Fig 1: The model of ATP production in Y-sperm and X-sperm under TLR7/8-ligand condition.In Y-sperm, mitochondrial and glycolytic ATP production occur regardless of the presence of TLR7/8 ligand (). Thus, Y-sperm showed high-speed motility. On the other hand, in X-sperm, the activation of TLR8 localized in the midpiece () suppressed mitochondrial ATP production. Additionally, the activation of TLR7 localized in the sperm tail () phosphorylated NF?B and GSK3a/ß and then suppressed the activity of hexokinase. As a result, ATP production in X-sperm was decreased under TLR7/8-ligand condition. Thus, only X-sperm showed low-speed motility under TLR7/8-ligand condition. GSK3a/ß, glycogen synthase kinase 3a/ß; TLR7/8, Toll-like receptor 7/Toll-like receptor 8; NF?B, nuclear factor-kappa B; X-sperm, X chromosome–bearing sperm; Y-sperm, Y chromosome–bearing sperm.
Fig 2: EPD suppressed the activation of the Toll-like receptor 7–myeloid differentiation primary response gene 88–nuclear factor-?B (TLR7/8–MyD88–NF-?B) signaling pathways. (A) The protein expressions of TLR7, TLR8, TRAF6, MyD88, p-IKKa, IKKa, p-IKBa, IKBa, p-NF-?B, and NF-?B were detected using Western blot assay. (B–H) The quantification of protein levels of TLR7, TLR8, TLAF6, MyD88, p-IKKa, IKKa, p-IKBa, IKBa, p-NF-?B, and NF-?B. Values are presented as the means ± SD of three independent experiments. ** P < 0.01 vs. control group, # P < 0.05, ## P < 0.01 vs. model group.
Fig 3: The expression of TLR7/8 encoded by the X chromosome in mouse sperm.(A) Presence of Tlr7/8 mRNAs in mouse sperm. mRNA was extracted from sperm and used for RT-PCR analyses using intron-spanning primer sets. To check the contamination of RNA extracted from epididymis in the sperm RNA sample, Hoxa1 and Hoxb1 were used as the marker of somatic cells. cDNA products were resolved on 2% (w/v) agarose gels. (B) Expression of TLR7/8 in mouse sperm. Sperm lysates were prepared for western analyses using an anti-TLR7 or anti-TLR8 antibody. ß- Actin was used as a loading control. Lysates prepared from spleen tissue were used as PCs. Results are representative of three biological experiments. (C,D) TLR7 histogram (C) and the density plot between TLR7 and acetylated tubulin (D) of mouse sperm. Sperm were incubated with the anti-TLR7 antibody conjugated with Alexa Fluor 647 and anti-acetylated tubulin antibody, which is a marker of sperm tail, and then were used for FCM. Dotted line and solid line indicated the borderline between positive cells and negative cells. (E) Percent of TLR7-positive and acetylated tubulin–positive sperm (TLR7+/Tubulin+) and TLR7-negative and acetylated tubulin–positive sperm (TLR7-/Tubulin+). The experiment was repeated with three biological replicates. Values represent the mean ± SEM of three replicates. Data associated with this figure can be found in the supplemental data file (S1 Data). (F,G) TLR8 histogram (F) and the density plot between TLR8 and acetylated tubulin (G) of mouse sperm. Sperm were incubated with the anti-TLR8 antibody and anti-acetylated tubulin antibody, which is a marker of sperm tail, and then were used for FCM. Dotted line and solid line indicated the borderline between positive cells and negative cells. (H) Percent of TLR8-positive and acetylated tubulin–positive sperm (TLR8+/Tubulin+) and TLR8-negative and acetylated tubulin–positive sperm (TLR8-/Tubulin+). The experiment was repeated three biological replicates. Values represent the mean ± SEM of three replicates. Data associated with this figure can be found in the supplemental data file (S1 Data). FCM, flow cytometry; TLR7/8, Toll-like receptor 7/Toll-like receptor 8.
Fig 4: The localization of TLR7/8 in mouse testis and sperm.(A) Localization of TLR7 in mouse testis. Cross-sections of mouse testes were stained with antibodies to visualize either TLR7 (red) or Sp56 (green), a marker of acrosome in round spermatid and sperm at 12 weeks. White lines delineate the border of the seminiferous tubule. Lower chart showed that the percent of Sp56 single positive cells or TLR7-Sp56 double positive cells in the seminiferous tubule. The number of positive cells in a section per testis was counted, and a total of three different samples were analyzed. Data associated with this figure can be found in the supplemental data file (S1 Data). *Leydig cell. Scale bar indicates 100 µm. (B) Localization of TLR8 in mouse testis. Cross-sections of mouse testes were stained with antibodies to visualize either TLR8 (red) or Sp56 (green), a marker of acrosome in round spermatid and sperm at 12 weeks. Lower chart showed that the percent of Sp56 single positive cells or TLR8-Sp56 double positive cells in the seminiferous tubule. The number of positive cells in a section per testis was counted, and a total of three different samples were analyzed. Data associated with this figure can be found in the supplemental data file (S1 Data). Scale bar indicates 100 µm. (C) Localization of TLR7 in mouse sperm collected from the epididymis. Sperm were collected from the epididymis into HTF medium and then air dried. Smears were incubated with the anti-TLR7 antibody and then a secondary antibody. P: the high magnification image of the sperm with a P signal of TLR7. N: the high magnification image of sperm with N for TLR7. Right figure showed that the percent of TLR7 positive/negative sperm. Scale bar indicates 10 µm. The number of positive sperm was counted, and a total of four different samples were analyzed. Data associated with this figure can be found in the supplemental data file (S1 Data). (D) Localization of TLR8 in mouse sperm collected from the epididymis. Sperm were collected from the epididymis into HTF medium and then air dried. Smears were incubated with the anti-TLR8 antibody and then a secondary antibody. P: the high magnification image of the sperm with a P signal of TLR8. N: the high magnification image of sperm with N for TLR8. Right figure showed that the percent of TLR8 positive/negative sperm. Scale bar indicates 10 µm. The number of positive sperm was counted, and a total of three different samples were analyzed. Data associated with this figure can be found in the supplemental data file (S1 Data). HTF, human tubal fluid; N, negative; P, positive; Sp56, formally known as a common name of zona pellucida 3 receptor (ZP3R); TLR7/8, Toll-like receptor 7/Toll-like receptor 8.
Fig 5: Paeonol decreases myeloid differentiation factor 88 (MyD88) and Toll-like receptor 8 (TLR8) proteins in the skin lesions of the psoriatic mouse model, and inhibits activation of the TLR7/8 signaling pathway. (A) The levels of MyD88 and TLR8 in paeonol-treated mice were assessed by western blotting. Immunoblotting showing the bands detected for various proteins. (B) Quantitation of A after normalization with ß-tubulin. Data are expressed as the mean ± SD (n=6/experiment). *p<0.05 and **p<0.01 vs. the mouse model.
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