Effects of Cryopreservation on Sperm with Cryodiluent in Viviparous Black Rockfish (Sebastes schlegelii)

Abstract

Black rockfish is an economically important fish in East Asia. Little mention has been paid to the sperm cryopreservation in black rockfish. In this study, the optimal cryodiluent was selected from 48 combinations by detecting various sperm parameters. Transcriptome and methylome analysis were further performed to explore the molecular mechanism of inevitable cryoinjuries. The results showed that cryopreservation had negative effects on the viability, DNA integrity, mitochondrial activity, total ATPase and LDH of sperm even with optimal cryodiluent (FBS + 15% Gly). Transcriptome and methylome analysis revealed that the expression of 179 genes and methylation of 1266 genes were affected by cryopreservation. These genes were enriched in GO terms of death, G-protein coupled receptor signaling pathway, response to external stimulus and KEGG pathways of phospholipase D signaling pathway and xenobiotic and carbohydrate metabolism pathways. The role of PIK3CA and CCNA2 were highlighted in the protein-protein interaction network, and the sperm quality-related imprinted gene mest was identified among the 7 overlapping genes between transcriptome and methylome. Overall, the cryodiluent for black rockfish sperm was optimized, providing a feasible method for cryopreservation. The transcriptome and methylome data further demonstrated the underlying molecular mechanisms of cryoinjuries, proving clues for improvement of cryopreservation method of black rockfish.

Keywords: black rockfish; methylome; sperm cryopreservation; sperm quality; transcriptome.

Figure 1

Effects of different cryodiluents on the post-thaw sperm motility parameters. (A) The motility of post-thaw sperm, (B) the VCL of post-thaw sperm, (C) the LIN of post-thaw sperm. VCL, curvilinear velocity; LIN (linearity, LIN = straight line velocity (VSL, µm/s) × VCL-1 × 100%). Statistical significance was accepted when p < 0.05 and indicated by different letters. FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethyl sulfoxide; EG, ethylene glycol; DMF, N, N-dimethylformamide; Gly, glycerol.

Figure 2

Effects of cryopreservation on sperm quality with the cryodiluent (FBS + 15% Gly). (A) Effects of cryopreservation on sperm plasma membrane integrity. (A1,A2) the eosin-aniline black staining. The dead sperm with damaged plasma membrane were stained to dark pink, whereas live sperm with plasma membrane integrity were not stained. (A3) Vitality of fresh and cryopreserved sperm. Sperm vitality measured as % of sperm without staining. (B) Effects of cryopreservation on sperm DNA integrity. (B1,B2) sperm chromatin dispersion (SCD) test. The sperm without fragmented DNA showed nucleoids with big halos of spreading of DNA loops, whereas those with fragmented DNA showed no halo or a small halo. (B3) DFI of fresh and cryopreserved sperm. DFI measured as % of sperm with no halo or a small halo. Scale bar = 20 µm. Statistical significance was accepted when p < 0.05 and indicated by different letters.

Figure 3

Effects of cryopreservation on sperm function with the cryodiluent (FBS + 15% Gly). (A) The location of mitochondria in black rockfish sperm. Scale bar = 5 µm. (B) Detection of mitochondrial membrane potential by Rh123. Scale bar = 50 µm. (C) Mitochondrial activity rate of fresh and cryopreserved sperm. Y-axis showed mitochondrial activity, measured as % of spermatozoa with green fluorescence in the mitochondrial area. (D) Detection of ATPase, LDH and SDH of fresh and cryopreserved sperm. Statistical significance was accepted when p < 0.05 and indicated by different letters.

Figure 4

Transcriptome analysis of sperm with the cryodiluent (FBS + 15% Gly). (A) DEGs in transcriptome profile of cryopreserved sperm. One hundred seventy-nine DEGs were identified, including 97 upregulated genes and 82 downregulated genes. (B) GO enrichment analysis of DEGs. Statistical significance was accepted when p < 0.05. (C) KEGG enrichment analysis of DEGs. Statistical significance was accepted when p-value < 0.05. (D) The PPIN of DEGs. The PPIN was constructed by STRING database and visualized using Cytoscape software. The node size is positively correlated with degree. (E) The expression pattern of key DEGs identified in PPIN. The x-axis shows sampled tissues, with the prefix F for sperm without cryopreservation and C for sperm with cryopreservation, and the y-axis shows genes. The color scale showed standardized RPKM values normalized by Z-score method.

Figure 5

Methylation analysis of sperm with the cryodiluent (FBS + 15% Gly). (A) DMGs in the methylome profile of cryopreserved sperm. One thousand two hundred sixty-six DMGs were identified, including 261 downregulated genes and 1005 upregulated genes. (B) GO enrichment analysis of DMGs. Statistical significance was accepted when q-value < 0.05. (C) KEGG enrichment analysis of DMGs. Statistical significance was accepted when q-value < 0.05. (D) The PPIN of DMGs. The PPIN was constructed by STRING database and visualized using Cytoscape software. The node size is positively correlated with degree. (E) The expression pattern of key DMGs with a degree of 10 or above identified in PPIN. The x-axis showed sampled tissues, with the prefix F for sperm without cryopreservation and C for sperm with cryopreservation, and the y-axis shows genes. The color scale showed standardized RPKM values normalized by Z-score method.