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. 2023 Dec 13;13(1):22126.
doi: 10.1038/s41598-023-48925-5.

Identification of hub genes and their expression profiling for predicting buffalo (Bubalus bubalis) semen quality and fertility

Divakar Swathi  1   2 , Laxman Ramya  1 , Santhanahalli Siddalingappa Archana  1 , Balaganur Krishnappa  1 , Bala Krishnan Binsila  1 , Sellappan Selvaraju  3
Affiliations

Identification of hub genes and their expression profiling for predicting buffalo (Bubalus bubalis) semen quality and fertility

Divakar Swathi et al. Sci Rep. .

Abstract

Sperm transcriptomics provide insights into subtle differences in sperm fertilization competence. For predicting the success of complex traits like male fertility, identification of hub genes involved in various sperm functions are essential. The bulls from the transcriptome profiled samples (n = 21), were grouped into good and poor progressive motility (PM), acrosome integrity (AI), functional membrane integrity (FMI) and fertility rate (FR) groups. The up-regulated genes identified in each group were 87, 470, 1715 and 36, respectively. Gene networks were constructed using up- and down-regulated genes from each group. The top clusters from the upregulated gene networks of the PM, AI, FMI and FR groups were involved in tyrosine kinase (FDR = 1.61E-11), apoptosis (FDR = 1.65E-8), translation (FDR = 2.2E-16) and ribosomal pathway (FDR = 1.98E-21), respectively. From the clusters, the hub genes were identified and validated in a fresh set of semen samples (n = 12) using RT-qPCR. Importantly, the genes (fold change) RPL36AL (14.99) in AI, EIF5A (54.32) in FMI, and RPLP0 (8.55) and RPS28 (13.42) in FR were significantly (p < 0.05) up-regulated. The study suggests that the expression levels of MAPK3 (PM), RPL36AL + RPS27A or RPL36AL + EXT2 (AI), RPL36AL or RPS27A (FMI) and RPS18 + RPS28 (FR) are potential markers for diagnosing the semen quality and fertility status of bulls which can be used for the breeding program.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Grouping of animals based on sperm functions and fertility rate. Sperm functions, such as progressive motility (a), acrosomal integrity (b), functional membrane integrity (c) and fertility rate (d) were higher in good as compared to poor semen-producing groups. ** denotes significant difference between the groups (p < 0.01); * denotes p < 0.05.
Figure 2
Figure 2
Volcano plots of the differentially expressed genes in each group. The genes from the transcriptome data having log2 fold change > ± 1 and p < 0.05 were considered as differentially expressed genes. There were 435, 744, 2170 and 117 genes differentially expressed in the progressive motility (a), acrosome integrity (b), functional membrane integrity (c) and fertility rate (d) groups, respectively.
Figure 3
Figure 3
Gene-set enrichment analysis of the differentially expressed genes. The enriched biological processes of the up-regulated and down-regulated genes were response to oxygen containing compound (a) and regulation of anatomical structure morphogenesis (b) in PM, lipid metabolic process (c) and cell cycle (d) in AI, glucose metabolic process (e) and RNA splicing (f) in FMI. There was no significant enrichment in the differentially expressed genes of the fertility rate group.
Figure 4
Figure 4
Hub genes identified in each of the four groups. The hub genes of both the up and down-regulated genes were identified in each of the groups progressive motility (a,e), acrosomal integrity (b,f), functional membrane integrity (c,g) and fertility rate (d,h). These nodes were identified from the intersection of all the analysis methods given in the Cytohubba. Variations in the colour from red to yellow represent more to less likely control points. Edges represent the association between the nodes.
Figure 5
Figure 5
Validation of the hub genes using RT-qPCR. The hub genes in each of the groups progressive motility (a), acrosomal integrity (b), functional membrane integrity (c), the fertility rate (d) and the overlapping gene EXT2 were validated using GAPDHS as a housekeeping gene. The hub genes RPL36AL, EIF5A, RPLP0 and RPS28 were significantly (p < 0.05) differentially expressed.
Figure 6
Figure 6
Receiver operating characteristic curve for the ability of the genes in predicting sperm functions and fertility rate. Univariate and multivariate analyses performed with the hub genes have a maximum prediction accuracy. The genes MAPK3, RPL36AL, RPS27A and RPS28 can influence sperm progressive motility (a), acrosome integrity (b), functional membrane integrity (c), and fertility rates, respectively. For fertility prediction, the combination model of RPS18 + RPS28 had a maximum sensitivity of 100%, specificity of 83.33% and likelihood ratio of 6 (d).
Figure 7
Figure 7
Graphical abstract depicting the identification of hub genes in sperm fertilization competence. The differentially expressed genes and their corresponding hub genes were identified in the bulls of different fertilization competence. The hub genes were validated using RT-qPCR and the receiver operating characteristic curve analysis identified MAPK3 (PM), RPL36AL + RPS27A or RPL36AL + EXT2 (AI); RPL36AL or RPS27A (FMI) and RPS18 + RPS28 (FR) can be used to select the high fertile bulls.
Figure 8
Figure 8
Methodological overview of the hub gene identification for bull fertility prediction. The methods include sperm function assessment, grouping based on sperm functions and FR, identification of differentially expressed genes in each group, up-regulated genes network construction for each group, identifying the intersection of all networks, identification of clusters, identification of hub genes, validation using RT-qPCR and receiver operating characteristic curve analysis.
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