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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.

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.

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.

With the increase in survival rates of cancer patients in recent years, infertility caused by anticancer treatments has become a significant concern for cancer survivors. Some studies have suggested that Sertoli cells play a key role in mediating testicular immunology in busulfan-induced aspermatogenesis. We recently demonstrated that Gosha-jinki-gan (TJ107), a traditional Japanese medicine, can completely recover injured spermatogenesis in mice 60 days after busulfan injection. In the present study, we sought to examine the levels of mRNA transcripts encoding markers of 25 Sertoli cell-specific products and 10 markers of germ cell differentiation. Our results demonstrated that only supplementation of TJ107 at day 60 after busulfan injection could significantly recover the increase in five mRNA species (Amh, Clu, Shbg, Testin, and Il1a) and the decrease in four mRNA species (Aqp8, CST9, Wnt5a, and Tjp1) in response to Busulfan (BSF) at day 120, with the increase of all examined spermatogenic markers. [ABSTRACT FROM AUTHOR]