Melphalan, alone or conjugated to an FSH-beta peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo

摘要

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the beta-chain of FSH beta. We hypothesized that conjugating melphalan to FSH beta peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSH beta peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSH beta consisting of amino acids 33 to 53 FSH beta (33-53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSH beta (33-53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSH beta (33-53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSH beta (33-53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSH beta (33-53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSH beta (33-53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.