In vitro spermatogenesis for toxicity testing

<p>Spermatogenesis is a long and complex cell differentiation process, consisted of three different phases; proliferation of spermatogonia, meiosis of spermatocytes, and morphological and nuclear structural changes of spermatids. Therefore, it is difficult to recapitulate the entire process of spermatogenesis in a culture condition. We have succeeded in reproducing spermatogenesis and producing fertile haploid cells, spermatids, by culturing mouse testicular tissue fragments on an agarose gel block, namely by gas-liquid interphase method. However, the efficiency is quite low compared to those in vivo. In addition, application of this culture method to animals other than mice has been unsuccessful for a long time. We have examined the culture condition carefully and analyzed the composition of the culture medium to identify critical substances. Then, we found that retinoic acid (Vit. A), triiodothyronine (T3), alpha-tocopherol (Vit. E), ascorbic acid (Vit. C), glutathione, and lysophospholipids, are among important substances. By using a new culture medium containing these substances, we cultured rat testicular tissue fragments and recently succeeded in inducing the spermatogenesis up to spermatids. On the other hand, we developed a culture method using a silicone tip made of dimethylpolysiloxane, by which the cultured testis tissue is made thin flat on the agarose gel. This allows the testicular tissue pieces to be flattened to a defined thickness, to the thickness of a seminiferous tubule in particular, and facilitated efficient and even distribution of nutrients and oxygen to the entire tissue. It also allowed precise and simple evaluation of testicular growth and extent of spermatogenic progression. We are now applying this method for the evaluation of testicular toxicity and will present some data in the presentation. </p>