Testosterone also promotes the synthesis of oleic acid from acetyl-CoA synthesized by ACLY. This is because there are many challenges to measuring the direct relationship between sperm metabolism and movement patterns. However, the probability of obtaining sperm without ICSI or IVF is extremely low, despite the presence of sufficient numbers of sperm with normal morphology (Murata et al., 2014; Stone et al., 2013; Frattarelli et al., 2008). We further confirmed that GLUT4 is the transporter that mediates this glucose uptake using indinavir, a GLUT4-specific inhibitor. ARs are known to directly regulate the expression of glycolytic and lipid metabolic genes (Wilson et al., 2013; Gonthier et al., 2019; Troncoso et al., 2021). Both the indinavir and testosterone treatments were significant.
(C) Percentage of Ki67 positive cells (top) and TUNEL positive (bottom) in Ctrl and Flut-treated seminal vesicle sections. Our previous studies (19–21) documented that seminal plasma also contains creatine and fatty acids taken up by sperm within minutes. TGFβ, a component of seminal plasma, increases antigen-specific Treg cells in the uterus of mice and humans, which induces immune tolerance, resulting in pregnancy (15, 16). Testosterone induces ACLY expression in seminal vesicles, a key factor in forming seminal plasma to acquire in vivo fertilization ability of sperm. In conclusion, the critical role of testosterone-induced metabolic changes in the seminal vesicles is to ensure the synthesis of oleic acid, which is important for sperm fertilization in vivo.
(B) Serum testosterone levels in seminal vesicle secretion donors. Cauda epididymis sperm was suspended with above artificial seminal fluid (1 × 106 sperm/mL), and a total of 40 µL (20 µL each per uterus) was injected through the cervix of the female mice. To examine the involvement of seminal vesicle secretions in fertilization, sperm from C57BL/6N mice were injected directly into the uterine bodies of superovulated CD1 mice by syringe, as previously described with minor modifications (17, 22). DEseq2 was used to identify differentially expressed genes (DEGs) with adjusted p- values less than 0.05 between the testosterone-treated groups in transcripts per million (TPM) between control and testosterone-treated cells. The seminal vesicle secretions from flutamide- treated mice were also collected similarly. In our previous study, the addition of fatty acids to the thawing medium of frozen bull sperm improved linear motility and viability (21). Differences in fatty acid concentrations, including oleic acid, in seminal plasma may provide a predictive marker for the fertilization potential of both human and domestic animal semen.
Primary human cells (HSVEpiC; 4460, ScienCell Research Laboratories, Carlsbad, CA, USA) were purchased from a commercial vendor. Densitometry analysis was performed using the ImageJ Gel Analysis tool, and the background of the gel was also removed individually for each band. Infection efficiency, calculated based on the number of GFP-positive cells using an APX100 Digital Imaging System, ranged between 70% and 90%. 100 ng/ml of testosterone was added to all procedures for the testosterone-treated group. Lentivirally transduced cells were prepared according to the manufacturer’s protocol. The sperm pellets were resuspended in HTF medium with or without an LM that included 2 μg/ml arachidonic acid, 10 μg/ml linoleic acid, 10 μg/ml linolenic acid, 10 μg/ml myristic acid, 10 μg/ml oleic acid, 10 μg/ml palmitic acid, and 10 μg/ml stearic acid (L0288, Sigma-Aldrich). Sperm recovered from the cauda epididymis of male mice were dispersed in 500 µl of mHTF without BSA and centrifuged at 300 × g for 3 min to wash out epididymis-derived factors.
The recovered cells as described above were fixed in 2–5 ml of cold (4°C) 70% methanol for at least 30 min on ice. The recovered cells were suspended in DMEM/F-12 medium, and cell numbers were determined using an Automated Cell Counter TC20TM (Bio-Rad Laboratories, Hercules, CA, USA) to calculate the number of cells contained per well (4 cm2). The cells were washed with HBSS (without CaCl2–2H2O/MgSO4) at the time of cell counting and collected using 0.25% (vol/vol) trypsin. A stromal cell marker (vimentin) was also used to confirm purity, but only a few of the cells were positive.
The recovered cells were suspended in DMEM/F-12 medium, and cell numbers were determined using an Automated Cell Counter TC20TM (BIO-RAD Laboratories, Hercules, CA, USA) to calculate the number of cells contained per well (4 cm2). The cells were washed with HBSS (without CaCl2- 2H2O/MgSO4) at the time of cell counting and collected using 0.25% (v/v) trypsin. Testosterone was dissolved in ethanol, and the final concentration of ethanol was adjusted to 0.1% (v/v) when used for cultured cells. Tissues were digested with 10 mL of HBSS containing 25 mg/mL pancreatin and 0.25% (v/v) trypsin EDTA for 1 hour at 4°C, 45 minutes at room temperature, and 15 minutes at 37°C, and epithelial cells were obtained. Sperm were collected from the cauda epididymis of 15 weeks or older C57BL/6N male mice in 100 μL of HTF medium. 3 weeks old CD1 female mice were injected intraperitoneally with 4IU eCG to stimulate follicle development and 5 IU hCG at 48 hours later. Testosterone levels in the serum samples were determined by a rodent testosterone ELISA test kit (ERKR7016, Endocrine Technologies, Inc., Newark, CA, USA) according to the manufacturer’s manual.