Co-Administration of the Traditional Medicines Hachimi-Jio-Gan and Hochu-Ekki-To Can Reverse Busulfan-Induced Aspermatogenesis.

Busulfan is used as a chemotherapeutic drug to treat childhood and adult chronic myelogenous leukemia, and as an immunosuppressive agent before bone marrow transplantation. A key side effect of busulfan is the alteration of male reproductive function. Infertility caused by anti-cancer treatments has become a significant concern, but there are currently limited treatments for this condition. Recently, we demonstrated that Gosha-jinki-gan, a traditional Japanese medicine, completely reversed the spermatogenesis defects caused by cancer treatment in mice. Hochu-ekki-to and Hachimi-jio-gan are commonly used to treat male infertility, and Hachimi-jio-gan shares herbal ingredients with Gosha-jinki-gan. Therefore, in the present study, we administered Hachimi-jio-gan and Hochu-ekki-to alone or in combination to mice with severe aspermatogenesis caused by busulfan treatment. We performed testis weight measurements, quantitative histological assessments of the testes and the epididymis, and evaluated sperm counts and morphology. We also assessed the expression of immune mediators and macrophage markers. Treatment with a combination of both the medicines significantly reduced busulfan-induced testicular toxicity when compared to the lone treatment with either medicine. We demonstrated that treatment efficacy was related to a differential impact on testicular inflammation, and that the synergistic effect of co-administration completely reversed the busulfan-induced damage to the reproductive functions.

The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice.

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.

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

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.

Induction of experimental autoimmune orchitis by immunization with xenogenic testicular germ cells in mice.

We previously showed that immunization of mice with syngeneic or allogeneic testicular germ cells (TGC) alone induces autoimmune inflammation in the testes without using any adjuvant. In the present study, we examined testicular autoimmune response against xenogenic TGC antigens in mice. The mice were immunized with murine, rat or guinea pig TGC and then the histopathology, delayed type hypersensitivity (DTH) response and humoral autoimmunity were investigated. The results showed that immunization with not only murine but also rat TGC caused experimental autoimmune orchitis (EAO) with hypospermatogenesis in mice, while that with guinea pig TGC could not. The DTH response to murine TGC was significantly elevated in mice that had been immunized with murine or rat but not guinea pig TGC. Serum autoantibody to murine TGC was immunohistochemically detected in the mice immunized with either murine, rat or guinea pig TGC, however, the level of autoantibody detected by ELISA revealed significant elevation when mice were immunized with murine and rat TGC. With the immunoblotting after electrophoresis, the murine TGC proteins at molecular masses around 55kDa and 70kDa can be detected when incubated with sera from m-TGC and r-TGC groups. These results represent the cross-reactivity among TGC of the mouse, the rat and the guinea pig at the levels of humoral immunity and also demonstrate that the rat TGC could elicit significant DTH response to murine TGC with the resultant EAO. This is the first to succeed in EAO induction by the use of xenogenic TGC.

Busulfan pretreatment for transplantation of rat spermatogonia differentially affects immune and reproductive systems in male recipient mice.

Testicular cell transplantation has generally been performed by using immune-deficient recipient mice to investigate the biology of spermatogonial stem cells (SSCs), the production of transgenic animals, and restoration of fertility. Recently, we demonstrated that rat spermatogenesis can occur in the seminiferous tubules of immune-competent recipient mice via pretreatment with busulfan (Myleran, 1, 4-butanediol methanesulfonate, 40 mg/kg) after transplantation of rat SSCs. However, considering the immunosuppressive effect of busulfan, there is a possibility that busulfan itself causes immune suppression in immune-competent recipient mice. The aim of this study was to determine the effects of busulfan on the immune system and spermatogenesis in immune-competent recipient mice. The results showed that at 60 days after busulfan treatment, just the same time as the transplantation, the recovery could be seen in the immune system including cell counts and functions of T and B lymphocytes in the spleen, but the spermatogenesis was more compromised. This study demonstrated that after busulfan pretreatment the immune system in immune-competent recipient mice had recovered by the time that rat spermatogenesis could occur in the murine testis. It became clear that xenogenic spermatogenesis can be tolerated in seminiferous tubules in the testes of immune-competent mice.