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Hyperhomocysteinemia lowers serum testosterone concentration via impairing testosterone production in Leydig cells

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Abstract

Hyperhomocysteinemia (HHcy) plays a salient role in male infertility. However, whether HHcy interferes with testosterone production remains inconclusive. Here, we reported a lower serum testosterone level in HHcy mice. Single-cell RNA sequencing revealed that genes related to testosterone biosynthesis, together with nuclear receptor subfamily 5 group A member 1 (Nr5a1), a key transcription factor for steroidogenic genes, were downregulated in the Leydig cells (LCs) of HHcy mice. Mechanistically, Hcy lowered trimethylation of histone H3 on lysine 4 (H3K4me3), which was bound on the promoter region of Nr5a1, resulting in downregulation of Nr5a1. Intriguingly, we identified an unknown cell cluster annotated as Macrophage-like Leydig cells (McLCs), expressing both LCs and macrophages markers. In HHcy mice, McLCs were shifted toward pro-inflammatory phenotype and thus promoted inflammatory response in LC. Betaine supplementation rescued the downregulation of NR5A1 and restored the serum testosterone level in HHcy mice. Overall, our study highlights an etiological role of HHcy in LCs dysfunction.

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Acknowledgements

We thank Professor Xuejiang Guo (State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Jiangning District, Nanjing, Jiangsu, China) for advice on experiments to detect animal subfertility.

Funding

This work was supported by grants from Nature and Science Foundation of China (81730019, 81521003, 82090020), Nature and Science Foundation of Guangdong province (2019B1515120075) and Outstanding Scholar Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110102004) to Dr. Jing Nie, grant from the Nature and Science Foundation of China (81900609) to Dr. Wenjing Lei.

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  1. Zhiyuan Su, Zhuoliang Liu, Wenjing Lei and Kai Xia contribute to this work equally.

Authors and Affiliations

  1. The State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Key Laboratory of Organ Failure Research (Ministry of Education), Division of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Zhiyuan Su, Zhuoliang Liu, An Xiao, Zheng Hu, Miaomiao Zhou, Fengxin Zhu, Jianwei Tian, Manqiu Yang & Jing Nie

  2. Department of Nephrology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230001, Anhui, China

    Wenjing Lei

  3. Center for Stem Cell Biology and Tissue Engineering, Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China

    Kai Xia & Andy Peng Xiang

  4. National-Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, Guangdong, China

    Kai Xia & Andy Peng Xiang

  5. Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, Guangdong, China

    Dong Wang

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Contributions

JN, ZS, ZL, WL and KX conceived the project and designed the study. ZS performed in vivo and in vitro experiment and data analysis with the assistance of WL. ZL conducted the scRNA-seq analysis and visualization with the assistance of DW. ZH, MZ, JT and MY provided reagents and technique support. JN, ZS, ZL and AX drafted the manuscript. ZS, ZL, KX, FZ, APX and JN edited and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jing Nie.

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Additional file 1: Fig. S1.

Tracking of serum testosterone in control and hyperhomocysteinemia (HHcy) mice. (A) Testosterone levels in serum (n≥5). Data are shown as mean ± SEM. Significance between the groups at the end of feeding according to two-tailed unpaired t test. *p<0.05. Fig. S2. Effect of HHcy on testes and epididymis. (A) Histopathology of testes. Scale bars 50 μm. (B) Abnormal tubule rate in testes (n=9). (C) Histopathology of epididymis. Scale bars 100 μm. (D) Malondialdehyde (MDA) level in the testes (n≥6). (E-G) RT-qPCR analysis of genes, Sod3 (E), Cat (F) and Mpo (G) in testes (n=6). The values were normalized to Actb. Data are shown as mean ± SEM. Significance according to two-tailed unpaired t test. *p<0.05, **p<0.01, ***p<0.001. Fig. S3. Assessment of cholesterol levels and the expression of cholesterol metabolism-related genes. (A) Total cholesterol (TC) levels in serum (n=10). (B) TC levels in testes (n=8). (C) RT-qPCR analysis of genes related to cholesterol metabolism in testes (n=6). The values were normalized to Actb. (D) RT-qPCR analysis of genes related to cholesterol metabolism in primary LCs (n=3). The values were normalized to Actb. Data are shown as mean ± SEM. Significance according to two-tailed unpaired t test. Fig. S4. scRNA-Seq profiling of cellular functions in testis. (A) Dot plots showing the enriched GO terms of each cell type. Fig. S5. Analysis of marker genes in the C02_LC. (A) Violin plots displaying expression level of LCs markers (Hsd3b1, Cyp17a1, Star and Cyp11a1) and spermatids markers (Prm2 andTnp2) in C01_LC, C02_LC and C17_ST. Fig. S6. Regulation of Nr5a1 by H3K4me3 and H3K36me3 in mice testis. (A) Schematic diagram showing the H3K4me3 and H3K36me3 binding sites around the Nr5a1 promoter region obtained from the UCSC genome browser (version mm9). Fig. S7. Hcy-induced alterations in factors regulating H3K4me3. (A) Ratio of SAM and SAH in testes of mice (n=9). Data represent mean ± SEM and analyzed with two-tailed unpaired t-test. (B) The mRNA expression levels of Kmt2a, Ash1l, Kmt2e and Kmt2c in LCs. Data was shown as mean ± standard deviation (SD) and analyzed with Wilcoxon rank sum test. (C) RT-qPCR analysis of Kmt2a, Kmt2e, Kmt2c and Ash1l in MLTC-1 cells treated with 400 μM Hcy for 60 hours (n=3). The values were normalized to Actb. Data are shown as mean ± SEM. Significance according to two-tailed unpaired t test. Fig. S8. Features and differences of LCs, McLCs and macrophages. (A) Violin plots displaying the distribution of detected gene numbers, total UMIs per cell and the percentage of mitochondrial genes. (B) Band-Altman plot (MA plot, log2 (Fold-Changes) versus the average expression) displaying the DEGs between McLCs and macrophages. Red dots represent genes highly expressed in McLCs and blue dots represent highly expressed in macrophages (|log2Fold-Change|>2). LCs markers labelled with red font; macrophages markers labelled with by blue font. (C) Band-Altman plot displaying the DEGs between McLCs and LCs. (D) Boxplot showing the AUCell scores of macrophage-associated GO-BP terms in three cell clusters. The comparison was based on the Wilcoxon rank sum test. ****p < 0.0001. Fig. S9. Intercellular communications from LCs and macrophages to LCs in mice testis. Sankey plot of intercellular communications from LCs to LCs in control and HHcy group. (B) Sankey plot of cellular communications from macrophages to LCs in control and HHcy group. Fig. S10. Distribution of Bhmt, Mthfr and Mtr expression in cell populations. (A) tSNE plots showing Bhmt, Mthfr and Mtr expressed in cell populations. LCs were denoted in circles. Table S1. Characteristics of the phenotype of mice. Table S2. Assessment of fertility of mice. Table S3. Luteinizing hormone (LH) levels in the serum. Table S4. Marker genes for each cell cluster. Table S5. Primer sequences for qPCR. (PDF 62311 KB)

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Su, Z., Liu, Z., Lei, W. et al. Hyperhomocysteinemia lowers serum testosterone concentration via impairing testosterone production in Leydig cells. Cell Biol Toxicol 39, 3077–3100 (2023). https://doi.org/10.1007/s10565-023-09819-4

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  • DOI: https://doi.org/10.1007/s10565-023-09819-4

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