Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep

Abstract

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

Keywords: FCGR1A; fertilization; multi-omics; programmed freezing; sheep; sperm.

FIGURE 1

Experimental design for integrated multi-omics analysis before and after the programmed freezing semen in sheep. (A) Experimental grouping and omics analysis methods. (B) Schematic diagram depicting programmed freezing and thawing of sheep semen. ddH₂O represents distillation–distillation H₂O.

FIGURE 2

Quality assessment of RNA-seq data and the subsequent analysis of DEGs. (A) Boxplot of RNA-seq differences between FS and PS groups. (B) PCoA distribution of the samples obtained from FS and PS groups. (C) Heatmap of expression comparison of DEGs and clustering of the samples obtained from FS and PS groups. (D) Volcano plot of various quantificational genes for RNA-seq compared with FS and PS groups. F_SR represents the sample from RNA-seq of the FS group. P_SR represents the sample from RNA-seq of the PS group. Sig_Up represents the upregulated DEGs. Sig_Down represents the downregulated DEGs.

FIGURE 3

GO classification and KEGG enrichment analyses of DEGs obtained from RNA-seq. (A) Bar plot of GO classification of DEGs. (B) Scatter plot of the KEGG enrichment pathways of DEGs.

FIGURE 4

GSEA of the quantificational genes from RNA-seq. Representative GSEA significance pathways of gene sets, including those of leishmaniasis (A), ferroptosis (B), cholinergic synapse (C), B-cell receptor signaling pathway (D), antifolate resistance (E), and alcoholism (F). (G) Heatmap of the relative expression of DEGs enriched in significant pathways by GSEA.

FIGURE 5

Quality assessment of TMT proteome data and analysis of DEPs. (A) Key information for each procedure of the proteome analysis. (B) PCA distribution of the samples from FS and PS groups. (C) Expression comparison of proteins and clustering of the samples from FS and PS groups. (D) Volcano plot of various quantificational proteins for proteome obtained from FS and PS groups. F_SP represents the sample from TMT proteome of the FS group. P_SP represents the sample from TMT proteome of the PS group. Sig_Up represents the upregulated DEPs. Sig_Down represents the downregulated DEPs.

FIGURE 6

Classification and enrichment analyses of different DEPs from TMT proteome. COG/KOG classification analysis (A), subcellular localization analysis (B), GO classification analysis (C), and KEGG enrichment analysis (D) of DEPs. CC represents cellular components.

FIGURE 7

Co-KEGG enrichment analysis and specific DEGs and DEPs contained in significant pathways. (A) Enriched genes and proteins in interesting pathway. (B) Scatter plot of the Co-KEGG enrichment pathways of DEGs and DEPs. Words in red represent interesting genes or proteins.

FIGURE 8

Pearson’s correlation analysis and the relational network between pathway-enriched DEGs and DEPs. (A) Pearson’s correlation analysis of DEGs and DEPs in significant pathways obtained by KEGG enrichment analyses of RNA-seq and TMT proteome. (B) Potential relationships and functional pathways of DEGs and DEPs.

FIGURE 9

O2PLS analyses of all the quantificational genes and proteins derived from RNA-seq and TMT proteome. (A) O2PLS analysis of all the quantificational genes and proteins. (B) Collection of various genes and proteins between Co-KEGG enrichment analysis and Pearson’s correlation analysis. (C) Collection of genes and proteins between Co-KEGG enrichment analysis, Pearson’s correlation analysis, and O2PLS analysis. KEGG_Method represents Co-KEGG enrichment analysis. Pearson_Method represents Pearson’s correlation analysis. O2PLS_Method represents O2PLS analysis.

FIGURE 10

Correlation of FCGR1A with sperm fertilization ability. (A) Experimental design for the evaluation of the fertilization ability of FCGR1A-blocked sperm. (B) Difference between the sperm motility and viability after IVF with sperm obtained from different treatments. (C) Difference between the cleavage rate after IVF with sperm obtained from the different treatments. (D) Difference between blastocyst rates after in vitro fertilization with sperm from different treatments. FS represents fresh sperm after floating. FS + anti-FCGR1A represents fresh sperm blocked by anti-FCGR1A. PS represents programmed freezing sperm after floating. PS + anti-FCGR1A represents fresh sperm blocked by anti-FCGR1A. *p < 0.05, **p < 0.001; ns, non-significance.

FIGURE 11

Representative sperm mRNA and protein response mechanisms in the frozen semen of sheep. (A) Located in the sperm acrosomal and cytoplasm membrane, FCGR1A, ITGA3, and BET1 proteins are downregulated related to the pathway and function. (B) Located in the sperm nucleus, SLX4 is downregulated related to the pathway and function. (C) Schematic diagram showing the differences in sperm motility, viability, cleavage rate blocked, and unblocked sperm.