GLP-1 receptor agonists show no detrimental effect on sperm quality in mouse models and cell lines

Abstract

Purpose: Glucagon-like peptide-1 receptor (GLP-1R) agonists exert multiple beneficial effects. However, their effects on reproduction system are controversial. Here, we aimed to investigate their effects on male reproduction and provide safety evidence for future clinical use.

Methods: Male diabetic mice and aged mice were treated with liraglutide or vehicle, and sperm concentration and motility were assessed. The expression and location of GLP-1R in testicular tissues and in four testicular cell lines (spermatogonia, spermatocytes, Leydig cells, and Sertoli cells) were detected. Cauda epididymis and testicular cells were treated with liraglutide, semaglutide or vehicle, and sperm motility and cell proliferation were detected to determine the direct effect of GLP-1R agonists. Global Glp1r knockout mice were constructed, and testicular morphology, sperm concentration and motility were detected to confirm the effects of GLP-1R signaling on male reproduction.

Results: Liraglutide significantly reduced blood glucose levels, but did not improve sperm parameters in diabetic mice. No significant differences were observed between liraglutide and control group in aged mice. GLP-1R was expressed in testicular tissues and all four cell lines, with the highest expression in Leydig cells. Liraglutide or semaglutide had no impacts on sperm count and motility in vitro, and had no effects on cell proliferation in four cell lines. The Glp1r knockout mice exhibited higher blood glucose levels and preserved normal testicular morphology, but their sperm concentration was higher than that in wildtype mice.

Conclusion: GLP-1R agonists have no detrimental effect on sperm concentration and motility in vivo and in vitro, while GLP-1R absence increase the sperm concentration.

Keywords: Aging; Diabetes; Glucagon-like peptide-1; Sperm concentration; Sperm motility.

Fig. 1

Liraglutide has no adverse effect on sperm concentration and motility in T2D mice. Six-week-old male C57BL/6 J mice were fed a high-fat diet for 3 months and injected intraperitoneally with 75 mg/kg streptozotocin to induce type 2 diabetes, and were treated for three weeks with liraglutide (Lira, 0.2 mg/kg, twice daily) or saline (as control [Ctrl]) via subcutaneous injection (n = 8 per group). A: Body weight. B: Random blood glucose level. C: Fasting blood glucose level. D: Glucose level during intraperitoneal glucose tolerance test (IPGTT). The arrowed indicates the upper detection limit (33.3 mmol/L) of the glucometer. E: Glucose level during insulin tolerance test (ITT). F: Fasting insulin level. G–O: The sperm count and motility assessment. G: Sperm concentration. H: Rapid progressive motility (grade A sperm). I: Progressive motility (grade A + B sperm). J: Velocity along the straight‑line path. K: Velocity along the curvilinear path. L: Velocity along the average path. M: Beat cross frequency. N: Straightness. O: Amplitude lateral head displacement. All data are presented as mean ± SD. Statistical analysis was conducted by Student’s t-test in A–O, ***P < 0.001

Fig. 2

Liraglutide has no negative impact on sperm concentration and motility in the aged mice. Male C57BL/6 J mice aged 18 months were treated for three weeks with liraglutide (Lira, 0.2 mg/kg, twice daily) or saline (as control [Ctrl]) via subcutaneous injection (n = 8 per group). A: Body weight. B: Random blood glucose level. C: Glucose level during intraperitoneal glucose tolerance test (IPGTT). D–L: Sperm count and motility assessment. D: Sperm concentration. E: Rapid progressive motility (grade A sperm). F: Progressive motility (grade A + B sperm). G: Velocity along the straight‑line path. H: Velocity along the curvilinear path. I: Velocity along the average path. J: Linearity. K: Straightness. L: Amplitude lateral head displacement. All data are presented as mean ± SD. Statistical analysis was conducted by Student’s t-test in A–L, *P < 0.05

Fig. 3

GLP-1R agonists have no direct effect on sperm motility and the testicular cell proliferation. A: Protein expression of GLP-1R was assessed in four testicular cell lines and testicular tissue by western blot. Mouse β-cell line Min6 cells were used as positive control. B: Immunohistochemical staining of GLP-1R in testicular tissue. Scale bar = 50 μm. The cells in the dashed box are enlarged on the right. C: Gene expression data of GLP-1R in human pancreatic islet, testis, testis germ cell, testis intersitial, Leydig cell. The data were generated by using BioGPS. D–L: Sperm suspensions collected from cauda epididymis of male C57BL/6 J mice were treated with liraglutide (Lira, 100 nmol/L), semaglutide (Sema, 100 nmol/L), or saline (as control [Ctrl]). n = 8 per group. Sperm motility was assessed. D: Rapid progressive motility (grade A sperm). E: Progressive motility (grade A + B sperm). F: Total motility (grade A + B + C sperm). G: Velocity along the straight‑line path. H: Velocity along the curvilinear path. I: Velocity along the average path. J: Beat cross frequency. K: Straightness. L: Amplitude lateral head displacement. M: Mouse spermatogonial cell line GC-1 cells, spermatocyte cell line GC-2 cells, Leydig cell line TM3 cells, and Sertoli cell line TM4 cells were treated with different concentrations of liraglutide or semaglutide (0–100 nmol/L) for 72 h. Cell proliferation was analyzed using CCK-8 kit (n = 4 per group). All data are presented as mean ± SD. Statistical analysis was conducted by one-way ANOVA in D–L, or by two-way ANOVA in M

Fig. 4

GLP-1R ablation has no effect on testicular morphology and sperm motility. The metabolic indicators and sperm parameters in eight-week-old male Glp1r^−/− mice and the littermate wildtype (WT) mice (n = 8 per group). A: Body weight. B: Random blood glucose level. C: Blood glucose during oral glucose tolerance test (OGTT). D: HE staining of the testicular tissue. Scale bar = 50 μm. The cells in the dashed box are enlarged on the right. E–M: The sperm count and motility assessment. E: Sperm concentration. F: Rapid progressive motility (grade A sperm). G: Progressive motility (grade A + B sperm). H: Velocity along the straight‑line path. I: Velocity along the curvilinear path. J: Velocity along the average path. K: Beat cross frequency. L: Straightness. M: Amplitude lateral head displacement. All data are presented as mean ± SD. Statistical analysis was conducted by Student’s t-test in A–C and E–M, ** P < 0.01, *** P < 0.001