The Hepatokine FGF21 Increases the Human Spermatozoa Motility

Abstract

Lifestyle, environment and excess body weight are not only associated with an increased risk of metabolic disorders, such as type 2 diabetes, but also to other pathological processes, such as infertility. A hormone produced mainly by the liver called fibroblast growth factor 21 (FGF21) is closely linked to the energy status and is increased in patients suffering from obesity or insulin resistance. Recently, FGF21 has been shown to be associated with female fertility disorders, but no or few data about the role of FGF21 on human male fertility has been described. In the present study, FGF21 was measured in the seminal fluid at a lower level in comparison to the blood level. Thus, in the present in vitro study, we aimed to decipher the FGF21 system in human semen. To evaluate the putative role of FGF21 on spermatozoa function, we incubated human spermatozoa with increasing concentrations of recombinant human FGF21. The FGF21 in seminal fluid is potentially produced by male reproductive tract tissues. In spermatozoa, the FGF21 signal was transduced by the two main receptors FGFR1-c and FGFR3 and the cofactor β-klotho, which are colocalized in the middle piece of spermatozoa and stimulated the PI3K/Akt and MAPK pathways. Finally, in vitro treatment by FGF21 significantly increased sperm motility and ATP levels. Concomitantly, exposure to FGF21 improved the oxidative stress, as a lower ROS level was observed. Overall, these results seem to indicate that the metabolic factor, FGF21, positively modifies the activity and quality of the parameters of human spermatozoa.

Keywords: FGF21 (fibroblast growth factor 21); fertility; human; metabolism diseases; sperm moTility; spermatozoa.

Figure 1

Presence of FGF21 in human plasma and seminal fluid. (A) FGF21 was determined, from two groups of fasting patients, in plasma (n = 10/BMI group) and in seminal fluid (n = 12/BMI group), in function of their body mass index (BMI). Control corresponding to a BMI ≤ 25 kg/m², and obesity corresponding to a BMI ≥ 30 kg/m². (B) Ratio of fasting FGF21 level between seminal fluid and plasma in the two groups of patients. (C) Immunoblot of a pool of human seminal fluid protein extracts (400 μg protein per sample) after immunoprecipitation with the FGF21 polyclonal antibody. Human recombinant FGF21 was loaded as the control to confirm the molecular weight of the immunoprecipitated FGF21 protein. *p < 0.05, **p < 0.01.

Figure 2

Localization of FGF21 in reproductive organs. FGF21 was localized by immunohistochemical staining in human testis (1 and 2), epididymis (4 and 5), seminal vesicles (7 and 8) and prostate (10 and 11). Negative controls (3, 6, 9, and 12) were sections incubated with IgG. We observed FGF21 expression in Leydig cells and the epithelia of the epididymis, prostate and seminal vesicles (see arrow), which contained all secretory cells.

Figure 3

Localization of FGF21 receptors on human spermatozoa. (A) Detection of sperm cofactor KLB and the FGFR1, FGFR3, and FGFR4 using Western immunoblotting. Red ponceau was used to check protein deposition on the membrane. Each lane represents extracts from different donors. Receptors and cofactor were tested on different gels. (B) FGF21 (1 and 2), FGFR1, FGFR3, FGFR4 (3, 6, and 9), and KLB (4 and 7) localization in human spermatozoa were analyzed by confocal microscopy after immunofluorescence. FGFR1, FGFR3 and FGFR4 were stained by Alexa Fluor 488 goat anti-rabbit IgG (green) and KLB (4 and 7) by Alexa Fluor 633 rabbit anti-mouse IgG (red). Merged picture of FGFR1 and FGFR3 with KLB showing the colocalization in the mid-piece of spermatozoa (5 and 8). Negative control was incubated with IgG (10). DNA was counterstained with DAPI.

Figure 4

Signaling pathways activated by the exposure of human spermatozoa to FGF21. (A, B) Western blot and analysis of phosphorylated (ser473) Akt and phosphorylated (Thr202/Tyr204) ERK in human spermatozoa exposed to recombinant FGF21 (0.01-10 ng/mL), as described in Materials and Methods section. Results are representative of at least four independent experiments. (C) Intracellular calcium responsiveness of sperm to recombinant FGF21 stimulation determined by fluorescence intensity relative to baseline (ΔF/F) at 30 min. *p < 0.05, **p < 0.01.

Figure 5

FGF21 treatments increase spermatozoa motility. (A) Sperm progressive motility analyzed by computer-assisted sperm analysis (CASA) after 30 min of stimulation with recombinant FGF21, was presented in percentage of control. A preincubation with the FGFR inhibitor PD173074 was also used in presence of absence of a 30 min of stimulation with recombinant 10 ng/mL FGF21, (n = 8 patients). (B, C) Concentration of ATP in sperm stimulated for 30 min with recombinant FGF21 (10^-8M per 2.10⁶ cells) and cAMP production in percentage of control, (n = 7 patients). (D) Percentage of acrosome-reacted sperm with (2) or without (1) calcium ionophore A23187 was quantified after PSA staining (percentage of PSA negative cells). Acrosome reaction of spermatozoa after 30 min of stimulation with recombinant FGF21 (n = 5 patients). (E) Total cholesterol concentration quantified in human spermatozoa after 30 min of stimulation with recombinant FGF21 (µg per 2.10⁶ cells, n = 7 patients). (F) ROS levels in human spermatozoa incubated with increasing concentrations of recombinant FGF21 for 30 min (relative luminescence units per 2.10⁶ cells, n = 7 patients). *p < 0.05, **p < 0.01.

Figure 6

Representative schema of FGF21 effects on human spermatozoa. Representative schema of the localization of FGFRs and KLB in spermatozoa and effects of FGF21 on human sperm. FGF21 induced phosphorylation of the Akt and ERK pathways and calcium levels, then increased the sperm motility associated with ATP content in a dose-dependent manner, and reduced oxidative stress.