Transcriptome Analysis Reveals Sertoli Cells Adapting Through Redox and Metabolic Pathways Under Heat Stress in Goats

Abstract

Background/objectives: Climate change-induced temperature elevations pose significant challenges to livestock reproduction, particularly affecting testicular function in small ruminants. This study investigates the acute heat-stress response in goat Sertoli cells (SCs), aiming to elucidate the molecular mechanisms underlying heat-induced damage to male reproductive tissues.

Methods: SCs were isolated from testes of 4-month-old black goats and exposed to heat stress (44 °C for 2.5 h). We employed transcriptome sequencing, CCK-8 assay, electron microscopy, ROS measurement, autophagy detection, Western blot analysis, and lactate concentration measurement. Bioinformatics analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction network analyses were performed on the transcriptome data.

Results: Heat stress significantly reduced SC viability, induced oxidative stress and autophagy, and altered gene expression profiles. We identified 1231 significantly differentially expressed genes, with significant enrichment in membrane-related processes and metabolic pathways. Metabolism-related genes, including PKLR, ACOT11, and LPCT12, were significantly downregulated. Protein-protein interaction network analysis revealed ten hub genes potentially crucial in the heat-stress response: HSP90AA1, HSPA5, BAG3, IGF1, HSPH1, IL1A, CCL2, CXCL10, ALB, and CALML4.

Conclusions: This study provides comprehensive insights into the molecular mechanisms underlying goat SC response to heat stress. The identified genes and pathways, particularly those related to metabolism and stress response, offer potential targets for developing strategies to mitigate heat-stress effects on livestock reproduction. These findings contribute to our understanding of climate change impacts on animal husbandry and may inform the development of heat-stress resistant livestock lines.

Keywords: Sertoli cell; autophagy; goat; oxidative stress; transcriptome analysis.

Figure 1

The isolation and identification of primary goat SCs. (A) Bright-field microscopy image of primary SCs. (B) H&E staining of primary SCs. (C) Oil Red O staining of primary SCs; black arrows indicate red lipid droplets. (D,E) Immunofluorescence staining of SC-specific proteins. (D) Vimentin (green fluorescence); (E) WT1 (red fluorescence). Cell nuclei were counterstained with DAPI (blue fluorescence). Scale bars are shown in the bottom right corner of each image.

Figure 2

The effects of heat stress on goat testicular SC viability and ultrastructure. (A) CCK-8 analysis of SC viability at different temperatures. (B) The impact of different heat-stress durations on SC viability. (C) The effect of various recovery times on SC viability after heat stress. Data are presented as mean ± SEM (n = 6). ** p < 0.01 compared to control. (D) Transmission electron microscopy images of SCs before and after heat stress. Left: control group at 32 °C; right: heat-stress group at 44 °C. Scale bars are shown in the bottom right corner of each image. N: Nucleus; M: Mitochondria; RER: rough endoplasmic reticulum; LD: lipid droplets; ASS: autophagic lysosome; AP: autophagosome.

Figure 3

Heat stress induces ROS production and autophagy in goat testicular SCs. (A) Representative fluorescence images of ROS levels in SCs at 32 °C and after heat-stress treatment. Green fluorescence indicates DCFH-DA probe staining. Scale bars are shown in the bottom right corner of each image. (B) The quantification of relative fluorescence intensity (OD488/525 nm) in the control and heat-stress groups. ** p < 0.01. (C) Representative fluorescence images of autophagy levels in SCs at 32 °C and after heat-stress treatment. Green fluorescence indicates MDA probe staining. Scale bars are shown in the bottom right corner of each image. (D) The quantification of relative fluorescence intensity (OD335/512 nm) in the control and heat-stress groups. ** p < 0.01. (E) Western blot analysis of autophagy-related proteins p62 and LC3-I/LC3-II. β-actin was used as an internal reference.

Figure 4

Heat stress induces gene expression changes and pathway enrichment in SCs. (A) A volcano plot of DEGs. Red dots represent upregulated genes in the heat−stress group, while blue dots represent downregulated genes. (B) RT−qPCR validation of DEGs. The bar graph shows the expression levels of 5 upregulated and 5 downregulated genes, randomly selected. *** p < 0.001. (C) A heatmap of the top 40 upregulated and 57 downregulated genes with the most significant expression changes. Color intensity indicates gene expression levels in the control and heat-stress groups. (D) GO enrichment analysis of DEGs. Shows the most significantly enriched biological processes, cellular components, and molecular functions. (E) KEGG pathway enrichment analysis of DEGs. Displays the most significantly enriched signaling pathways.

Figure 5

Heat stress activates multiple metabolic pathways in goat testicular SCs and affects lactate production. (A) A metabolic pathway map of DEGs based on iPath3 website analysis. Red lines indicate metabolic pathways activated by heat stress. (B) A bar graph of DEGs related to various metabolic processes in the heat-stress group. Shows expression changes in genes associated with glucose metabolism, lipid metabolism, amino acid metabolism, energy metabolism, and nucleotide metabolism. (C) The effect of heat stress on lactate content in SCs’ culture supernatant. The bar graph compares lactate concentrations between the control and heat-stress groups. *** p < 0.001.

Figure 6

STRING interaction analysis of all differentially expressed genes DEGs between 32 °C and heat-stressed SCs. Protein–protein interaction network visualized using STRING, with interactions shown at a confidence level of 0.4, while edges between nodes indicate various types of interactions, color-coded and defined in the figure legend. Isolated nodes without edges were removed from the visualization.

Figure 7

Core genes related to heat-stress injury associated with SCs. Node scores were calculated using Cytoscape software, and the top 10 nodes ranked by node scores were selected.