Supernatants from epithelial cells of the testosterone-added group induced motility and linear motility (highest VSL and LIN; Figure 7K–N). Qualitative and quantitative analysis of fatty acid synthesis was performed by gas chromatography–mass spectrometry (GC–MS). Although Hmgcr, which encodes the rate-limiting enzyme involved in cholesterol synthesis, was unchanged, the expression of Acc, which encodes the rate-limiting enzyme (ACC1; ACACA) for fatty acid synthesis, was significantly upregulated by testosterone. Interestingly, testosterone significantly increased the expression of Acly, which encodes a cytoplasmic enzyme that converts citrate transported from the TCA cycle into acetyl-CoA, a substrate that is essential for fatty acid synthesis. (A) The localization of the androgen receptor (AR; NR3C4) in seminal vesicle of control (Ctrl) mice and those treated with flutamide (Flut). The revised version of the manuscript significantly strengthens the role of ACLY as a central regulator of seminal vesicle epithelial cell metabolic programming. While the connection between media fatty acids and sperm motility patterns is still not fully conclusive, the authors have taken substantial steps to clarify and tone down their conclusions. They have also addressed the ambiguous references to the strength of the relationship between fatty acids and sperm motility, making the manuscript more balanced and nuanced. In this revised report, Yamanaka and colleagues investigate a proposed mechanism by which testosterone modulates seminal plasma metabolites in mice. The evidence supporting the upregulation of metabolic enzymes and the role of ACLY is solid, highlighting the potential contributions of fatty acids to sperm motility. All cell cultures used in this study were routinely tested for mycoplasma contamination by MycoAlert Mycoplasma Detection Kit (Cat. #LT07-218, Lonza, Basel, Switzerland). HSVEpiC (4460, ScienCell Research Laboratories, Carlsbad, CA, USA) were purchased, seeded in 2 μg/cm2 poly-l-lysine-coated plates, and cultured in epithelial cell medium (4101, ScienCell Research Laboratories). To avoid false-positive or false-negative results, DNase-treated sections were used as positive controls, and TdT-untreated sections were used as negative controls for apoptosis. After 60 min of incubation with or without treatments (Ctrl, Flu, or HTF), the sperm were added into HTF medium at a final concentration of 2 × 105 spermatozoa/mL, and coincubated with the COCs. The sperm suspension was centrifuged and washed twice with the base medium. Briefly, sperm were incubated with 200 µl of working solution containing 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) dye at 37°C for 30 min in the dark. Mitochondrial activity of sperm was measured using a MitoPT JC-1 Assay Kit (911, Immuno Chemistry Technologies, LLC, Bloomington, MN, USA) according to our previous study (Zhu et al., 2019). Studies show that men who engage in regular aerobic exercise have significantly higher sperm concentration and motility than sedentary men. Physical activity is one of the most effective ways to boost male fertility. Whether you are dealing with infertility or you want to donate sperm to help a single woman or a couple have a baby, here are 14 evidence-based ways to increase sperm count. Then, we work together in order to develop a treatment plan that delivers the maximum benefit (restored testosterone levels) with the smallest risk of unwanted side effects possible. For men who experience drop-offs in sperm production due to TRT, several therapeutic remedies can effectively restore spermatogenesis and improve fertility. It is true that abstinence increases sperm count. Men trying to conceive are often advised to remain sexually abstinent for several days to allow their sperm count to build up. That means a man’s age is not necessarily the same as the age of his sperm. When it comes to reproduction, female biology is often described in terms of a ticking clock. The cells were normalized to 10,000 viable cells/well immediately before analysis. Unexpectedly, when measuring mitochondrial respiratory metabolism normalized by the total number of live cells with the flux analyzer, the basal respiratory rate (the electron transport chain activity) was significantly decreased by testosterone (Figure 4F–J). Because extracellular pyruvate levels simultaneously declined, we inferred that the cells had an increased pyruvate demand and, at that time, hypothesized that the excess pyruvate would enter the mitochondria to support enhanced oxidative metabolism. These data indicate that testosterone enhances glucose utilization. Genes largely upregulated by testosterone included those already reported in seminal vesicles (Kawano et al., 2014; Noda et al., 2019; Smyth et al., 2022), such as the Seminal Vesicle Secretion (Svs) family (Svs1–6), Pate4, Spinkl, Ceacam10, and Sva (Figure 3B, C). Each replicate experiments with 3–6 wells containing pooled cells from 3 to 5 mice.
