The SP600125-induced tetraploid (SP4N) cells were come from State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University (Zhou et al., 2016). Diploid crucian carp fibroblast (2N) cells were obtained from the caudal fin of C. auratus red var., and tetraploid fish (CC4N) cells were derived from the caudal fin of a tetraploid hybrid of C. auratus red var. (♀) × C. carpio L. (♂).

Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Life Technologies, CA, United States), supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, United States), 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, United States), 0.1% 2-mercaptoethanol (2-ME, Invitrogen, Carlsbad, CA, United States), 1 mM sodium pyruvate (Invitrogen, Carlsbad, CA, United States), and 1 mM nonessential amino acids (Invitrogen, Carlsbad, CA, United States). And all of the cells used were from the 16th to 18th passage.

Confluent cells were treated with 0.1 μg/ml colchicine for 2–4 h before chromosome preparation. The cells were digested into single-cell by 0.25% trypsin (Invitrogen, Carlsbad, CA, United States). After hypotonic treatment with 0.075 mol/L KCl for 30–60 min, the cells were fixed twice with cold Carnoy’s fixative (methanol: glacial acetic acid = 3:1, v/v) for 20 min each time. Then, the cells were dropped onto the cold slides and stained with 5% Giemsa solution (Solarbio Inc. United States) for 15 min. More than 30 metaphases were examined for each cell line.

Analysis of DNA ploidy levels and nuclear C values were performed with a flow cytometer (Sysmex-partec, Germany). Cells were digested into single-cell suspension by 0.25% trypsin. After being filtered through a 40 μm cell strainer, the cells were incubated with 2 μg/ml Hoechst 33342 (Invitrogen) and 50 μM Verapamil (Sigma) for about 15 min.

After fixed in 4% paraformaldehyde for 30 min, cell samples were treated with 0.3% Triton X-100 for 3 min and blocked for 1 h with 0.2% bovine serum albumin (Calbiochem, San Diego, CA, United States). Anti-α-tubulin antibody (dilution ratio was 1:100; GeneTex, Inc. North America) was used as the primary antibodies. Anti-mouse IgG fluorescent secondary antibody (dilution ratio was 1:200) was purchased from Abways Biotechnology Co., Ltd. (Shanghai, China). DNA and mitochondria were stained with Hoechst 33342 (Invitrogen) and MitoTracker Green FM (Invitrogen), respectively.

Cells were fixed in 2.5% glutaraldehyde for 2 h, and then treated with 1% OsO4 for 2 h. After dehydration through an ethanol gradient, the specimens were embedded in Epon812 resin (TAAB, United States). The sections were double-stained with uranyl acetate and lead citrate, and were examined by an electron microscope (JEM-1230, JEOL, Tokyo, Japan).

Wound healing assay was used to examine the cell migration capacity. The wound was created perpendicular to the marking on 60-mm dish, then, washed with PBS until no cells at the scratch. Upon the pre-experiment basis, the sampling time point was 24 h. Images acquired were analyzed quantitatively by ImageJ software. For each sample, experiments were repeated at least 3 times.

Cells were digested by 0.25% trypsin into a single-cell solution, washed 3 times with sterile physiological saline. After centrifugation, 500 μL ddH₂O was added and homogenate was broken in a hot bath (95°C). Cellular ATP content was assessed using an ATP Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. For each sample, experiments were repeated at least 3 times.

The copy number of mitochondrial DNA was determined by real-time fluorescence quantitative PCR (qPCR). The mtDNAs was extracted with DNA extraction kit (D3396-01, Omega). The enc1 gene (nuclear single-copy gene) and cytb gene (mitochondrial gene) were selected for qPCR test. According to the multiple of cytb relative to enc1 (Li et al., 2007), the number of mtDNA copies in a single cell was converted according to the number of template genomes. For each sample, experiments were repeated at least 3 times.

For this research, we obtained mRNA sequencing (seq) data of in vitro diploid C. auratus red var. and tetraploid C. auratus red var. (♀) × C. carpio L. cultured cells from the NCBI SRA database (SRR7640867, SRR7640866, SRR7640869, and SRR7640868) (Ren et al., 2020). Next, we submitted the mRNA-seq data of SP600125-induced tetraploid cells to the NCBI SRA (SRR9964682 and SRR9964683).

Total RNA was extracted using RNAiso Plus reagent (Takara, Bio. Beijing). The qRT-PCR was conducted as described in our previous research (Mo et al., 2019; Ren et al., 2020). Primers were designed using Primer Premier 5.0 software (Supplementary Table S2), and used to detect expression with the following amplification conditions: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The average threshold cycle (Ct) was calculated for each sample using the 2−ΔΔCt method (Livak and Schmittgen, 2001). The housekeeping gene β-actin was used as the reference gene. For each sample, qRT-PCR analysis was conducted three times.

As shown in Figure 1, the SP4N cells and CC4N cells were coincident in size, where the long diameters were 103.5 ± 20.0 µm, and the short diameters were 45.5 ± 12.5 µm). In contrast, the long and short diameters of the 2N cells were 100.0 ± 12.0µm and 25.5 ± 8.5µm, respectively. The nuclei diameters of the SP4N cells and CC4N cells were about 42.5 ± 8.0 µm, while 22.5 ± 5.0 µm in that of the 2N cells (Figure 1A). In the SP4N cells and CC4N cells, there were about 70% of metaphases with 200 chromosomes and others were between 187 and 198. And the majority of the 2N cells (90%) contained 100 chromosomes, while for the other 10% of them, the number of chromosomes was between 93 and 98 (Figure 1B). Moreover, the relative DNA contents (C value) were about 4C in the SP4N cells and CC4N cells, while that in the 2N cells was 2C (Figure 1C). Flow cytometry analysis further demonstrated that there existed diploid (2x, 100) and tetraploid (4x, 200) peaks in the 2N cells, while the tetraploid (4x, 200) and octoploid (8x, 400) peaks display in the SP4N and CC4N cells (Figure 1C).

By immunofluorescence staining, normal chromosome behaviors were observed in the mitosis of SP4N cells (Figures 2A–F). The growth of SP4N cells was stable during subsequent subculture such that more than 38 passages were expanded (Supplementary Figure S1).

The results of wound healing assay indicated that the migration ability of SP4N cells was the weakest among the three fish cell lines, and that of the 2N cells was stronger than that of the CC4N cells (Figure 3A). There was no significant difference in the levels of ATP contents under unit protein concentration among the three types of cells (Figure 3B). However, the mtDNA copy number of SP4N cells was significantly lower than those of the 2N and CC4N cells (Figure 3C). The 2N cells had more mitochondria than the SP4N and CC4N cells, but giant mitochondria were observed in the SP4N cells (Figures 3D,E).

The transcriptome data of SP4N cells were compared with those of the 2N cells or the CC4N cells, collected from the NCBI SRA database. By comparison between the SP4N cells and the 2N cells, 663 differentially expressed genes (DEGs) were obtained. Among these genes, 255 (38.46%) were significantly up-regulated and 408 (61.54%) significantly down-regulated (Figure 4A). Comparing the SP4N cells with the CC4N cells, a total of 35,599 differentially expressed genes were obtained, among which 30,449 (85.53%) genes were significantly up-regulated and 5,150 (14.47%) genes were significantly down-regulated (Figure 4B). These results showed that the transcription difference between the SP4N cells and the 2N cells was smaller than that between the SP4N cells and the CC4N cells.

According to the GO and KEGG database, the DEGs of SP4N cells and 2N cells were significantly different in the cell adhesion and migration (alcam, tn, jami), immunity (nik, stat1, cd2), calcium ion regulation (ncx, cacna1a, cacna2d3), energy metabolism (egr2, hk2, adora1) (Supplementary Table S3). While the DEGs of SP4N cells and CC4N cells were among the tumor related genes (cldn, fn1, twist), transcription factors (eif2s2, eif4e), cycle regulation related genes (cdc25b, cdc20), and energy metabolism (uqcrc2, pfka, ndufs6) (Supplementary Table S4).

The transcriptome data demonstrated that the DEGs were involved in cell cycle regulation pathways in the SP4N and CC4N cells, but not in the SP4N and 2N cells (Figure 5A, Supplementary Figure S2 andTables S3, S4). Eight genes of cell cycle pathway were chosen to be further determined by qRT-PCR. As shown in Figure 5B, in the SP4N cells, the expression levels of these eight genes were similar to those in the 2N cells, but significantly different from those in the CC4N cells.

As shown in Supplementary Table S3, S4, many energy metabolism-related genes were annotated to KEGG related pathways from transcriptome data among the SP4N cells, 2N cells and CC4N cells. We selected six glycolytic genes (glut1, hk1, pfkfb, pgam, eno, ldh) and seven oxidative phosphorylation genes (nd1, nd2, nd5, ndufa4, ndufs8, sdhd, uqcr) for further detection by qRT-PCR. As shown in Figure 6A, comparing with the 2N cells, most of the genes in the SP4N cells related to glycolysis and mitochondrial oxidative phosphorylation pathway were down-regulated. The transcription levels of glycolysis and mitochondrial oxidative phosphorylation related genes in the two tetraploid fish cells were significantly different where the expression level in the CC4N cells was higher than the SP4N cells. Except for pgam and ndufa4, which were slightly lower than the 2N cells, the expression levels of other detected genes in the CC4N cells were higher than the 2N cells and SP4n cells (Figure 6A).

Sirtuin family belongs to the third type histone deacetylase, especially Sirt3, Sirt4 and Sirt5, localized in mitochondria and closely associated with metabolism and oxidative respiration (Haigis et al., 2006; Jin et al., 2009; Nakagawa et al., 2009). The transcription levels of sirt3, sirt4 and sirt5 genes in the SP4N cells were the lowest among the 3 cells (shown as SP4N < 2N < CC4N) (Figure 6B).