Gosha-Jinki-Gan Recovers Spermatogenesis in Mice with Busulfan-Induced Aspermatogenesis

Abstract

Busulfan is an anti-cancer chemotherapeutic drug and is often used as conditioning regimens prior to bone marrow transplant for treatment of chronic myelogenous leukemia. Male infertility, including spermatogenesis disturbance, is known to be one of the side effects of anticancer drugs. While hormone preparations and vitamin preparations are used for spermatogenesis disturbance, their therapeutic effects are low. Some traditional herbal medicines have been administered to improve spermatogenesis. In the present study, we administered Gosha-jinki-gan (TJ107; Tsumura Co., Ltd., Tokyo, Japan) to mice suffering from severe aspermatogenesis after busulfan treatment to determine whether TJ107 can recover spermatogenesis. Male 4-week-old C57BL/6J mice were administered a single intraperitoneal injection of busulfan, and they were then fed a normal diet for 60 days and then a TJ107 diet or TJ107-free normal diet for another 60 days. After busulfan treatment, the weight of the testes and the epididymal sperm count progressively decreased in the normal diet group. On the other hand, in the TJ107 group, these variables dramatically recovered at 120 days. These results suggest that busulfan-induced aspermatogenesis is irreversible if appropriate treatment is not administered. Supplementation of TJ107 can completely recover the injured seminiferous epithelium via normalization of the macrophage migration and reduction of the expressions of Tool-like receptor (TLR) 2 and TLR4, suggesting that TJ107 has a therapeutic effect on busulfan-induced aspermatogenesis.

Keywords: anticancer treatment; aspermatogenesis; oriental medicine; testicular immunology.

Figure 1

Three-dimensional high-performance liquid chromatography profile of Gosha-jinki-gan. Gal: galloyl.

Figure 2

Histological examination of the testes (n = 10) at day 120 in each group. Intact seminiferous tubules showing all maturation stages of the germinal epithelium from spermatogonia to spermatozoa are seen in the control group (a) and the control+TJ107 group (b). Atrophic seminiferous tubules with azoospermia are seen in the BSF group (c). Normal-appearing seminiferous tubules are seen in the BSF+TJ107 group (d). Bar = 50 µm.

Figure 3

Detection of proliferating cells in the testes using antibodies against Ki67 nuclear antigen at day 120 in the control (a), control+TJ107 (b), BSF (c), and BSF+TJ107 groups (d). The enlarged part (Bar: 40 μm) of the seminiferous tubules is bounded by solid frames and is shown in the top right corner in each testis section. Ten testes from each group were examined. Dark brown spots indicating Ki67-positive nuclei of proliferating spermatogonia are detected in almost all seminiferous tubules in the control (a), control+TJ107 (b), and BSF+TJ107 (d) groups at day 120. Most germ cells are destroyed and few seminiferous tubules with Ki67-positive cells are sporadically observed in the BSF group (c). Bar: 40 μm. (e) Expression of Ki67 in testicular tissues of mice from each group (n = 10). Asterisks indicate p < 0.05.

Figure 4

Histological detection of TUNEL staining in testicular sections of mice from the control (a), control+TJ107 (b), BSF (c), and BSF+TJ107 (d) groups at day 120. Ten testes from each group were examined. Dark brown spots indicating TUNEL-positive nuclei of apoptotic germ cells are seen. Some TUNEL-positive germ cells are detected in the seminiferous tubules in the control (a), control+TJ107 (b), and BSF+TJ107 (d) groups at day 120. Most germ cells are destroyed at day 120 in the BSF group; thus, TUNEL-positive cells (arrowhead) are hardly detected (c). Bar: 40 μm. (e) Expressions of Fas, FasL, Caspase3, Caspase8, and Caspase9 in testicular tissues of mice from each group (n = 10). Asterisks indicate p < 0.05.

Figure 5

TLR expression and macrophage migration at day 120 in each group. A: Expressions of TLR2 and TLR4 in testicular tissues of mice from each group (n = 10). Asterisks indicate p < 0.05. B: F4/80 antigen-positive cells in testicular sections (n = 10) of mice from the control (a), control+TJ107 (b), BSF (c), and BSF+TJ107 (d) groups. Many macrophages are seen infiltrating the interstitium around the atrophic seminiferous tubules in the BSF group (c). Bar = 20 µm. (e) Expression of the macrophage marker gene in testicular tissues of mice from each group (n = 10). Asterisks indicate p < 0.05.

Figure 6

Time schedule of treatment applied to the mice in each group.