Effects of seminal vesicle fluid and prostate fluids on sperm motility

(A) The effects of secretions from the prostate and seminal vesicles on sperm motility were directly compared. (B-F) The effects of secretions from the prostate (Pros) or seminal vesicles (SV) on the motility parameters of epididymal sperm were tested: (B) Motile, (C) Progressive motile, (D) Curvi-Linear Velocity; VCL, (E) Straight-Line Velocity; VSL and (F) Linearity; LIN is the ratio of VSL to VCL of sperm that was incubated with the mixture of seminal vesicle secretions or prostate extracts. (G) Viscosity of a solution containing prostate or seminal vesicle secretion with a protein concentration of 10 mg/mL. (H) Experimental design to evaluate the effect of seminal vesicle secretions collected from male mice treated with or without flutamide (50 mg/kg subcutaneously every day for 7 days) on sperm. Note that the donor mice for sperm and seminal vesicle secretions were different. (I-L) Performed bioassay using seminal vesicle fluid after treatment with vehicle (Ctrl) or flutamide: (I) Motile, (J) Progressive motile, (K) LIN, (L) VSL. (M) The high mitochondrial membrane potential (hMMP) was checked by the JC-1 kit. Antimycin (Anti) was used as the negative control for hMMP. Data are mean ± SEM. n=3-9 independent replicates. The viscosity measurement data were from n=3 repeated experiments using pooled prostate or seminal vesicle extracts from at least 20 mice. Percentage data were subjected to arcsine transformation before statistical analysis. (B-G, I-L) Significance was tested in comparison using Student’s t-test. (M) Since the results of the Bartlett test were significant, a two-way ANOVA using a generalized linear model was performed. Both the flutamide administration and antimycin addition were significant. The interaction was not significant. Games-Howell was performed as a post hoc test. Different letters represent significantly different groups.

The testosterone-androgen receptor pathway inhibits the cell proliferation of seminal vesicle 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 from 10× magnification for left panel and 20 µm from 20× magnification 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 (c), 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. Percentage data were subjected to arcsine transformation before statistical analysis. Significance was tested in comparison to Ctrl using Student’s t-test. The cell number data at d8 was compared with the control using 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) Extracellular acidification rate (ECAR) analysis by an extracellular flux analyzer in seminal vesicle epithelial cells cultured with 100 ng/mL testosterone (Testo) or vehicle (Ctrl) for 7 days: (A) ECAR kinetics of seminal vesicle epithelial cells using an extracellular flux analyzer. (B) Glucose induced ECAR and (C) Oligomycin-sensitive ECAR. (D,E) The concentrations of pyruvate and lactate in the culture supernatant. These measurements were normalized based on cell count and viability. (D) Pyruvate concentration in the medium where seminal vesicle epithelial cells were cultured with or without testosterone for 24 hours. (E) Lactate concentration. (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) FCCP uncoupled respiration, (I) Spare respiratory capacity and (J) Oligomycin-sensitive respiration. 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. The cells were normalized to 10,000 viable cells/well immediately before analysis. The viability of the cells before experiments was 86-93%.

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 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.

Testosterone enhances glucose uptake and fatty acid synthesis via GLUT4 trans-localization in seminal vesicle epithelial cells.

(A) The glucose uptake ability of seminal vesicle epithelial cells was detected using fluorescence-tagged glucose. The cells were cultured with 100 ng/mL testosterone and/or indinavir (100 µM) for 7 days and then further treated with glucose uptake probe-green for 15 min, and intracellular fluorescence was measured by flow cytometry (n=4). (B) Lipid accumulation in the medium where seminal vesicle epithelial cells were incubated for 24 hours (n=6). (C) Immunostaining of GLUT4 in flutamide-treated or vehicle mice derived seminal vesicle. Scale bar is 50 µm. (D) Western blot images of GLUT4 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. Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. (E) Fatty acid composition in the medium was analyzed using gas chromatography. (F) Quantification of Elovl family gene expression by RT-qPCR. (G) Pathway of glucose assimilation to oleic acid. Data are mean ± SEM. n=3-6 independent replicates. Each replicate experiments with 3-6 wells containing pooled cells from 3-5 mice or medium. (A,B) The glucose uptake and lipid measurements were normalized based on cell count and viability. A two-way ANOVA was performed. Both the Indinavir and testosterone treatment were significant. The interaction was significant. Tukey’s Honest Significant Difference test was performed as a post hoc test. Different letters represent significantly different groups. Student’s t-test was used for comparison between the two groups.

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-G) OA improved sperm motility parameters. Sperm collected from mouse epididymis was incubated with OA for 60 min: (D) Motile, (E) Progressive motile, (F) Straight-Line Velocity; VSL and (G) Linearity; LIN (H) Increase in the Oxygen consumption rate (OCR) of cultured sperm was measured using flux analyzer after treatment with 10 nM OA or 10 nM OA + 40 nM Etomoxir (Eto). Ctrl: the sperm cultured in HTF medium. Each well was seeded with 180 μL of NaHCO3-Free HTF medium containing 3,000,000 sperm. (I) 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. (J,K) The sperm, after 1 hour of incubation with or without 10 nM OA, were used for the IVF test and analyzed for the rate of oocytes reaching cleavage. and blastocyst stage. (J) Cleavage rate. (K) Blastocyst rate. (L) Artificial insemination using sperm treated for 1 hour with “pseudo” seminal plasma from control (Ctrl), flutamide-treated (Flu) mice and supplement of 10 nM OA (Flu+OA) followed by an evaluation of the fertilization rates. Note that the donor mice for sperm and seminal vesicle secretions were different. Data are mean ± SEM. n=3-11 independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons. Different letters represent significantly different groups. 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) Number of cells at 7 days after incubation. (E,F) Changes in oxygen consumption of seminal epithelial cells were analyzed by a flux analyzer: (E) Oxygen consumption rate (OCR) kinetics of seminal vesicle epithelial cells transfected with scrambled shRNA or ACLY shRNA. (F) Basal OCR. (G) The impact of ACLY knockdown on testosterone-induced fatty acid synthesis was measured. These measurements were normalized based on cell count and viability. The viability of the cells before experiments was 67-81%. (H-I) Fatty acid composition in the cultured supernatants was analyzed using gas chromatography. (H) Palmitate (C16:0). (I) Stearic acid (C18:0). (J) Oleic acid (C18:1). (K-N) The effect of culture supernatants of testosterone-treated cells on sperm motility was evaluated, especially after ACLY knockdown. (K) Motile. (L) Progressive motile. (M) Straight-Line Velocity; VSL. (N) 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. Percentage data were subjected to arcsine transformation before statistical analysis. (B) Significance was tested in comparison using Student’s t-test. (D,G-M) A two-way ANOVA was performed. Tukey’s Honest Significant Difference test was performed as a post hoc test. (D) Both the ACLY knockdown and testosterone treatment were significant. The interaction was significant. (G) Both the ACLY knockdown and testosterone treatment were significant. The interaction was not significant. (H,I) Only the ACLY knockdown were significant. The interaction was not significant. (J) Both the ACLY knockdown and testosterone treatment were significant. The interaction was significant. (F,N) Since the results of the Bartlett test were significant, a two-way ANOVA using a generalized linear model was performed. Games-Howell was performed as a post hoc test. (E) Only the ACLY knockdown were significant. The interaction was not significant. (N) Testosterone treatment were significant. The interaction was significant. Different letters represent significantly different 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 (Testo) or without (Ctrl). (C-H) Extracellular acidification rate (ECAR) kinetics and Oxygen consumption rate (OCR) kinetics of human seminal vesicle epithelial cells after 6 days of culture with or without 100 ng/mL testosterone using a flux analyzer. (D) Glucose induced ECAR and (E) Oligomycin-sensitive ECAR. (G) Basal OCR, (H) FCCP uncoupled respiration. (I) The glucose uptake ability of seminal vesicle epithelial cells was detected using fluorescence-tagged glucose. (J) Lipid accumulation in the medium where human seminal vesicle epithelial cells were incubated for 24 hours. (H-I) Fatty acid composition in the cultured supernatants was analyzed using gas chromatography. (H) Palmitate (C16:0). (I) Stearic acid (C18:0). (J) Oleic acid (C18:1). Data are mean ± SEM. n=3 independent replicates. Student’s t-test was used for comparison between the two groups. (C-J) These measurements were normalized based on cell count and viability. The viability of the cells before experiments was 88-96%. (I,K,L) Since the results of the Bartlett test were significant, a two-way ANOVA using a generalized linear model was performed. Games-Howell was performed as a post hoc test. Both the Indinavir and testosterone treatment were significant. The interaction was significant. (J) A two-way ANOVA was performed. Tukey’s Honest Significant Difference test was performed as a post hoc test. Both the Indinavir and testosterone treatment were significant. The interaction was significant. Different letters represent significantly different groups.

Metabolic changes in the seminal vesicle epithelial cells by testosterone improve the fertility of sperm.

Left part: Testosterone promotes glucose uptake by changes in the localization of GLUT4 in seminal vesicle epithelial cells. ATP-citrate-lyase (ACLY) activated by testosterone promotes fatty acid synthesis by skipping some tricarboxylic acid (TCA) cycle steps. Simultaneously, cell proliferation in the epididymal epithelial cells is suppressed. Testosterone also promotes the synthesis of oleic acid from acetyl-CoA synthesized by ACLY. Right part: Oleic acid, incorporated into the sperm midpiece, increases the oxygen consumption rate (OCR) in sperm mitochondria and increases ATP production. Therefore, oleic acid in seminal plasma is important for male fertility because it improves sperm linear motility and fertilization ability.

Sperm quality control data and exploratory research into the effects of seminal vesicle secretions on sperm fertilization

(A) The baseline information for the epididymal sperm used in Fig 1 and Supplemental Fig 2A. Curvi-Linear Velocity; VCL, Straight-Line Velocity; VSL and Linearity; LIN is the ratio of VSL to VCL of sperm incubated in HTF medium one hour. (B) Serum testosterone levels in seminal vesicle secretion donors. (C) A representative flow cytometry pattern of JC-1 staining of sperm that was not treated with drugs (HTF). (D) The effect of various drugs on the proportion of high mitochondrial membrane potential (hMMP; R7). Data are mean ± SEM. At least three independent replicates. (B) Significance was tested in comparison using Student’s t-test. (D) Percentage data were subjected to arcsine transformation before statistical analysis. Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons. Different letters represent significantly different groups.

Characteristics of the seminal vesicle in aging mice

(A) The effects of secretions from aged seminal vesicle fluid on sperm motility. Straight-Line Velocity; VSL, Motile, and Progressive motile. (B) Serum testosterone (T4) levels in young (3 months old) or aged (12 months) mice. (C) The high mitochondrial membrane potential (hMMP) was checked by the JC-1 kit. (D) Hematoxylin/Eosin (HE) staining in seminal vesicles of young and aged mice. Scale bar=50 µm for left panel and 20 µm for right panel. (E) Representative images staining for Ki67. Scale bar=50 µm. (F) The localization of the androgen receptor (AR; NR3C4) in the seminal vesicles of aged mice. Scale bar=50 µm. (E,F) Images were obtained at 10× magnification. Data are mean ± SEM. At least three independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. (A,B) Significance was tested in comparison using Student’s t-test. (C) A two-way ANOVA was performed. Tukey’s Honest Significant Difference test was performed as a post hoc test. Both the age and antimycin treatment were significant. The interaction was not significant. Different letters represent significantly different groups.

ACLY expression in in vivo seminal vesicle

(A) Representative Western blot images of ACLY and α-tubulin in three sets of seminal vesicle collected from 50 mg/kg flutamide subcutaneously for 7 days (Flut) or vehicle treated mice (Ctrl). (B) ACLY immunostaining: seminal vesicles from wild-type mice, flutamide-treated mice, and 12-month-old aging mice. Scale bar is 50 µm.

Oleic acid synthesis in vivo regulated by the testosterone-androgen receptor system

(A) Fatty acid composition of seminal vesicle extracts in flutamide-treated (Flu) or vehicle mice (Ctrl) pretreated with a methylation kit was analyzed by gas chromatography. *Moles contained per unit wight of seminal vesicle secretions. (B) Quantification of Elovl6 gene expression by RT-qPCR. Data are mean ± SEM. Student’s t-test was used for comparison between the two groups (n=5).

The effects of oleic acid and seminal vesicle secretions on sperm function

(A) Tracing of oxygen consumption rate (OCR) basal and injection with 10 nM oleic acid (OA) or OA + 40 nM Etomoxir (Eto). (B) The sperm, after 1 hour of incubation with or without seminal vesicle secretions (SV) from nontreated (Ctrl) or flutamide-treated mice(Flu), were used for IVF, and the cleavaged oocytes were observed. The absolute number of oocytes collected from the oviduct and the cleavage rate are shown. (C) Western blot analysis for phosphotyrosine (P-Tyr) in capacitated spermatozoa (Ctrl; 1-hour incubation in HTF medium) and treated with SV from healthy mice or 10 nM oleic acid (OA). (D) Quantitative analysis of GLUT4 relative to α-tubulin obtained from Western blot. (E) Flow cytometric analysis of the sperm after 1 hour incubation in control medium (HTF) and medium containing SV or 10 nM OA, using fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; to distinguish between acrosome-reacted and non-reacted cells) and propidium iodide (PI; to distinguish between dead and viable cells). (F) The percentage of viable sperm with an acrosome reaction (PI-, PNA-FITC+) was evaluated by flow cytometry in the 4th quadrant (4Q; red square). Data are mean ± SEM. At least three independent replicates. Percentage data were subjected to arcsine transformation before statistical analysis. (B) Dunnett’s test was used to analyze the cleavage rate. Different letters represent significantly different groups. (D,F) Differences between groups were assessed by one-way analysis of variance (ANOVA). When ANOVA was significant, differences among values were analyzed by Tukey’s Honest Significant Difference test for multiple comparisons.

Gating strategy of flow cytometry for sperm

(A) Gating strategy for the selection of single sperm. Using forward scatter (FSC)-A and side scatter (SSC)-A dot plots, similar size and complexity cells were firstly selected (R1). In FSC-A and FSC-H dot plots and FSC-A and FSC-W dot plots, similar-size cells were accumulated near area; thus, using these plots, again similar-size cells were selected (R2, R3). The cells in R3 were used for below analysis. (B) The dot plots of 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) green (x-axis) and red (y-axis). The percentage of JC-1 red-positive sperm (R7) was used for the analysis. (C) Histograms of BioTracker Red staining. Blue line indicates the range of positive cells used in Figure 7I. (D) The dot plots of fluorescein isothiocyanate-conjugated peanut agglutinin (PNA-FITC; x-axis) and propidium iodide (PI; y-axis).

Full-length blotting images of western blotting

(A) Full-length blotting images of GLUT4 and α/β-TUBLIN in seminal vesicle epithelial cells cultured with 100 ng/mL testosterone (Testo) or in vehicle (Ctrl) in Figure 6D. (A) Full-length blotting images of ACLY and α/β-TUBLIN in seminal vesicle epithelial cells cultured with 100 ng/mL testosterone (Testo) or in vehicle (Ctrl) in Figure 8A. (B) Full-length blotting images of ACLY and α/β-TUBLIN in seminal vesicle epithelial cells in Figure 8C. 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.

Gating strategy of flow cytometry for seminal vesicle epithelial cells

Gating strategy for the selection of single sperm. Using forward scatter (FSC)-A and side scatter (SSC)-A dot plots, similar size and complexity cells were firstly selected (R1). In FSC-A and FSC-H dot plots and FSC-A and FSC-W dot plots, similar-size cells were accumulated near area; thus, using these plots, again similar-size cells were selected (R2, R3). The cells in R3 were applied to the propidium iodide (PI) histogram plot. Labelling DNA with PI allows fluorescence-based analysis of the cell cycle. The intensity of PI fluorescence correlates with the amount of DNA in each cell. Comparing the G1 and G2 phases, the amount of DNA doubles in the G2 phase, so the fluorescence intensity of the cell population also doubles.

Primer sequences used for quantitative real-time PCR.