SPERMATOCYTE TOXICITY OF 2-METHOXYETHANOL IN-VIVO AND IN-VITRO - REQUIREMENT FOR AN INTACT SEMINIFEROUS TUBULE STRUCTURE FOR GERM-CELL DEGENERATION

Cultures of isolated testicular cells are widely used for evaluating m echanisms of action of direct-acting testicular toxicants. However, fo r testicular cells, the expression of toxicity in vitro is frequently different from that found in vivo. 2-Methoxyethanol (ME) produces test icular lesions in rats which are characterized by pachytene spermatocy te degeneration 24 hr after dosing. As part of a study to evaluate mec hanisms of male germ cell toxicants, we compared the morphological asp ects of spermatocyte toxicity after in vivo exposure to ME with those found in various testicular cell culture systems after in vitro exposu re to the active ME metabolite, 2-methoxyacetic acid (MAA). Immature r ats were used because they respond to in vivo ME treatment in the same way as adults, but their testes are relatively enriched in pachytene spermatocytes. 24-day-old rats were given a single dose of 250 mg ME/k g body weight by gavage and the testes were evaluated 24 hr after dosi ng. In vitro, cultured seminiferous tubules, Sertoli-germ cell co-cult ures, and enriched mixed germ cells, all prepared from 24-day-old rats , were evaluated after 24-hr in vitro exposure to 5 mM MAA (a level of MAA found after ME exposure in vivo). Testes from ME-exposed rats sho wed the expected pachytene spermatocyte degeneration 24 hr after dosin g. Similar changes were observed in cultured seminiferous tubules afte r 24 hr of exposure to MAA in vitro. However, without the intact semin iferous tubule structure in vitro, the expression of MAA spermatocyte toxicity was different. In conventional Sertoli-germ cell co-cultures, spermatocyte detach ment from the Sertoli cell monolayer occurred as expected, although no significant morphological degeneration was obser ved in these detached germ cells. Similarly, no increase in degenerati ng spermatocytes was noted in isolated enriched mixed germ cells after in vitro MAA exposure. Instead, toxicity in these germ cell fractions was expressed only by increased uptake of trypan blue dye, revealing an increase in plasma membrane permeability. In summary, this in vivo/ in vitro comparison of the spermatocyte toxicity of ME/MAA showed that the expression of toxicity is different in the different culture arch itectures and that an intact seminiferous tubule structure is required for full expression of the morphological degeneration produced by ME/ MAA, and suggests the usefulness of culture seminiferous tubules in fu ture mechanistic studies.