphology
To assess the influence of exogenous Nacr on the morphology and growth status of bovine fibroblasts, a series of experiments were conducted. Bovine fibroblasts were exposed to varying concentrations of Nacr at 2.5mM, 5mM, 7.5mM, and 10mM in growth medium for 4 hours, 8 hours, and 12 hours, respectively. The objective was to observe the possible alterations in cell morphology over the specified time periods. The results, illustrated in Fig. 1a, revealed no significant differences among cells in each group after 4 hours of treatment. Following 8-hour treatment, however, a noticeable decrease in cell density was observed in groups treated with 7.5mM and 10mM Nacr, accompanied by a limited occurrence of cell death. Subsequently, 12 hours later, the fibroblast morphology changed significantly in all Nacr concentrations (2.5mM, 5mM, 7.5mM and 10mM). The cells were irregular in shape, diminished three-dimensionality, and the number of dead cells increased (Fig. 1b).
Impact of Nacr on Bovine Fibroblast Proliferation and Gene Expression
As the alteration in cell morphology became evident with higher concentrations of Nacr and extended co-culture periods, we postulated that these changes might influence the proliferative capacity of bovine fibroblasts. Therefore, we evaluated the proliferation capacity of the fibroblasts treated with 2.5mM, 5mM, 7.5mM, and 10mM Nacr for 4 hours, 8 hours, and 12 hours using Edu labeling. The results showed that 2.5mM and 7.5mM Nacr had no significant effect on cell proliferation after 4 and 8-hour treatment. Notably, the addition of 5mM Nacr increased the proliferation rate (Fig. 2a, b, d and e). However, after a 12-hour treatment, only 10mM Nacr led to a decrease in cell proliferation, while 2.5mM, 5mM, and 7.5mM Nacr had no discernible effect, which may indicate cytotoxicity of higher Nacr concentrations affecting cell proliferation (Fig. 2c, f).
Further analysis showed that 8-hour treatment of Nacr with different concentrations significantly increased the cell proliferation capacity, indicating that the cell growth state were improved. To delve into the molecular mechanism of this effect, the fibroblasts were treated with Nacr for 8 hours, and the expression of key proliferation-related genes were examined. Thes include Proliferating cell nuclear antigen (PCNA), Cyclin-dependent kinases (CDK)1–2, Marker of Proliferation Ki67 (MK167), and Cyclin-dependent kinase inhibitor (CDKN)1-2A. The results, depicted in Fig. 2g, demonstrated that 2.5mM, 5mM, and 7.5mM Nacr significantly upregulated the mRNA expression levels of PCNA, CDK1, MK167, and CDK2 (P < 0.05), while suppressing the expressions of CDKN1A and CDKN2A (P < 0.05). Notably, the effect of 5mM Nacr was the most significant (P < 0.01). In contrast, 10mM Nacr decreased the expressions of PCNA, CDK1, MK167, and CDK2, and promoted the expressions of CDKN1A and CDKN2A. Collectively, 5mM Nacr incubated with cells for 8 hours effectively enhanced the expression of cell proliferation marker genes, thereby promoting cell proliferation.
Effect of Nacr on Cell Cycle and Related Gene Expression
Cell proliferation, intricately linked to the cell cycle, prompted an investigation into whether Nacr influences cell proliferation by modulating cell cycle. Bovine fibroblasts cultured with 2.5mM, 5mM, 7.5mM, and 10mM Nacr for 4 hours, 8 hours, and 12 hours, respectively underwent flow analysis to scrutinize the cell cycle. After 4 hours of treatment, 5mM Nacr significantly reduced the proportion of G0/G1 phase cells (65.05% vs 72.84%, P < 0.05), increased the proportion of S phase cells (10.58% vs 7.11%, P < 0.05), while 10mM Nacr decreased the proportion of S phase cells, other Nacr concentrations exhibited no notable effect on cell cycle. After 8-hour treatment, 5mM and 7.5mM Nacr increased the proportions of S-phase cells to 8.84% and 8.37% (7.98% and 7.89%), respectively, and the proportion of cells in G2/M phase to 15.59% and 15.16% (12.93% and 12.93%), respectively. Additionally, the proportion of G0/G1 phase cells significantly decreased compared to untreated fibroblasts. After 12 hours of treatment, all Nacr concentrations increased the proportion of cells in G0/G1 phase, while decreasing the proportion of cells in the S and G2/M phases, possibly due to the toxic effect that inhibited cell proliferation and blocked the G0/G1 phase (Fig. 3a-d). Notably, treatment of bovine fibroblasts with 5mM Nacr for 8 hours exhibited a more favorable effect on the normal cell cycle, with significant increases in S and G2/M phase cells, thus promoting cell proliferation.
To confirm these findings, we analyzed the mRNA expression levels of cell cycle-related genes using RT-qPCR. Compared to untreated cells, 2.5mM, 5mM, and 7.5mM Nacr upregulated the mRNA expressions of Cyclin-dependent kinase 4 (CDK4), CDK6, Cyclin B1 (CCNB1), Cyclin A2 (CCNA2), Cyclin E2 (CCNE2), and Cyclin D1 (CCND1). However, 10mM Nacr did not affect the expression of these cell cycle-related genes (Fig. 3e). These results further support the conclusion that an 8-hour treatment with different concentrations of Nacr has positive effect on bovine fibroblasts growth by modulating cell cycle and influencing proliferation.
Effect of Nacr on Apoptosis-Related Gene Expression in Bovine Fibroblasts
The results described above indicate that Nacr may enhance the growth and proliferation of bovine fibroblasts, its effect on apoptosis has been unclear. To address this, Annexin V-FITC/PI double staining was used to examine the apoptosis of the cells treated with different concentrations of Nacr at different time intervals. After 4 hours of treatment, compared to cells without added Nacr, the total apoptotic cell percentage significantly decreased from 18.6–12.61% in the 5mM Nacr-treated group (P < 0.01). In the 7.5mM Nacr-treated group, the apoptotic rate decreased from 18.6–13.18% (P < 0.01), and from 18.6–13.6% in the 10mM Nacr-treated group (P < 0.05). After 8-hour treatment, the total apoptosis rate in the 5mM Nacr-treated group was significantly reduced compared to untreated cells (10.42% vs 21.44%, P < 0.001). The early apoptosis decreased from 3.67–2.53%, while the late apoptosis decreased from 17.77–7.89%. In the 7.5mM Nacr-treated group, the total apoptosis rate was significantly reduced (13.18% vs 21.44%, P < 0.01), the early apoptosis decreasing to 2.05% and the late decreasing to 11.13%. After 12-hour treatment, the apoptotic cell death increased, with the highest percentages in 2.5mM, 5mM, and 7.5mM Nacr-treated groups (Fig. 4a-d). Continuous treatment of bovine fibroblasts with 5mM Nacr for 8 hours significantly reduced the apoptosis rate.
To explore the regulatory effect of Nacr on cell apoptosis, RT-qPCR was employed to analyze the expression of apoptosis-related genes after 8 hours of treatment with 2.5mM, 5mM, 7,5mM, and 10mM Nacr. Treatments with 2.5mM and 5mM Nacr down-regulated the expressions of pro-apoptotic genes BAD, CYCS, CASP3, and CASP9, while up-regulating the expressions of apoptosis suppressor genes BCL2 and BCL-XL. Conversely, 7.5mM and 10mM Nacr up-regulated the expressions of pro-apoptotic genes and down-regulated apoptosis suppressor genes (Fig. 4e). Continuous treatment with 5mM Nacr for 8 hours was particularly effective in reducing the expression of apoptotic-promoting genes, inhibiting cell apoptosis, and maintaining the normal growth of bovine fibroblasts.
Effect of Nacr on Histone Crotonylation Modification in Bovine Fibroblasts
In this experiment, we try to investigate whether Nacr regulate the overall Kcr modification level in cells and modulate cell proliferation by Cr-coA content. According to the previous results, 5mM Nacr treatment for 8 hours is optimal to maintain cell morphology, promote cell proliferation, and inhibit cell apoptosis. We examined the Cr-coA content in fibroblasts treated with 5mM Nacr for 8 hours. The results showed that the content of Cr-coA in cells was significantly increased (Fig. 5a). Furthermore, Nacr increased the overall modification level of Kcr in fibroblasts, especially significantly elevated H3K9cr modification (Fig. 5b-e). To unravel the mechanism of Nacr regulating Kcr modification, we detected and analyzed the expression of the enzymes related to Kcr modification. Nacr treatment with 2.5mM, 5mM, 7.5mM, and 10mM up-regulated the expression of Kcr-modified writer genes. These include E1A binding protein p300 (EP300), Cbp/p300-interacting transactivator with Glu/Asp-rich carboxyterminal domain 1 (CITED1), and Acyl-coA synthetase 2 (ACSS2). Simultaneously, Nacr down-regulated the expression of Kcr-modified erasers genes such as Histone deacetylase 2 (HDAC2), HDAC3, SIRT1, and SIRT3. Specifically, Nacr at 2.5mM and 5mM up-regulated the expression of Kcr-modified readers genes, including double PHD fingers 2 (DPF2), Chromodomain Y-Like protein (CDYL), MLLT3 super elongation complex subunit (MLLT3), and YEATS domain containing 2 (YEATS2) (Fig. 5f).
In summary, 5mM Nacr has the best effect on the regulation of Kcr-modified genes of writers, readers, and erasers. These results suggest that Nacr, as a small molecule additive regulating Kcr modification, can specifically modulate the level of Kcr modification in bovine fibroblasts by influencing the expression of Kcr-modified writers and erasers genes. To eliminate the interference from other modifications, we also assessed the enzymes associated with histone methylation such as SUV39H1 histone lysine methyltransferase, SUV39H2, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), Euchromatic histone lysine methyltransferase 1 (EHMT1), lysine demethylase 4A (KDM4A), KDM4B, KDM4C, and KDM5A. The mRNA expressions of these enzymes were reduced but with no significant differences after treatment with different concentrations of Nacr for 8 hours (Fig. 5g). These results indicate that Nacr treatment of bovine fibroblasts specifically influence histone crotonylation, but do not affect the expression of histone methylation genes.
Enhancement of Somatic Cell Nuclear Transfer Efficiency by The Use of Nacr-Treated Cells
The aforementioned results have proved that the introduction of Nacr induce a notable augmentation of high level histone lysine crotonylation (Kcr) modification in bovine fibroblasts, which stand out as a pivotal factor to significantly increase cellular activity and cell proliferation in bovine fibroblasts. Then, the fibroblasts treated by 5mM Nacr for 8 hours were used as donor cells to produce cloned embryos by somatic cell nuclear transfer (SCNT). The Nacr-treated cells resulted in 38.1% of the embryos developed to blastocysts and total cell number of SCNT blastocyst, which was significantly higher than the control (25.2%) (Table 1, Fig. 6B-C). These findings confirm that Nacr-treated fibroblasts can significantly improve SCNT embryo development, which suggest that Nacr induce cell upsurge of Kcr modification and then increase cellular developmental potential.
Table 1
Development of SCNT embryos from different donor cell
Items
|
Donor cells
|
con Bovine fibroblasts
|
5mM Nacr Bovine fibroblasts
|
The number of replicates
|
3
|
3
|
Number of fused clones
|
477a
|
511a
|
2-cell rate (%)
|
81.7 ± 0.94a
|
83.8 ± 1.29a
|
8-cell rate (%)
|
71.7 ± 1.55a
|
72.7 ± 2.08a
|
Blastocyst rate (%)
|
25.2 ± 0.62a
|
38.1 ± 1.25b
|
Note: Different letters indicate significant difference, a, b in the same column (P < 0.05). |