The world is facing a growing health crisis with obesity, diabetes, and non-alcoholic steatohepatitis (NASH) on the rise. These conditions are not only affecting the overall fitness and well-being of individuals but also have a significant impact on reproductive health, particularly in men. Alarmingly, there is a positive correlation between metabolic diseases and male infertility, especially among younger populations. This correlation is primarily attributed to the overconsumption of high-calorie diets, which negatively affects male reproductive function.
Spermatogenesis, the process of sperm production, is governed by germ cells and Sertoli cells. Sertoli cells play a crucial role in providing nutrients, maintaining cellular junctions, and supporting the meiosis of germ cells. Recent studies have highlighted the importance of Sertoli cells in testicular function, and their impairment can lead to reproductive disorders.
Research has shown that a high-fat diet (HFD) can disrupt the balance of gut microbiota and metabolic homeostasis, ultimately leading to male infertility. This connection between gut microbiota and spermatogenesis has been confirmed through fecal microbiota transplantation (FMT) experiments. Additionally, previous studies have identified Vitamin A metabolism as a key player in the gut-testis axis, with its disruption hindering spermatogenesis in metabolic syndrome models.
L-citrulline, a non-essential amino acid synthesized in the small intestine, is a precursor to arginine and has been found to promote the recovery of spermatogenesis and improve sperm quality. In this study, we investigated the potential of L-citrulline as a crucial metabolite in the gut-testis axis to enhance the functionality of Sertoli cells.
Our findings revealed that a high-concentrate diet (HCD) caused DNA damage in Sertoli cells and disrupted arginine biosynthesis, resulting in a significant decrease in L-citrulline abundance. Furthermore, we discovered that L-citrulline, a metabolite derived from gut microbiota, can rescue DNA damage in Sertoli cells and enhance the blood-testis barrier (BTB) function, ultimately improving sperm quality induced by HCD. These results highlight the potential of L-citrulline supplementation as a promising strategy to address male infertility caused by HCD.
To investigate the impact of HCD on spermatogenesis, we established a HCD sheep model. We found abnormal testicular morphology and partial shedding of germ cells in the spermatogenic tubules of the HCD group. The expression of the marker gene SOX9, which is associated with Sertoli cells, was significantly reduced in the HCD group compared to the control group.
Sertoli cells are the exclusive somatic cells in the testicular microenvironment that interact directly with germ cells and play a crucial role in establishing the BTB. Therefore, we examined BTB-related adhesion genes in testicular tissue, specifically Claudin11 and Occludin. The expression of these genes was markedly reduced in the HCD group, indicating a disruption in the BTB structure.
To determine the mechanism responsible for the reduction of Sertoli cells caused by HCD, we performed scRNA-sequencing and analysis on sheep testes from both the control and HCD groups. We identified differentially expressed genes (DEGs) that were mainly enriched in processes related to reproductive processes, DNA metabolism, DNA biosynthesis, and regulation of ATP-dependent activity signaling pathways. Notably, within the genes associated with DNA metabolism and DNA biosynthesis, a significant proportion (36.21%) were dedicated to the regulation of DNA damage and repair mechanisms.
Further analysis revealed that L-citrulline exhibited the most significant alteration among the differential metabolites associated with arginine biosynthesis and arginine and proline metabolism. We also observed a high negative correlation between the levels of L-citrulline and DNA damage repair-related genes, Pcna and Gtf2h5. These findings suggested that L-citrulline metabolism was disturbed, closely associated with the DNA damage process of Sertoli cells in the HCD model.
Since the homeostasis of gut microbiota is essential for host metabolism, and citrulline is synthesized in the small intestine, we investigated the effect of gut microbiota on L-citrulline metabolism. We performed 16S rDNA analysis on small intestinal digesta samples and found that the gut microbiota profiles in the HCD group were significantly different from the control group. We identified 36 bacteria that exhibited alterations between the two groups, with most of these bacteria belonging to the Bacillota phylum.
To validate the disruption of gut microbiota homeostasis in the HCD group, we analyzed three differential bacterial genera: RuminococcaceaeNK4A214group, ChristensenellaceaeR-7group, and LachnospiraceaeNK3A20group. Spearman correlation analysis revealed that RuminococcaceaeNK4A214group and Flexilinea were significantly correlated with four metabolites associated with L-citrulline metabolism. Furthermore, functional prediction analyses indicated that these bacterial genera were significantly involved in L-citrulline metabolism, consistent with the findings from metabolomic enrichment analysis.
To further investigate the effect of gut microbiota on Sertoli cells development, we optimized the FMT from sheep in the control and HCD groups to mice. We observed abnormal morphology of the seminiferous tubule and partial detachment of germ cells in the HCD-FMT group compared to the Con-FMT group. Immunofluorescence staining of SOX9 revealed a significant reduction in the number of SOX9-positive cells in the seminiferous tubules of the HCD-FMT group. Additionally, we found a substantial decrease in SOX9 expression and a significant reduction in the expression of BTB-related genes Claudin11 and Occludin in the HCD-FMT group.
To test the hypothesis that L-citrulline is essential for Sertoli cell function, we performed culture experiments on a TM4 cell line, a Sertoli cell line. We observed a significant increase in reactive oxygen species (ROS) signaling in the sucrose group, whereas there was a notable decrease in the sucrose + citrulline group. Assessment using the MTT kit revealed that L-citrulline significantly improved the proliferation of Sertoli cells treated with sucrose. Evaluation of γH2AX protein levels indicated that L-citrulline significantly attenuated DNA damage in Sertoli cells.
Furthermore, we isolated and cultured primary Sertoli cells from mouse testicular tissue. Immunofluorescence staining for SOX9 confirmed high Sertoli cell purity. MTT assays demonstrated that L-citrulline significantly restored sucrose-impaired cell viability. Immunoblotting revealed reduced γH2AX levels in the sucrose + citrulline group, indicating attenuation of DNA damage. qPCR analysis further supported this finding, showing normalized expression of DNA repair genes. Importantly, L-citrulline upregulated tight junction-associated genes, suggesting enhanced BTB integrity.
Based on these findings, we propose that the administration of L-citrulline has a therapeutic effect on DNA damage in Sertoli cells and maintains BTB integrity in the HCD model. Therefore, we administered a 9-week treatment of L-citrulline via gavage in the HCD mouse model. The results demonstrated that the rescued group exhibited significantly elevated L-citrulline levels compared to the HCD group.
In addition, we found that spermatozoa morphology and count were significantly improved in the rescued group, with a significant decrease in the number of spermatozoa head losses, acrosome abnormalities, and tail curling. H&E staining showed well-organized seminiferous tubule morphology in the rescued group compared to the HCD group. Furthermore, IHC staining and Western blotting demonstrated a substantial enhancement in the SOX9-positive signal quantity in the rescued group.
ROS detection revealed a significant increase in ROS signaling in the HCD group, while it significantly decreased in the HCD + L-citrulline group. We assessed Claudin11 and Occludin expression in the testis and found that they were significantly improved in the rescued group. Additionally, the levels of γH2AX, Pcna, and Gtf2h5 were also improved in the rescued group.
In conclusion, our study provides compelling evidence that L-citrulline supplementation has beneficial effects on attenuating DNA damage in Sertoli cells. These findings not only advance the identification of clinical biomarkers for male infertility but also position dietary L-citrulline supplementation as a viable strategy to restore reproductive function. Further research is needed to explore the potential of L-citrulline as a targeted metabolite for treating male infertility caused by long-term high-concentrate diets.
