Miyuki Kuramasu

Low-dose exposure to di-(2-ethylhexyl) phthalate (DEHP) increases susceptibility to testicular autoimmunity in mice

Exposure to di-(2-ethylhexyl) phthalate (DEHP) induces spermatogenic disturbance (SD) through oxidative stress, and affects the immune system by acting as an adjuvant. Recently, we reported that in mice, a low dose of DEHP, which did not affect spermatogenesis, was able to alter the testicular immune microenvironment. Experimental autoimmune orchitis (EAO) can be induced by repeated immunization with testicular antigens, and its pathology is characterized by production of autoantibodies and SD. In the present study, we investigated the effect of a low-dose DEHP on the susceptibility of mice to EAO. The exposure to DEHP-containing feed (0.01%) caused a modest functional damage to the blood-testis barrier (BTB) with an increase in testicular number of interferon gamma (IFN-γ)-positive cells and resulted in the production of autoantibodies targeting haploid cells, but did not affect spermatogenesis. While only single immunization with testicular antigens caused very mild EAO, the concurrent DEHP exposure induced severe EAO with significant increases in number of interferon gamma-positive cells and macrophages, as well as lymphocytic infiltration and serum autoantibody titer accompanied by severe SD. To summarize, the exposure of mice to the low-dose DEHP does not induce significant SD, but it may cause an increase in IFN-γ positive cells and modest functional damage to the BTB in the testis. These changes lead to an autoimmune response against haploid cell autoantigens, resulting in increased susceptibility to EAO.

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.

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.