Effects of seminal vesicle fluid and prostate fluids on sperm motility

(A) To determine the effect of seminal plasma on sperm motility, we prepared “pseudo” seminal plasma from the mixture of seminal vesicle secretions or prostate extracts. (B-D) Tested various concentrations of these fluids on epididymal sperm: (B) Curvi-Linear Velocity; VCL, (C) Straight-Line Velocity; VSL and (D) Linearity; LIN is the ratio of VSL to VCL of sperm that was incubated with the mixture of seminal vesicle secretions and/or prostate extracts. (E) Experimental design to evaluate the effect of seminal secretions collected from male mice treated with or without flutamide (50 mg/kg subcutaneously for 7 days) on sperm. (F-H) Performed bioassay using seminal vesicle fluid after treatment with vehicle (Ctrl) or flutamide: (F) LIN, (G) VSL and (H) the activity of mitochondria were checked by JC-1 kit. Data are mean ± SEM. n=3-5 independent replicates. Significance was tested in comparison to HTF or Ctrl (vehicle-treated animals) using Student’s t-test.

The testosterone-androgen receptor pathway inhibits the cell proliferation of seminal vesicle gland epithelial cells.

(A) The localization of the androgen receptor (AR; NR3C4) in seminal vesicle of control (Ctrl) mice and those treated with flutamide (Fult). Scale bar=50 µm for left panel and 20 µm for right panel in each group. (B,C) Cell proliferation and apoptosis in seminal vesicle of control (Ctrl) mice and those treated with flutamide (Fult): (B) Representative images of Ctrl and Flut staining for Ki67 (top) and TUNEL (bottom). Scale bar=20 µm. (C) Percentage of Ki67 positive cells (top) and TUNEL positive (bottom) in Ctrl and Flut-treated seminal vesicle sections. (D) Experimental design to evaluate the effects of testosterone on the proliferation of seminal vesicle epithelial cells in vitro. The epithelial cells were culutured with or without testosterone for 8 days. (E) Growth curves of seminal vesicle epithelial cells that cultured with 0, 1, 10 and 100 ng/mL of testosterone. (F,G) Cell cycle status was determined by flow cytometry. Data are mean ± SEM. n=3-5 mice or independent replicates. Each replicate experiments with 3-6 wells containing pooled cells from 3-5 mice. Significance was tested in comparison to Ctrl using Student’s t-test. The cell proliferation data were compared to Ctrl by Dunnett’s test.

Testosterone changes the expression of genes involved in metabolic pathway in seminal vesicle epithelial cells.

(A) Volcano plot of differentially expressed genes. RNA sequencing was performed using RNA extracted from the seminal vesicle epithelial cells cultured with or without 100 ng/mL testosterone. Genes with a significant expression change are highlighted as red dots. It found 4460 significant genes for a cutoff of P<0.05. (B) MA plot of differential expression results. The seminal vesicle specific genes are highlighted as red dots rather than significant genes. (C) Top 10 genes upregulated or downregulated by testosterone, respectively, for altered gene expression. (D) Conducted KEGG analysis to identify differences in pathway enrichment by testosterone, identifying the most variability in metabolic pathway genes. It shows the number of differential expressed genes annotated to Gene Ontology shows (x axis). p-value which measure the statistical significance of a possible functional enrichment for each term.

Testosterone regulates glucose metabolism and mitochondrial ATP production in seminal vesicle epithelial cells.

(A-C) Glycolytic pathway analysis by an extracellular flux analyzer in seminal vesicle gland epithelial cells cultured with 100 ng/mL testosterone (Testo) or vehicle (Ctrl) for 7 days: (A) Extracellular acidification rate (ECAR) kinetics of seminal vesicle epithelial cells using an extracellular flux analyzer. (B) Glycolysis and (C) Glycolytic capacity. (D) Pyruvate concentration in the medium where seminal vesicle epithelial cells were cultured with or without testosterone for 24 hours. (E) Lactate concentration in the medium where seminal vesicle epithelial cells were cultured with or without testosterone for 24 hours. (F-J) Mitochondrial respiration measurement by an extracellular flux analyzer: (F) Oxygen consumption rate (OCR) kinetics of seminal vesicle epithelial cells with or without 100 ng/mL testosterone. (G) Basal OCR, (H) Maximum respiration, (I) Spare respiratory capacity and (J) ATP production. Data are mean ± SEM of n = 3 replicate experiments with 3-6 wells containing pooled cells from 3-5 mice or medium. Student’s t-test was used to compare Ctrl and Testo.

Effect of testosterone on gene expression of enzymes involved in the glucose metabolic pathway in seminal vesicle epithelial cells.

To elucidate the effects of testosterone on gene expression of enzymes involved in glucose catabolism and anabolism in seminal vesicle gland epithelial cells. ctrl: 7 days of culture with vehicle. testo: 7 days of culture with 100 ng/mL testosterone. Data are mean ± SEM of n = 3 replicate experiments with 3-6 wells containing pooled cells from 3-5 mice. Student’s t-test was used to compare Ctrl and Testo.

Oleic acid is incorporated to sperm, which enhances linear motility and mitochondrial activity.

(A) Epididymal sperm were incubated with fluorescently labeled oleic acid (OA) and the fluorescence intensity after quenching was observed by a fluorescence microscope. (B) The fluorescence intensity of fluorescence-conjugated OA in sperm was detected by flow cytometry. (C) Average fluorescence intensity of sperm at different concentrations of fluorescence-conjugated OA. (D,E) OA improved sperm motility parameters. Sperm collected from mouse epididymis was incubated with OA for 60 min: (D) Straight-Line Velocity; VSL and (E) Linearity; LIN (F) Increase in the Oxygen consumption rate (OCR) after treatment with 10 nM OA or 10 nM OA + 40 nM Etomoxir (Eto). (G) The percentage of ATP-Red positive sperm after 1 hour incubation with or without 10 nM OA, measured using BioTracker ATP-Red staining and flow cytometry. (H) Artificial insemination using sperm treated with “pseudo” seminal plasma from control (SV), flutamide-treated (Flu) mice and supplement of OA (Flu+OA) followed by an evaluation of the fertilization rates. Data are mean ± SEM. n=3-6 independent replicates. Student’s t-test was used for comparison between the two groups.

Testosterone-regulated ACLY induces metabolic shifts in seminal vesicle epithelial cells.

(A) Western blot images of ACLY and α/β-tubulin in three sets of seminal vesicle epithelial cells cultured with 100 ng/mL testosterone (Testo) or in vehicle (Ctrl) for 7 days. (B) Quantitative analysis of ACLY relative to α-tubulin obtained from Western blot. (C) siRNA knockdown experiments of ACLY in seminal vesicle epithelial cells. ACLY protein levels in scrambled shRNA or ACLY shRNA-transfected seminal vesicle epithelial cells cultured with or without 100 ng/mL testosterone were determined by Western blot. (D-F) Changes in oxygen consumption of seminal epithelial cells were analyzed by a flux analyzer: (D) Oxygen consumption rate (OCR) kinetics of seminal vesicle epithelial cells transfected with scrambled shRNA or ACLY shRNA. (E) Basal OCR and (F) Maximum respiration. (G) The impact of ACLY knockdown on testosterone-induced fatty acid synthesis was measured. (H) The effect of culture supernatants of testosterone-treated cells on sperm motility was evaluated, especially after ACLY knockdown. Curvi-Linear Velocity; VCL, Straight-Line Velocity; VSL, Linearity; LIN. Data are mean ± SEM. n=3-6 independent replicates. Repeated experiments were performed with cells recovered from 3 wells containing pooled cells from 3 to 5 mice. Student’s t-test was used for comparison between the two groups.

Testosterone regulates the metabolic activity of human seminal epithelial cells.

(A) Representative image of human seminal vesicle epithelial cells. (B) Growth curves of seminal vesicle epithelial cells with 100 ng/mL testosterone. (C) Oxygen consumption rate (OCR) kinetics and (D) Extracellular acidification rate (ECAR) kinetics of human seminal vesicle epithelial cells after 6 days of culture with or without 100 ng/mL testosterone using a flux analyzer. (E) Effects of testosterone on fluorescent glucose uptake and inhibitors of GLUT4 function. (F) Effects of testosterone and GLUT4 inhibitors on fatty acid synthesis. Data are mean ± SEM. n=3 independent replicates. Student’s t-test was used for comparison between the two groups.