Glucagon receptor blockade protects spermatogenesis by enhancing PFKFB3-mediated lactate production in Sertoli cells

Male fertility has declined globally. Diabetes and aging are major contributors to the reduced sperm production and testicular dysfunction. Elevated glucagon signaling leads to diabetic complications, yet its impact on male reproductive function remains unclear.

Streptozotocin-induced type 1 diabetic mice, naturally aged mice, and healthy young male mice were treated with a glucagon receptor (GCGR) monoclonal antibody or control. Sperm quality and testicular outcomes, including histology, oxidative stress, and apoptosis, were assessed. We then generated global Gcgr knockout mice and compared their sperm parameters and testicular outcomes with those of wild-type mice. Mouse spermatogonia cells, spermatocyte cells, Leydig cells, and Sertoli cells were treated with different concentrations of glucagon, and cell proliferation was evaluated. In Sertoli cells, maturation markers and blood-testis barrier (BTB)-associated genes were examined. The histology and function of Sertoli cells were further measured in diabetic or aged mice after GCGR antibody treatment. To investigate the underlying mechanism, we treated Sertoli cells with glucagon, a GCGR antibody, or a GLP-1R antagonist, and measured the lactate content, lactate dehydrogenase activity, and the expression of glucose transporters, glycolysis-associated genes, and lactate dehydrogenase isoforms. We focused on the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). The lactate production and PFKFB3 level were further determined in diabetic or aged mice. Next, by using over-expression or knockdown of Pfkfb3 in glucagon-treated Sertoli cells and constructing Pfkfb3 heterozygous knockout mice, we confirmed the role of PFKFB3 in lactate production and Sertoli-cell function.

In diabetic mice, the GCGR antibody increased sperm concentration (P < 0.001) and reduced testicular oxidative stress and apoptosis. In aged mice, the GCGR antibody improved progressive motility (P = 0.004), with minimal effects on reproductive hormone levels, testicular oxidative stress, and apoptosis. In healthy young mice, the GCGR antibody increased sperm concentration (P = 0.003), progressive motility (P = 0.022), and total motility (P = 0.020). Global Gcgr knockout impaired spermatogenesis despite improved metabolic status, indicating the participation of GCGR signaling in embryonic testicular development and spermatogenesis. Glucagon did not directly alter isolated sperm motility or proliferation of testicular cell lines, but it suppressed Sertoli-cell maturation markers, BTB-associated genes, lactate production, and PFKFB3 expression. These effects were rescued by the GCGR antibody, whereas GLP-1R blockade showed partial or variable protection. Besides, these inhibitory effects of glucagon were rescued by Pfkfb3 overexpression while Pfkfb3 knockdown showed the similar effects with glucagon. In diabetic and aged mice, the GCGR antibody administration restored Sertoli-cell maturation markers and BTB markers, upregulated testicular lactate levels and increased the abundance of PFKFB3. Pfkfb3 heterozygous deletion reduced total motility (P = 0.007) and testicular lactate content (P = 0.013), accompanied by the lower Sertoli-cell markers and BTB-associated genes expression.

Pharmacological GCGR blockade reverses dysfunctional Sertoli cells and restores spermatogenesis in models of diabetes and aging, and PFKFB3-mediated lactate production participated this process. Our study presents a promising therapeutic strategy for male infertility.