Abstract Male infertility has emerged as a global issue, partly attributed to psychological stress. However, the cellular and molecular mechanisms underlying the adverse effects of psychological stress on male reproductive function remain elusive. We created a psychologically stressed model using terrified-sound and profiled the testes from stressed and control rats using single-cell RNA sequencing. Comparative and comprehensive transcriptome analyses of 11,744 testicular cells depicted the cellular landscape of spermatogenesis and revealed significant molecular alterations of spermatogenesis suffering from psychological stress. At the cellular level, stressed rats exhibited delayed spermatogenesis at the spermatogonia and pachytene phases, resulting in reduced sperm production. Additionally, psychological stress rewired cellular interactions among germ cells, negatively impacting reproductive development. Molecularly, we observed the down-regulation of anti-oxidation-related genes and up-regulation of genes promoting reactive oxygen species (ROS) generation in the stress group. These alterations led to elevated ROS levels in testes, affecting the expression of key regulators such as ATF2 and STAR, which caused reproductive damage through apoptosis or inhibition of testosterone synthesis. Overall, our study aimed to uncover the cellular and molecular mechanisms by which psychological stress disrupts spermatogenesis, offering insights into the mechanisms of psychological stress-induced male infertility in other species and promises in potential therapeutic targets. Keywords: MT: Bioinformatics, single-cell RNA sequencing, psychological stress, spermatogenesis, reproductive damage Graphical abstract graphic file with name fx1.jpg [41]Open in a new tab __________________________________________________________________ Yang and colleagues revealed the cellular and molecular mechanisms underlying the interference of psychological stress with spermatogenesis and the initiation of reproductive impairments. These findings contribute insights into the etiology of psychological stress-induced male infertility across diverse species, while also holding promise as prospective therapeutic targets. Introduction Male infertility is a common condition that affects at least 7% of men worldwide and is on the rise.[42]^1 Moreover, male infertility is the sole factor in 20%–30% of infertility cases, but the pathogenesis of 70% of male infertility cases remains unclear.[43]^2 Currently, numerous studies have shown that stress is an important factor affecting the physical and mental state of healthy individuals and can disturb the internal homeostasis of the body. With the development of society and the increase of competitive pressure in all aspects of life, long-term stress can affect the physiological functions of the human body, including adverse effects on metabolism, immune function, the nervous system, the cardiovascular system, and the reproductive system, leading to disease.[44]^3^,[45]^4 Psychological stress has also emerged as one of the important potential risk factors affecting male fertility.[46]^4^,[47]^5^,[48]^6^,[49]^7 It has been reported that severe physical and psychological stress under the stimulation of external factors can cause a decrease in the quality of gonads such as prostate, seminal vesicles, testes, and epididymis, as well as a disturbance in the secretion of sex hormone levels. This impaired the function of the male gonadal axis, resulting in abnormal sperm production and decreased quality.[50]^8 Psychological stress may also lead to significantly lower plasma testosterone (T) levels,[51]^9^,[52]^10 thereby reducing sperm count and motility.[53]^11 In addition, studies have shown that the sperm quality of patients with male infertility caused by psychological stress improved significantly after psychotherapy, indicating that psychological stress is an important factor contributing to male infertility,[54]^12^,[55]^13 but the mechanisms involved have not been thoroughly described. The development of single-cell omics has greatly facilitated the exploration of specific mechanisms in male infertility. As one of the key technologies in single-cell omics research, single-cell transcriptome sequencing is currently widely used. Since Tang et al.[56]^14 completed the first single-cell RNA sequencing (scRNA-seq) in 2009, scRNA-seq has been continuously improved and has become a common method for whole-cell expression analysis with the emergence of some large commercial platforms.[57]^15 Over the past decade, scRNA-seq has also been widely used to study gene expression patterns of spermatogenesis and to reveal the dynamic process and key regulatory factors of spermatogenesis, which produces mature male gametes.[58]^16^,[59]^17^,[60]^18^,[61]^19^,[62]^20 Especially, single-cell resolved transcriptome profiling can greatly promote the exploration of the specific effects of chronic psychological stress on the complex process of spermatogenesis. Furthermore, animal studies have shown that psychological stress can disrupt the function of the hypothalamic-pituitary-testis axis by reducing the release of luteinizing hormone and follicle-stimulating hormone (FSH),[63]^21^,[64]^22 resulting in testicular cell apoptotic death.[65]^23 Our previous study also revealed that male rats exposed to psychological stress showed slow growth, reduced sperm quality, and abnormal levels of reproductive endocrine hormones.[66]^24 Understanding the effects of psychological stress on the infertility of animal models can provide strong support for the discovery of similar mechanisms in humans. Therefore, in this work, we used our previously created terrified-sound stressed model in Sprague-Dawley (SD) rats (Rattus norvegicus)[67]^24 and performed scRNA-seq of the testicular tissues from the stressed and normal rats ([68]Figure 1A). Through the comparative and comprehensive analyses of the transcriptomes of the two groups, we revealed the specific mechanism by which the psychological stress of terrified-sound affects the process of spermatogenesis and ultimately leads to reproductive damage in male rats. The findings provide a theoretical basis for the study of psychological stress on male reproductive impairments for other animals and humans, and further promise in finding therapeutic targets for male infertility caused by psychological stress. Figure 1. [69]Figure 1 [70]Open in a new tab The single-cell atlas for spermatogenesis of rats (A) The schematic view of the experimental workflow. (B) Temporary slide microscopy shows lower sperm count in the caudal epididymis of psychologically stressed rats compared with controls. (C) The UMAP plot shows testicular cell distributions of the normal and stressed rats (n = 3 for each group). Each dot represents a testicular cell and is colored according to its sample source, wherein C1–C3 refers to three rats from the control group, and S1–S3 refers to the three rats from the stressed group. (D) The UMAP plot shows testicular cell distributions of the normal and stressed rats (n = 3 for each group). Each dot represents a testicular cell and is colored according to its group source. (E) The UMAP plots show the 11 clusters with annotated cell types for normal (n = 3) and stressed (n = 3) rats, respectively. Each dot represents a testicular cell and is colored according to its cell type. (F) The top 25 differentially expressed genes and their enriched GO terms for each cell cluster. (G) Comparison of distribution profiles for each cell attribute across 11 cell types. Results Single-cell transcriptomics delineates the cellular landscape of spermatogenesis To investigate the effect of terrified-sound psychological stress on reproductive damage in male rats, we constructed a model of terrified-sound psychological stress with male SD rats. Histologically, we observed (by H&E staining) a reduction in the number of germ cells in the testes of rats subjected to terrified-sound psychological stress ([71]Figure S1A). The sperm density was also decreased in rats in the stress group ([72]Figure 1B). To further explore the effect of terrified-sound on the testis of male rats, we performed scRNA-seq on testicular tissues from three control and three terrified-sound-stressed male rats, respectively ([73]Figure 1A). A total of 9,000 and 8,998 cells for the control group and the stress group, respectively, were captured from the raw data. After quality control (see [74]materials and methods for details), 11,744 cells (6,596 cells from the control group and 5,148 cells from the stress group) ([75]Table S1) and 23,170 genes were retained for subsequent analyses. Next, we calculated the cell cycle score for each cell. Principal-component analysis (PCA) plots show a good overlap between different periods ([76]Figure S2A). UMAP plots show that different cell types contain various periods of the cell cycle ([77]Figure S2B). These findings indicate that our data have a lower cell cycle dependency. Therefore, our subsequent analysis was not corrected for cell cycle effects. After batch correction with Harmony[78]^25 ([79]Figures 1C and 1D), we built the cell atlas of rat spermatogenesis with 11 cell clusters using unsupervised clustering. To assign identities to the clusters, we determined the expression of known cell-type-specific marker genomes and identified 11 germ cells at various stages ([80]Figures 1D and 1E), covering the sperm differentiation progression: Undifferentiated spermatogonia (Undiff_SPG), differentiated spermatogonia (Diff_SPG), primary spermatocytes (Pri_SPT), leptotene, zygotene, pachytene, diplotene, second secondary meiotic cells (Meiosis_II), round sperm cells (RoundSpermatid), elongated sperm cells (Elongating), and sperm cells. We confirmed the correct clustering assignment by evaluating the expression of specific marker genes ([81]Figure S2C; [82]Table S2). Furthermore, we performed Gene Ontology (GO) enrichment of the marker genes for each cell cluster, which further confirmed the assigned cell types ([83]Figure 1F). In addition, we further examined the distribution of cell types in each sample and quantified their relative proportions at different stages of spermatogenesis ([84]Figure S1E). To characterize our cellular atlas, we show the proportion of mitochondrial-encoded RNA, the total number of genes detected, the total number of unique transcripts, and the proportion of cells from control vs. stress in each cell type ([85]Figure 1G). During spermatogenesis, mitochondria undergo significant changes in morphology, size, localization, and number. Most of the mitochondrial DNA of male germ cells is eliminated during spermatogenesis, and mitochondrial DNA is reduced to one-tenth of its initial number.[86]^26 It has been reported that the reduction of mitochondrial DNA copy number in sperm may reduce the frequency of mitochondrial DNA damage. We found that mitochondrial mRNA levels gradually decreased with the spermatogenesis process. In particular, the mitochondrial mRNA level decreased significantly after entering meiosis. This is consistent with previous reports. Furthermore, the total number of genes detected, as well as the total number of unique transcripts, did not vary significantly across most cell types. But these are reduced in sperm cells compared with other cells. Psychological stress induces cell-type-specific transcriptomic alterations during spermatogenesis To understand how psychological stress with terrified-sound will affect the transcriptome rat germ cells, we performed differential expression analysis and functional enrichment analysis for each cell type during spermatogenesis ([87]Figure S3; [88]Table S3). We found that most differentially expressed genes (DEGs) are specific to a single cell type (819 up-regulated genes and 768 down-regulated genes), indicating the cell-type-specific effects of psychological stress. In addition, there are also many DEGs (both the up- and down-regulated genes) that are shared across multiple cell types, including 48 up-regulated genes and 62 down-regulated genes shared by at least two cell types. Functional enrichment analysis of the commonly up-regulated genes revealed significant changes in reactive oxygen species (ROS)-related processes, including “regulation of nitric-oxide synthase activity,” “positive regulation of ROS metabolic process,” etc. ([89]Figure 2B). A list of representative ROS genes shows significant up-regulation after psychological stress ([90]Figure 2C), including Lcn2 (lipocalin 2) and Mif (macrophage migration inhibitory factor), which are related to ROS metabolism, and Acvr2a (activin A receptor type 2A), Apoe (apolipoprotein E), which are related to nitric oxide (NO) synthesis.[91]^27^,[92]^28 Notably, in addition to the up-regulated ROS genes in the stress group, we also found ROS scavenging-related genes such as Gstp1, Gsr, etc.[93]^29 were down-regulated in the stress group. These results imply that external psychological stress may cause excessively elevated ROS levels in the testes of rats to further induce intracellular oxidative stress (OS). Numerous previous studies have shown that OS will reduce fertility through lipid peroxidation, sperm DNA damage, and apoptosis.[94]^30^,[95]^31^,[96]^32 Intriguingly, we also found that the commonly up-regulated genes were enriched in DNA damage-related processes such as "intrinsic apoptotic signaling pathway in response to DNA damage." Figure 2. [97]Figure 2 [98]Open in a new tab Transcriptomic alterations of the testicular germ cells (A) The shared and cell-type-specific DEGs between the control and stressed rats. (B) The enriched GO biological processes of the shared up-regulated (top) or down-regulated (bottom) DEGs between the control and stress groups. (C) The differential expressions of the representative genes for given biological processes. (D) The volcanoplots of the cell-type-specific DEGs after psychological stress. (E) Ten enriched GO terms for up-regulated cell-type-specific DEGs. (F) Ten enriched GO terms for down-regulated cell-type-specific DEGs. Moreover, we also investigated those cell-specific DEGs ([99]Figure 2D). GO enrichment analyses were performed on all up-regulated and down-regulated DEGs, respectively ([100]Figures 2E and 2F). Correspondingly, we noticed DNA damage repair-related enrichments such as "nucleotide-excision repair" in the stressed group, wherein some important genes (Rap2, Slx4, Dclre1a, and Rad52) were significantly down-regulated after psychological stress ([101]Figure 2C). Altogether, the psychological stress of terrified-sound may cause enhanced ROS generation ability and weakened the scavenging ability of rat testis. This leads to OS, which in turn affects some biochemical reactions during spermatogenesis, such as DNA damage repair, apoptosis, etc. Psychological stress disrupts the cellular dynamics of spermatogenesis To further reveal the stress-induced effects on cellular transitions during the course of rat spermatogenesis, we investigated the transient cellular dynamics by RNA velocity analysis. We set undifferentiated spermatogonia (Undiff_SPG) as the starting point of spermatogenesis. The velocity streamlines show that spermatogenesis in the control samples starts from Undiff_SPG cells and then directs to differentiated spermatogonia (Diff_SPG), primary spermatocytes (Pri_SPT), leptotene, zygotene, pachytene, diplotene, second meiotic cells (Meiosis_II), round spermatids (RoundSpermatid), elongated spermatids (Elongating), and finally to spermatids. In contrast, for the stressed group, we observed apparent differentiation arrest during the differentiation of diplotene to pachytene cells ([102]Figure 3A). Besides, latent time suggested that this differentiation arrest may also exist at the transition from spermatogonia to primary spermatocytes ([103]Figure S4A). Additionally, the pseudotime analysis confirmed that the psychological stress retarded the course of spermatogenesis in the stress group as early as in Diff_SPG cells and then the diplotene phase during the differentiation ([104]Figure 3B). Figure 3. [105]Figure 3 [106]Open in a new tab Psychological stress induces spermatogenesis retardations (A) RNA velocities derived from scVelo kinetic models for normal and stress groups. The streamlines show significant changes between the control and stress groups, indicating spermatogenesis retardation at differentiated spermatogonia and diplotene phase. (B) The cumulative plots of pseudotime highlight spermatogenesis retardations at differentiated spermatogonia and diplotene phases. Each dot represents a cell. (C) The functional enrichment of hypervariable genes for normal and stress groups. (D) Temporal expression of ROS-related genes over differentiation course. (E) Representative ROS genes that are differentially expressed between control and stressed groups. To further evaluate the underlying changes in gene expression throughout spermatogenesis subjected to psychological stress, we performed functional enrichment analysis on the chronologically hypervariable genes over the pseudotime ([107]Figure 3C). Interestingly, we again observed significant enrichments in biological processes related to OS for both groups, such as "cellular response to OS," "response to hydrogen peroxide," etc. Besides, there are some enriched processes related to abiotic stimuli, such as "cellular response to abiotic stimulus" and "cellular response to environmental stimulus." Moreover, changes in some genes with pivotal roles for "chromosome condensation," "protein folding," and "regulation of protein stability" in the stress group may be more helpful to understand the specific mechanism of sperm damage in male rats caused by psychological stress ([108]Figure 3C). Consistent with the group-wise comparisons, the temporally hypervariable genes were also differentially enriched in the biological processes related to ROS between the control and stress groups ([109]Figure S4B). We found that the enrichments of "cellular response to OS," "protein folding," and other related functions in the control group were more significant than that in the stress group. The enrichments of functions such as "cellular response to abiotic stimulus" and "cellular response to environmental stimulus" were more significant in the stress group. Furthermore, we performed a trajectory-based differential expression analysis of genes specifically enriched in "cellular response to OS" ([110]Figure S4C). We examined the expression of these oxidative stress-related genes over the differentiation course. We found that the expression of genes related to ROS generation, such as Fos and F3, was up-regulated, while the expression of genes encoding antioxidant proteins, such as Hspa8, Nqo1, and Prdx2, was down-regulated. This further points to the fact that psychological stress may retard spermatogenesis by raising ROS in rat testes ([111]Figures 3D, 3E, and [112]S4D). Psychological stress retards spermatogenesis at the spermatogonia and pachytene phases by elevating ROS To further explore the phase-specific effects of psychological stress on different segments of spermatogenesis, we divided the course of spermatogenesis into three successive phases: the spermatogonia phase (including Undiff_SPG, Diff_SPG, and Pri_SPT cells), the meiosis phase (including Leptotene, zygotene, pachytene, diplotene, and Meiosis_II cells), and the sperm phase (including RoundSpermatid, Elongating, and Sperm cells). We revisited the cellular dynamics of each phase in more detail. We first reclustered cells from the spermatogonia phase into more subpopulations and annotated them as "UnSPG.1," "UnSPG.2," "DiSPG.1," "DiSPG.2," "DiSPG.3," "DiSPG.4," and "SPT" ([113]Figures 4A and [114]S5A). RNA velocity analysis revealed "UnSPG.1" as the starting point of the differentiation process. Velocity streamlines indicate that samples in the stress group differ from the control group when "DiSPG.1" points to "DiSPG.2." "DiSPG.1" in the stress group seemed to have more cells flowing toward and into a similar direction to "UnSPG.2" ([115]Figure 4A). We assessed the cellular composition at the spermatogonia phase and found an increased number of cells in "UnSPG.2," "DiSPG.1," "DiSPG.2," and "DiSPG.3" in the stress dataset. However, "UnSPG.1" cells at the beginning of differentiation and "DiSPG.4" at the end of differentiation at the spermatogonia phase, as well as "SPT" cells, were reduced compared with the control dataset ([116]Figure S5B). We speculate that spermatogenesis in male rats after stress is blocked during the differentiation of "DiSPG.1" into "DiSPG.2." Additionally, we performed differential expression analysis for each cell type during the spermatogonia phase ([117]Table S4). Then, we performed gene set enrichment analysis on the DEGs of "DiSPG.1" cells. We found that the “response to hydrogen peroxide” pathway was down-regulated in the stress group ([118]Figure S5C). Notably, HDAC6, GPX1, and STAR genes were all down-regulated in the stress group. Among them, HDAC6 and GPX1 encode antioxidant proteins.[119]^33^,[120]^34 Their down-regulated expression may be one of the reasons accounting for the excessive ROS caused by psychological stress. Moreover, it is interesting to note that high ROS levels lead to down-regulation of STAR expression, resulting in the inhibition of testosterone synthesis, which in turn leads to reproductive toxicity.[121]^35 Figure 4. [122]Figure 4 [123]Open in a new tab Spermatogonia and pachytene retardation in stressed rats (A) Streamline plot of RNA velocity for the spermatogonia phase. Retardation in streamline flows can be observed in the stress group. (B) Streamline plot of RNA velocity for the meiosis phase. Retardation in streamline flows can be observed in the stress group. (C) Twin heatmaps showing normalized expression of oxidative stress (OS)-related genes identified by trajectory-based differential expression analysis at the spermatogonia phase. These genes are differentially expressed in the stress group. Cells are ordered by pseudotime, with normal cells toward the left and stressed cells toward the right. The arrows indicate the differentiation directions. (D) Twin heatmaps showing normalized expression of OS-related genes identified by trajectory-based differential expression analysis at the meiosis phase. These genes are differentially expressed in the stress group. Cells are ordered by pseudotime, with normal cells toward the left and stressed cells toward the right. The arrows indicate the differentiation directions. (E) The heatmap shows the pairwise comparisons of activities for the selected regulator at the spermatogonia phase. Regulon activities are represented as the scaled AUC scores (area under the recovery curve, defined by SCENIC). (F) The heatmap shows the pairwise comparisons of activities for the selected regulator at the meiosis phase. Regulon activities are represented as the scaled AUC scores (area under the recovery curve, defined by SCENIC). Similarly, we also performed RNA velocity analysis for the meiotic phase, wherein cells from the control group differentiate from "zygotene" to "pachytene", and then from "pachytene" to "diplotene." In the stressed group, the flows of the velocity streamlines were opposite that of the control group ([124]Figure 4B), which indicates that the process of rat spermatogenesis was retarded in meiosis after the psychological stress of terrified-sound. In addition, we also performed the same RNA velocity analysis for the sperm phase. However, no significant difference was observed between the stressed and control groups ([125]Figure S6A). To further reveal the upstream regulatory basis of the DEGs and the hypervariable genes between the stress and control groups, we used SCENIC to identify “regulons,” of which each is a group of genes collectively regulated by a specific transcription factor. Overall, we identified 255 regulons in the two datasets, each containing up to 4,999 genes. We performed functional enrichment analysis on these 255 regulators and found the GO term “cellular response to OS” ([126]Figure S5D). To assess potential changes in gene expression observed during spermatogenesis in stressed samples, we used trajectory-based differential expression analysis. Cells were aligned along the latency time, and cell annotation indicated a sequence of spermatogenesis progression ([127]Figures 4C and 4D). We found that the expression of genes that promote ROS production, such as Fos, were up-regulated during the spermatogonia phase and down-regulated during the meiosis phase in the stressed group. Furthermore, the results of the SCENIC analysis showed that "DiSPG.1" cells displayed significantly enhanced activation of four regulons in stressed samples: ATF2 (77 genes), XBP1 (20 genes), BRF2 (106 genes), and RELA (71 genes) ([128]Figure 4E). Relatively, the RELA regulon was significantly activated in "pachytene" cells, while the ATF2 and BRF2 regulons were inactivated during the meiosis phase ([129]Figure 4F). Notably, these four regulators did not show this trend in other cell types during the spermatogonia phase. They even exhibit down-regulation of activity during early and late differentiation. Additionally, among the genes regulated by ATF2, we identified RELA. However, other transcription factors do not appear to be mutually regulated. Interestingly, activation of ATF2 regulon was significantly enhanced in “DiSPG.1” cells, but the expression of the gene itself was down-regulated ([130]Figure S6B). According to the reported studies, elevated levels of ROS do lead to a decrease in ATF2 expression. However, ATF2 and its regulated genes act as a regulon, and its increased activity may cause unfavorable spermatogenesis, such as apoptosis.[131]^36^,[132]^37^,[133]^38 Altogether, the above results suggest that psychological stress could cause rat spermatogenesis retardation, specifically at the spermatogonia and pachytene phases, by elevating the ROS. Psychological stress rewires testicular interactions in a manner detrimental to reproductive development The process of spermatogenesis is very complex but is highly organized, involving a series of intricate cell-cell interactions between germ cells.[134]^39 We then investigated the cell-cell communications between germ cells to reveal how psychological stress could affect the testicular interactions during spermatogenesis. Compared with the control group, the number and strength of interactions between germ cells in the testes of the stress group showed attenuation ([135]Figure 5A). In particular, compared with the control group, we noticed that the number and strength of interactions between pachytene cells and zygotene, diplotene, and other cells were significantly reduced ([136]Figure 5B). We then identified differences in signaling pathways enriched for ligand-receptor pairs linking cell interactions between the two groups. We enriched these signals into four clusters according to the functional similarity of signaling pathways ([137]Figure 5C). Notably, activin signaling may help regulate spermatogonia self-renewal and differentiation. Compared with the control group, activin signaling was significantly reduced in the stress group, especially in the interaction between Diff_SPG cells and Pri_SPT cells ([138]Figure 5E). This is consistent with previous work.[139]^40 Additionally, the KIT signaling pathway, which is also known to play a key role in maintaining the balance of spermatogonia self-renewal and differentiation, was significantly reduced in the stress group ([140]Figure 5D). Moreover, VEGF, EGF, WNT, and other pathways were down-regulated in the stress group, and these pathways all contributed to development, proliferation, and differentiation. In particular, the number and strength of interactions between the VEGF signaling pathway and Undiff_SPG with other cells were significantly reduced ([141]Figure 5E). Furthermore, signaling pathways related to the immune system were also altered in the stress group, including a weakening of the CD6 signaling pathway and an enhancement of the complement signaling pathway ([142]Figure S6D). Figure 5. [143]Figure 5 [144]Open in a new tab Global alterations of the germ cell interactions induced by psychologic stress (A) Both the CellChat-inferred interaction number (left) and strength (right) between germ cells are attenuated by psychological stress. (B) Circular network diagrams depicting differences in the interaction number (left) or strength (right) in the cell-cell communication network between control and stress groups. Red or blue edges represent increased or decreased signals in the stress group compared with the control group, respectively. (C) Inferred differential signaling networks clustered by functional similarity. Each dot represents the communication network of one signaling pathway. The dot size is proportional to the overall communication probability. Different colors represent different clusters of signaling pathways. Different shapes represent different groups. (D) The representative information flow for the control (red) and stress (cyan) groups. (E) The selected differential signaling networks inferred by CellChat. The edge width represents the communication probability. (F) Psychologic stress significantly altered the sending and receiving of signals for different cell types. We also identified cell clusters that varied significantly between the two groups for sending or receiving signals. We found that all cell types in the stress group had significantly lower signal strengths in both sending and receiving. Compared with different cell types in each group, unlike the control group, Undiff_SPG, Diff_SPG, Pri_SPT, and Leptotene cells that were earlier in the spermatogenesis process became the main source of sending and receiving signals in the stress group ([145]Figure 5F). Altogether, these results suggest that psychological stress perturbed the cell-cell interactions between testicular germ cells in a manner detrimental to reproductive development. Discussion Male infertility has become a global problem, for which psychological stress has been proven to be an important cause. In this work, we pioneered the study using our previously built rat model to reveal the putative mechanism by which psychological stress causes defective spermatogenesis and reproductive damage ([146]Figure 6). We performed single-cell transcriptome profiling on 11,744 rat testicular cells to provide normal and defective cellular references of spermatogenesis