High Vulnerability of Brandt’s Voles to Heatwaves: 39°C as a Threshold for Male Reproductive Dysfunction

1. Introduction

Rising global temperatures pose a significant threat to wild animal health and population persistence [1,2]. In male mammals, spermatogenesis is highly temperature sensitive and requires scrotum maintained approximately 2–4°C below core body temperature. Scrotal temperature is easily elevated by the environment, which can disrupt spermatogenic homeostasis, resulting in impaired sperm quality and male infertility [3]. Brandt’s vole (Lasiopodomys brandtii) is a representative small mammal species of the Eurasian temperate steppe. It dominates the typical steppes of Inner Mongolia (China), Mongolia, and the Trans-Baikal region of Russia, where its population dynamics exert substantial influences on grassland structure and ecosystem functioning [4]. In the temperate steppe of Inner Mongolia, regional warming has accelerated to approximately 0.37°C per decade, which is three times the global average [5,6]. Extreme temperatures reached 42.3°C in 2021, as recorded by the Inner Mongolia Meteorological Observatory. In reality, ambient temperature is measured inside a shaded instrument shelter (Stevenson screen) at a standard height above the ground. However, the actual temperature within a few centimeters of the ground is significantly higher. It is precisely this near-surface microclimate temperature that influences Brandt’s vole. Climate change has been closely associated with changes in the population dynamics and geographical distribution of prairie-dwelling small mammals, including Brandt’s vole, with these changes being heavily driven by reproductive capacity [7,8,9]. Due to its high sensitivity to thermal stress, Brandt’s vole provides an ideal model for investigating how climate warming affects wild small mammals.

Endoplasmic reticulum (ER) stress signaling plays a critical role in spermatogenesis, sperm maturation, and fertilization [10]. Although heat stress is widely recognized to impair male reproductive function, the molecular mechanisms linking heat stress, ER stress, and reproductive dysfunction in wild small mammals remain poorly understood. Here, we hypothesize that climate warming impairs male reproductive function in Brandt’s voles via ER stress. Using integrated transcriptomic and proteomic analyses, we aim to elucidate the molecular basis of heat-induced testicular dysfunction in Brandt’s voles.

2. Materials and Methods

2.1. Experimental Animals

Adult male Brandt’s voles (body weight: 45 ± 5.5 g; age: 9–10 weeks) were utilized for this study. These animals were the offspring of a wild-captured population originally sourced from the grasslands of Xilinhot, Inner Mongolia, China (43° 02’–44° 52’ N, 115° 13’–117° 06’ E). Voles were housed individually in a controlled environment maintained at a temperature of 22 ± 2°C and relative humidity of 35%–50%, under a 12:12 h light/dark cycle. Animals were provided ad libitum access to standard laboratory chow and water, with daily monitoring for health status and sanitary cage maintenance. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Approval No. ZZUIRB2023-008) and were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals of China.

2.2. Heat Stress Treatment and Sample Collection

Male Brandt’s voles were randomly assigned to four experimental groups (n=12 per group): a control group (Ctrl) maintained at room temperature, and three heat stress groups subjected to temperatures of 37°C (C37), 39°C (C39), and 41°C (C41), respectively. Prior to heat exposure, all animals were anesthetized via intraperitoneal injection of 5 mg/kg Xylazine (J329BA0031, Sangon Biotech, Shanghai, China). Based on an established protocol [11]. Heat stress was induced by immersing the lower abdomen and scrotum in a water bath at the corresponding temperature for 30 min. Following treatment, animals were allowed to recover at room temperature for 24 h. The animals were subsequently euthanized, and the testes were immediately dissected and weighed. For histological analysis, intact testes were fixed in a specialized testis fixative (Servicebio, Wuhan, China). For molecular analysis, testicular tissues were snap-frozen in dry ice and stored at -80 °C until RNA and protein extraction.

2.3. Histological Analysis

Following fixation, testes were dehydrated and embedded in paraffin. Six micrometer-thick sections were prepared using a microtome (Thermo Fisher Scientific, San Jose, CA, USA) and subjected to standard hematoxylin and eosin (H&E) staining protocols. Slides were then scanned and documented using an Olympus VS200 SLIDEVIEW imaging system to evaluate histological alterations.

2.4. Biochemical Analysis of Oxidative Stress

Testicular tissues were homogenized in an ice-water bath using 0.9% NaCl at a mass-to-volume ratio (w/v) of 1:5 to 1:10. The resulting homogenates were centrifuged at 8000 × g for 10 min at 4°C, and the supernatants were collected for subsequent biochemical analysis. The activities of catalase (CAT) and superoxide dismutase (SOD), as well as the concentrations of malondialdehyde (MDA) and reduced glutathione (GSH), were quantified using assay kits (Solarbio Science & Technology Co., Ltd., Beijing, China; Cat. Nos. BC4780, BC5160, BC0020, and BC1170).

2.5. Transmission Electron Microscopy

Testicular tissues were primarily fixed in 0.1 M cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde and 1.4% sucrose at 37°C for 1.5 h. Samples underwent post-fixation in 1% osmium tetroxide and were subsequently dehydrated and embedded in Epon-812 resin. Ultra-thin sections (90 nm) were prepared using an ultramicrotome and double-stained with uranyl acetate and lead citrate. Testicular ultrastructure was visualized using a TEM (JEM-1400, JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 80 kV.

2.6. Quantitative Real-Time PCR Analysis

Total RNA was isolated from testicular tissue using the Trizol reagent (Solarbio Science & Technology Co., Ltd., China; Cat. No. P1011). RNA concentration and purity were assessed using a NanoDrop spectrophotometer. First-strand cDNA was synthesized using the HiScript® III All-in-One RT SuperMix Perfect kit (Vazyme Biotech Co., Ltd., China; Cat. No. R333-01). Quantitative real-time PCR was performed using the SYBR green method on an Applied Biosystems 7500 real-time PCR system. Species-specific primers for Brandt’s voles were synthesized by Sangon Biotech (Shanghai, China), and all primer sequences are provided in supplementary Table S1.

2.7. RNA Sequencing and Data Analysis

Transcriptome sequencing was performed by Feisha Biotechnology (Wuhan, China). Raw sequence data were quality-filtered using fastp (v1.0.1) to remove low-quality reads and adapter sequences. High-quality clean reads were assembled de novo using Trinity (v2.5.1). Temporal or pattern-based gene expression profiles were analyzed using the Mfuzz R package (v2.60.0) with default fuzzy c-means clustering parameters. Differentially expressed genes (DEGs) were identified using a threshold of fold change ≥ 2 and P-adj ≤ 0.01. The False Discovery Rate (FDR) was applied to correct for multiple testing. Functional annotation of DEGs was conducted against multiple databases, including the NCBI non-redundant (NR) protein sequences, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), Swiss-Prot, and Pfam.

2.8. Liquid Chromatography Tandem Mass Spectrometry and Data Analysis

Testicular protein expression profiles were characterized using data-independent acquisition (DIA) mass spectrometry performed with Orbitrap Astral mass spectrometer (Thermo Scientific) provided by SanshuBio Co. Ltd. (Shanghai, China). Samples were analyzed in DIA mode. Raw MS data were processed using DIA-NN (v1.9). Protein identifications were filtered at a 1% FDR at both the precursor and protein levels. Differentially expressed proteins (DEPs) were functionally annotated against GO and KEGG databases using eggNOG-mapper.

2.9. Statistical Analysis

Data are presented as the mean ± SEM. Statistical evaluations were conducted using GraphPad Prism (version 9.1.0, GraphPad Software, San Diego, CA, USA). For comparisons between two groups, an unpaired Student’s t-test was employed. A value of P < 0.05 was defined as the threshold for statistical significance.

3. Results

3.1. Heat Stress Impaired Testicular Histological Structure

As illustrated in Figure 1 and 2A. Following heat exposure at 37°C, 39°C, and 41°C, a significant reduction in both testicular size and index was observed (P < 0.05). Morphological assessment of spermatozoa revealed normal structures in the Ctrl group, whereas heat-stressed groups exhibited marked sperm abnormalities, including absent tail, multi-headed, and coiled-tail phenotypes (Figure 2B). Histological examination of the Ctrl group revealed a healthy testicular architecture characterized by well-organized spermatogonia layers and abundant luminal spermatozoa. In contrast, the heat stressed groups displayed progressive morphological deterioration, including vascular congestion, irregular seminiferous tubule contours, and a disrupted spermatogenic epithelium (Figure 2C). Furthermore, ultrastructural analysis via TEM demonstrated that heat stress induced significantly impaired mitochondrial integrity and pronounced swelling of the ER (Figure 2D).

3.2. Heat Stress Induced Oxidant Stress in Testis

The effects of heat stress on the antioxidant capacity and lipid peroxidation of testes are summarized in Figure 3. Compared to the Ctrl group, C39 and C41 groups showed significantly reduced GSH content (P < 0.05), whereas no significant alterations were observed in the C37 group (Figure 3A). Conversely, SOD activity was upregulated in the C39 and C41 groups (P < 0.05) but remained unchanged under the 37°C treatment (Figure 3C). Interestingly, MDA levels showed no fluctuation across all heat-stressed groups (P > 0.05; Figure 3B). Regarding CAT activity, only the C41 group showed a reduction (P < 0.05) (Figure 3D).

3.3. Heat Stress Disturbed the Barrier Function in Testis

Compared to the Ctrl group, claudin 1 (Cldn1) mRNA abundance remained unchanged in the C37 and C39 groups, but was significantly upregulated in C41 group (P < 0.05; Figure 4A). In contrast, heat stress induced a robust inhibitory effect on the expression of occludin (Ocln) and tight junction protein 1 (Tjp1) (P < 0.05; Figure 4B,C).

3.4. Heat Stress Induced ER Stress in Testis

The expression profiles of key ER stress markers were evaluated at both the transcriptional (Figure 5) and translational (Figure 6) levels. Compared to the Ctrl group, heat stress resulted in a downregulation of DNA-damage inducible transcript 3 (Ddit3) and glutamine-rich 1 (Qrich1) mRNA abundance (P < 0.05; Figure 5A,B). Notably, the mRNA levels of activating transcription factor 4 (Atf4) and X-box binding protein 1 (Xbp1) were significantly upregulated only in the C41 group, whereas no significant changes were observed at C37 and C39 group (Figure 5C,D).

To validate these findings, the protein abundance was analyzed via western blotting (Figure 6A). Consistent with the transcriptional data for the C41 group, the protein expression of ATF4, ATF6B, α-subunit of eukaryotic initiation factor 2 (eIF2α), and XBP1 was elevated (P < 0.05; Figure 6B–E). Similar to the mRNA patterns, heat stress at 37°C and 39°C did not induce a significant increase in this protein abundance.

Figure 6. Analysis of ER stress-related protein expression in testicular tissue of Brandt’s voles. Representative Western blot images of ATF4, ATF6B, eIF2α and XBP1(A). Quantitative analysis of relative protein levels for ATF4 (B), ATF6B (C), eIF2α (D) and XBP1 (E) in the testicular tissue of Brandt’s voles. Data are expressed as mean ± SEM (n = 6 per group). ns means no significance, * means P < 0.05, ** means P < 0.01.

Figure 6. Analysis of ER stress-related protein expression in testicular tissue of Brandt’s voles. Representative Western blot images of ATF4, ATF6B, eIF2α and XBP1(A). Quantitative analysis of relative protein levels for ATF4 (B), ATF6B (C), eIF2α (D) and XBP1 (E) in the testicular tissue of Brandt’s voles. Data are expressed as mean ± SEM (n = 6 per group). ns means no significance, * means P < 0.05, ** means P < 0.01.

3.5. Results of RNA-Sequencing Data Filtering

Following stringent quality control, the proportion of high-quality filtered reads ranged from 99.92% to 99.93% (supplementary Table S2). The sequencing depth yielded between 57.02M and 91.85M clean reads per sample, with a minimum Q30 score of 92.97%. A total of 673474 transcripts were identified, and the average mapping rate of reads to the reference genome was 86.66% (supplementary Tables S3 and S4).

3.6. Functional Annotation

350367 unigenes were successfully annotated in at least one database (supplementary Table S5). Specifically, 63463 unigenes were identified in the NCBI NR database, followed by 60107 in Swiss-Prot, and 59484 in Pfam. Furthermore, functional classification and pathway analysis assigned 57631 unigenes to GO terms, 43964 to eggNOG clusters, and 40680 to KEGG pathways. Specialized annotations included 12637 unigenes in TMHMM, 9130 in SignalP, and 3271 in the CAZy database.

Table S5. Unigenes annotations.

Databases Annotation	Number of Unigenes
Nr	63463
Swiss-Prot	60107
KEGG	40680
Gene_Ontology	57631
eggNOG	43964
Pfam	59484
CAZy	3271
SignaIP	9130
TMHMM	12637

Table S5. Unigenes annotations.

Databases Annotation	Number of Unigenes
Nr	63463
Swiss-Prot	60107
KEGG	40680
Gene_Ontology	57631
eggNOG	43964
Pfam	59484
CAZy	3271
SignaIP	9130
TMHMM	12637

3.7. Identification of Differentially Expressed Genes

In the C37 group, 1344 DEGs were identified, comprising 645 upregulated and 699 downregulated genes. The C39 group exhibited the highest transcriptional response with 3248 DEGs (1876 upregulated; 1372 downregulated), while the C41 group contained 1580 DEGs (763 upregulated; 817 downregulated) (supplementary Figure S1A). Overlap analysis via a Venn diagram revealed a core set of 340 common DEGs consistently altered across all three heat treatments (supplementary Figure S1B). The distribution and significance of these expression profiles were further visualized through volcano plots for each pairwise comparison (supplementary Figure S1C–E).

3.8. DEGs Cluster Analysis Based on Temperature Course

As shown in Figure 7, these clusters, ranging in size from 6,919 to 11,893 genes. Cluster 1 (6,919 genes) remained at basal levels at 37°C but showed a transient peak at 39°C before declining at 41°C. In contrast, genes in clusters 2, 4, and 5 exhibited V-shaped trajectories, characterized by a sharp downregulation at 37°C or 39°C followed by a robust induction at 41°C. Cluster 3 genes demonstrated a progressive, temperature-dependent upregulation throughout the 37-41°C. Conversely, cluster 6 genes were consistently repressed across all thermal treatments. Clusters 7 and 9 displayed inverted V-shaped profiles, with marked transcriptional enhancement at 37°C or 39°C followed by a subsequent reduction. Finally, cluster 8 was characterized by an initial upregulation at 37°C followed by a decrease at 39°C.

3.9. GO and KEGG Enrichment Analysis of DEGs

GO analysis revealed that 833, 1,734, and 995 DEGs were categorized into 54, 53, and 52 functional subcategories in the C37 (supplementary Figure S2A), C39 (supplementary Figure S2B), and C41 (supplementary Figure S2C) groups, respectively. Predominantly enriched terms included “response to stimulus”, “membrane part”, and “antioxidant activity”. KEGG pathway mapping further identified 1311, 2412, and 1536 DEGs associated with diverse biological functions. These were classified into six primary KEGG categories, with the highest DEG distribution observed in environmental information processing, organismal systems, and human diseases (supplementary Figure S3).

3.10. Validation of DEGs with qRT-PCR

To verify the accuracy of the RNA-sequencing findings, 15 representative DEGs involved in ER stress, inflammation, protein quality control, and metabolic regulation were selected for qRT-PCR validation (supplementary Figure S4). Consistent with the transcriptomic profiles, the mRNA expression levels of CCAAT enhancer binding protein delta (Cebpd), Suppressor of cytokine signaling 3 (Socs3), Heat shock protein beta-1 (Hspb1), Activating transcription factor 3 (Atf3), Membrane associated ring-CH-type finger 10 (Marchf10), Spermatogenesis associated 21 (Spata21), Meiosis specific nuclear structural 1 (Mns1), SEC11 homolog A (Sec11a), Coiled-coil domain containing 33 (Ccdc33), Prostaglandin-endoperoxide synthase 2 (Ptgs2), IQ motif and ubiquitin domain containing gene (Iqub), and Uromodulin (Umod) were significantly upregulated across the heat-stress cohorts. Conversely, Patatin-like phospholipase domain-containing 3 (Pnpla3), GLI Family Zinc Finger 1 (Gli1), and Aquaporin 2 (Aqp2) exhibited marked downregulation following thermal exposure. Although the magnitude of fold changes varied slightly between the two platforms, the directional trends of gene expression were highly concordant. These results provide robust validation of the transcriptomic data and confirm the reliability of the sequencing-based expression profiles.

3.11. Protein Identification and Quantification

A total of 5,808 proteins were high-confidently identified across three biological replicates (supplementary Table S6).

Table S6. Overview of protein and peptide identification and quantification.

Sample	Identified Peptides	Quantified Peptides	Identified Protein	Quantified Protein
Ctrl_1	67725	51407	5808	5298
Ctrl_2	67725	55594	5808	5465
Ctrl_3	67725	54412	5808	5446
C39_1	67725	54421	5808	5394
C39_2	67725	55591	5808	5531
C39_3	67725	55773	5808	5518

Principal component analysis (PCA) revealed that the first two components accounted for 56.3% of the total variance, with the PC1 clearly separating the Ctrl and C39 group (supplementary Figure S5).

Table S6. Overview of protein and peptide identification and quantification.

Sample	Identified Peptides	Quantified Peptides	Identified Protein	Quantified Protein
Ctrl_1	67725	51407	5808	5298
Ctrl_2	67725	55594	5808	5465
Ctrl_3	67725	54412	5808	5446
C39_1	67725	54421	5808	5394
C39_2	67725	55591	5808	5531
C39_3	67725	55773	5808	5518

Principal component analysis (PCA) revealed that the first two components accounted for 56.3% of the total variance, with the PC1 clearly separating the Ctrl and C39 group (supplementary Figure S5).

Hierarchical clustering analysis further confirmed these results (Figure 8).

Differentially expressed proteins (DEPs) were identified using a fold change ≥ 2.0 and P_adj < 0.01. A total of 394 DEPs were identified between the two groups (supplementary Figure S6). Among these, 233 proteins were upregulated and 161 proteins were downregulated.

3.12. Functional Categorization Analysis

The DEPs were predominantly localized to protoplasm, cytoplasm, cytoplasmic granule lumen, cytoplasmic granule membrane, cytoplasmic granule (supplementary Figure S7).

Within the Biological process category, DEPs were significantly enriched in reproduction. Cellular component analysis revealed high enrichment in the nuclear chromosome, golgi membrane, and ubiquitin ligase complexes. In the molecular function category, proteins associated with nucleotide binding and transcription cis-regulatory region binding predominated (supplementary Figure S8).

KEGG pathway mapping further contextualized the proteomic response to heat stress (supplementary Figure S9A,B). Significant enrichment was observed in the MAPK signaling pathway and tight junctions. Notably, stratified analysis of directional expression revealed that Heat Stress-Up DEPs were primarily associated with protein processing in the ER, neurotrophin signaling pathway, and Salmonella infection pathways. Conversely, Heat Stress-Down DEPs were predominantly enriched in pathways related to aldosterone-regulated sodium reabsorption and various cancer-related signaling modules (supplementary Figure S9C).

4. Discussion

As a representative steppe rodent, Brandt’s vole provides an ideal model for examining how climate warming compromises the fertility of wild small mammals [12]. We identified ER stress as a central mediator of heat-induced testicular dysfunction in Brandt’s voles. These findings establish a mechanistic framework linking climate warming to male reproductive vulnerability in wild small mammals. Spermatogenesis is strictly temperature dependent, and germ cells with high mitotic activity are particularly vulnerable to heat stress [13]. Consistent with previous studies [14,15,16], we observed that in Brandt’s voles, a single 30-min heat exposure at 39°C or 41°C was sufficient to markedly reduce the testicular index, induce structural sperm abnormalities, and cause widespread degeneration of the seminiferous epithelium (Figure 1 and Figure 2A–C). Heat stress is known to exacerbate ROS production, leading to oxidative stress when antioxidant defenses are overwhelmed. Although MDA levels did not increase significantly, the increasing trend across heat-stressed groups indicates an early shift toward oxidative damage (Figure 3B). Heat exposure also caused a marked depletion of GSH, reflecting its rapid consumption in ROS scavenging and impaired redox homeostasis [17]. In parallel, SOD activity increased, whereas CAT activity declined, indicating a disruption of the antioxidant cascade (Figure 3C,D). As SOD converts superoxide radicals into H₂O₂, reduced CAT activity would limit H₂O₂ detoxification, favoring its accumulation. Given that H₂O₂ is a potent inducer of testicular cell apoptosis under heat stress, this imbalance likely underlies the reduced testicular index and histopathological damage observed at 39°C and 41°C [18]. The blood–testis barrier (BTB), formed by specialized junctions between Sertoli cells, is essential for maintaining the microenvironment required for spermatogenesis. In Brandt’s voles, heat stress markedly suppressed the expression of Ocln and Tjp1, consistent with Sertoli cell dysfunction and BTB disruption reported in other rodent models (Figure 4B,C) [19]. Notably, Cldn1 expression was significantly upregulated (Figure 4A). We therefore propose that Cldn1 upregulation in Brandt’s voles reflects a cytoprotective but ultimately insufficient response to counteract BTB breakdown under heat stress.

To comprehensively characterize the testicular response to heat stress, we integrated transcriptomic and proteomic analyses. Transcriptome profiling revealed extensive heat-induced gene reprogramming, with functional enrichment predominantly associated with the ER and protein proteostasis pathways, including folding, sorting, and degradation (supplementary Figure S2). These transcriptional signatures were supported by ultrastructural evidence of ER swelling, indicating pronounced ER perturbation following heat exposure. Given the cellular heterogeneity of the testis, DIA-based quantitative proteomics was employed to resolve the executive molecular responses, focusing on the C39 group, which exhibited the most pronounced transcriptional shift (supplementary Figure S1A). Proteomic analysis identified differentially expressed proteins enriched within the endomembrane system and inner cell membranes, with functional annotations highlighting roles in reproduction and stress adaptation (supplementary Figures S8 and S9). The strong consistency among transcriptomic, proteomic, and ultrastructural evidence identifies ER stress as the central mediator of heat-induced testicular dysfunction in Brandt’s voles, providing a mechanistic framework for the reproductive impairment observed under heat stress.

The ER is central to cellular proteostasis, maintaining an oxidative environment essential for protein folding and post-translational modification. Heat-induced ROS impair ER folding capacity, while the accumulation of misfolded proteins further amplifies ROS generation, establishing a self-reinforcing cycle of cellular stress [20,21]. To counteract this stress, cells activate the UPR, a conserved signaling network that initially limits protein synthesis and enhances chaperone expression (Figure 6) [22]. The downregulation of Qrich1 and pro-apoptotic factor Ddit3 validates the activation of early protective mechanisms of the UPR (Figure 5A,B) [23,24]. This pattern suggests that germ cell loss in heat-stressed Brandt’s voles may proceed via alternative pathways, such as IRE1-dependent RNA decay or mitochondria-mediated apoptosis [25,26].

This study demonstrates that heat exposure severely disrupts testicular function in Brandt’s voles, leading to seminiferous degeneration and impaired sperm quality. Integrative omics analyses and protein abundance analyses reveal that oxidative stress–driven activation of ER stress is a central mechanism underlying this pathology. These findings highlight that 39°C represents a critical threshold for the reproductive vulnerability of Brandt’s voles even following short-time exposure.