Table 1.
Primers sequences used as target and reference genes used in qPCR reactions.
Table 2.
Antibody used as target and reference used in immunohistochemistry and Western Blot reactions.
Table 3.
Effects of NMN treatment on body weight, testis weight and semen quality of the mice.
Fig 1.
Effects of NMN intervention on testosterone, FSH and LH in mice exposed to aluminum.
A) Serum testosterone; B) Serum testosterone FSH; C) Serum LH. Testosterone (T), Follicle-stimulating hormone(FSH), luteinizing hormone (LH); **p < 0.01.
Fig 2.
Effect of NMN intervention on testis and epididymis of mice exposed to aluminum.
A) HE staining of mouse testis; B) HE staining of mouse epididymis; C) Diameter of mouse spermatogenic tubule; D)Mouse spermatogenic tubule area; E-F) Spermatogonium and spermatocyte count. **p < 0.01.
Fig 3.
Effect of NMN intervention on function of supporting cells in mice exposed to aluminum.
A–C) qPCR validation of NMN intervention effects on GATA4, WT-1 and Vimentin mRNA expression in aluminum-exposed mouse testes; D–G) Western Blotting assessment of NMN intervention effects on testicular supporting cell function in aluminum-exposed mice; H) Immunohistochemical detection of Vimentin, ZO-1, and Occludin expression in mouse testicular tissue; I–L) qPCR validation of NMN intervention effects on ZO-1, Occludin, TNF-α, and IL-6 in aluminum-exposed mouse testes. *p < 0.05, **p < 0.01.
Fig 4.
NMN Modulates Testis Gene Expression in Aluminum-Exposed Mice.
Testicular tissues from the control, high aluminum dose, and NMN intervention groups were analyzed through RNA sequencing. A) PCA analysis of testis gene expression is shown, with each point representing a sample. B) Volcano map comparison of gene expression between pairs (n = 3). C) Venn diagrams illustrate the differentially expressed genes between groups, and heat maps of the differentially expressed genes in clusters. D) Cluster analysis of genes related to the NOD-like receptor signaling pathway. E) KEGG pathway enrichment analysis of differentially expressed genes across the three groups.
Fig 5.
Effect of NMN intervention on NLRP3 and inflammation related genes in testis of mice exposed to aluminum.
A) Effect of NMN intervention on the expression of NLRP3,GSDMD, caspase-1 and IL-1β mRAN in the testis of aluminium-exposed mice; B) Western Blotting detected the effects of NMN intervention on the expression of NLRP3 and inflammation-related gene proteins in the testis of mice exposed to aluminum; C)Image J Analysis of the effects of NMN intervention on the expression of NLRP3,GSDMD, caspase-1 and IL-1β proteins in the testis of aluminium-exposed mice; D) Immunohistochemical detection of NMN intervention on the expression of NLRP3 and inflammation-related gene proteins in testis of aluminum-exposed mice.
Fig 6.
Effect of NMN Intervention on Marker Expression at Different Developmental Stages of Spermatogenic Cells in AlCl3-Exposed Mice.
Immunofluorescence staining was used to detect the expression of germ cell markers at different developmental stages. A) Shows that the pan-germ cell marker MVH was significantly downregulated in the AlCl3-exposed group, with expression restored in the AlCl3 + NMN group; B) The pro-meiotic marker STRA8 showed no significant differences among the control, AlCl3, and AlCl3 + NMN groups; C) Spermatocyte meiosis marker SCP3 showed significantly reduced expression in the AlCl3 group, with expression restored in the AlCl3 + NMN group following NMN intervention. Results indicate aluminum exposure primarily impacts the meiosis stage of germ cells, while NMN maintains spermatocyte meiosis integrity to exert protective effects.