Beneficial Effect of Selenium Doped Carbon Quantum Dots Supplementation on the in vitro Development Competence of Ovine Oocytes

Figures & data

Figure 1 The characteristic f Se/CDs. (A) The TEM characteristic of Se/CDs. (B) The 200 nm, 10 nm morphology and lattice of Se/CDs. (C) XPS spectrum of Se-CDs. (D) The FT-IR spectrum scanning from 4000 cm-1 to 800 cm-1. (E) The UV absorption (blue), excitation (black), and emission (red) spectra of Se-CDs (λex =340 nm, λem = 360 nm). (F) The normalized excitation-dependent fluorescence emissions of Se/CDs (340–480 nm).

Notes: Scale bar= 20 nm. The red circles in the panel indicated the Se/CDs. The red arrows in the panel indicated Se 3d at 55.78 eV. The chemical bonds located in the Se/CDs were marked with the green dotted box.

Figure 2 The effect of Se/CDs’ supplementation on the IVM efficiency of ovine oocytes. (A) Morphology of COCs before IVM, COCs after IVM and MII oocytes. (B) Maturation rates.

Notes: NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The red scale bar of COCs before IVM=500 μm. The red scale bar of COCs after IVM=500 μm. The red scale bar of MII oocytes=100 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05).

Figure 3 The effect of Se/CDs’ supplementation on the cytoplasmic maturation, mitochondrial activities and ROS production levels of ovine MII oocytes.

Notes: (A) CGs’ dynamics staining of ovine MII oocytes. (B) MitoTracker staining of ovine MII oocytes. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. LCA-FITC represents the LCA-FITC staining specific for the CGs dynamics of ovine MII oocytes. DIC, differential interference contrast microscope, represents the bright field of ovine MII oocytes after LCA-FITC staining. MERGED represents the merged results of LCA-FITC staining and bright field. Scale bar=50 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. MitoTracker represents the MitoTracker staining of different groups. DIC represents the bright field of ovine MII oocytes after MitoTracker staining. MERGED represents the merged results of MitoTracker staining and DIC. Scale bar=50 μm. (C) DCFH-DA staining of ovine MII oocytes. (D) Relative fluorescence intensity of LCA-FITC. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. DCFH-DA represents the DCFH-DA staining of different groups. DIC represents the bright field of ovine MII oocytes after DCFH-DA staining. MERGED represents the merged results of DCFH-DA staining and DIC. Scale bar=50 μm. (E) Relative fluorescence intensity of MitoTracker. (F) Relative fluorescence intensity of DCFH-DA NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05).

Figure 4 The effect of Se/CDs’ supplementation on the histone methylation levels of ovine MII oocytes. (A) H3K9me3 immunofluorescence staining of ovine MII oocytes. (B) H3K27me3 immunofluorescence staining of ovine MII oocytes. (C) Relative fluorescence intensity of H3K9me3. (D) Relative fluorescence intensity of H3K27me3.

Notes: NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. HOECHST represents the Hoechst 33,342 staining of different groups. H3K9me3 represents the H3K9me3 immunofluorescence staining of different groups. MERGED represents the merged results of H3K9me3 staining and Hoechst 33,342 staining. Scale bar=5 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. HOECHST represents the Hoechst 33,342 staining of different groups. H3K27me3 represents the H3K27me3 immunofluorescence staining of different groups. MERGED represents the merged results of H3K27me3 staining and Hoechst 33,342 staining. The scale bar=5 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05). NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05).

Figure 5 The effect of Se/CDs’ supplementation on the DNA methylation levels of ovine MII oocytes. (A) 5mC immunofluorescence staining of ovine MII oocytes. (B) 5hmC immunofluorescence staining of ovine MII oocytes. (C) Relative fluorescence intensity of 5mC. (D) Relative fluorescence intensity of 5hmC.

Notes: NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. HOECHST represents the Hoechst 33,342 staining of different groups. 5mC represents the 5mC immunofluorescence staining of different groups. MERGED represents the merged results of 5mC staining and Hoechst 33,342 staining. Scale bar=5 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. HOECHST represents the Hoechst 33,342 staining of different groups. 5hmC represents the 5hmC immunofluorescence staining of different groups. MERGED represents the merged results of 5hmC staining and Hoechst 33,342 staining. Scale bar=5 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05). NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05).

Figure 6 The effect of Se/CDs’ supplementation on the embryonic development potentials of ovine MII oocytes. (A) Morphology of 2-cells after IVF. (B) Fertilization rates. (C) Blastocyst development rates.

Notes: NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. Scale bar =100 μm. NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05). NC represents the negative control group. LD, MD and HD represent the Se/CDs groups supplemented with 50, 100 and 200 μg/mL of Se/CDs, respectively. The different lowercase letters in each column indicate significant differences between different groups (p<0.05).

Figure 7 The effect of Se/CDs’ supplementation on the transcript profiles of ovine MII oocytes. (A) Numbers of differentially expressed genes (DEGs) that are abnormally repressed (blue) or upregulated (red) in the Se/CDs group compared to the NC group. (B) Heatmap illustration showing the DEGs in the Se/CDs group compared to the NC group. (C) Top 20 KEGG pathways enriched in the Se/CDs group compared to the NC group. (D) GO analyses of the DEGs in the Se/CDs group compared to the NC group. (E) Expression levels of DEGs related to oocyte maturation, mitochondrial functions and epigenetic modifications in the NC and Se/CDs groups.

Notes: NC represents the negative control group. Se/CDs represent the Se/CDs group supplemented with 100 μg/mL of Se/CDs, respectively.