Effect of liver growth factor on both testicular regeneration and recovery of spermatogenesis in busulfan-treated mice

Abstract

Background: Some adult stem cells persist in adult tissue; however, we do not know how to stimulate stem cells in adults to heal injuries. Liver growth factor (LGF) is a biliprotein with hepatic mitogen activity. Its concentration increases markedly in the presence of any type of liver injury, and it shows in vivo therapeutic biological activity at extrahepatic sites.

Methods: We have analyzed the effect of LGF on the replenishment of germinal cells in the testes of mice injected with busulfan, a common cancer drug that also specifically affects germ line stem cells and spermatogonia. We determined the testicular and epididymal weight, spermatozoal concentration in the epididymis and sperm motility, and performed a histological analysis.

Results: Intraperitoneal administration of LGF was able to partially restore spermatogenesis, as well as sperm production and motility, in mice sterilized with busulfan. LGF treatment in busulfan-treated animals that have suffered a disruption of spermatogenesis can accelerate the reactivation of this process in most of the tubules, as shown in the histological analysis.

Conclusions: Our results suggest a potential use of LGF in the mobilization of testicular stem cells and in the restoration of spermatogenesis after busulfan-induced damage to the testicular germinal epithelium.

Figure 1

Differences in testis and epididymis weight observed between experimental groups. Epididymal (A) and testicular (B) weight in the four groups of animals analyzed (control, LGF treatment, busulfan treatment, busulfan plus LGF treatment). ^{a, b, c}Different letters indicate significant differences between groups based on one-way ANOVA (P ≤ 0.01). Error bars, SEM. LGF: liver growth factor.

Figure 2

Differences in sperm motility and concentration observed between experimental groups. Spermatozoal concentration (×10⁶ sperm per ml) (A) and motility (B) in sperm samples recovered from cauda epididymidis and vasa deferentia of the four groups of males analyzed (control, LGF treatment, busulfan treatment, busulfan plus LGF treatment). ^{a, b, c}Different letters indicate significant differences between groups based on one-way ANOVA (P ≤ 0.01). Error bars, SEM. LGF: liver growth factor.

Figure 3

Histological comparison of seminiferous tubules from testes of experimental groups. Pictures of the control group (A); 63 days after busulfan treatment (B) with Sertoli cell-only tubules (sterisk) and nodules of hyperplastic Leydig cells (star); and 63 days after the injection of busulfan and liver growth factor (C) with tubules showing vacuolization (arrow). Hematoxylin & eosin. Bar 50 μm.

Figure 4

Seminiferous tubule diameters from the three groups of mice analyzed. Control, busulfan treatment, busulfan plus LGF treatment. ^{a, b, c}Different letters indicate significant differences between groups based on one-way ANOVA (P ≤ 0.01). Error bars, SEM. LGF: liver growth factor.