Although it is perhaps still premature to attempt to assign the different reactivities of the numerous cytotoxic agents that have been tried in spermatogenesis to the different categories of damage they produce, as outlined in the introduction to this review, a tentative grouping may nevertheless be considered worthwhile, if only to be criticized and proven invalid by further work.
Kinetic damage. This is difficult to recognize, since it requires a tedious examination of the distribution and proportion of the phases of the testis present in any transverse section of the tubule at different times after treatment with the drug. The only satisfactory demonstration of true kinetic damage is probably the observation of the changing pattern of mitoses observed after treatment of mice with 6-azauracil. From inadequate data, it may be inferred that this type of damage may be present after treatment with antiandrogens, bis(dichloroacetyl)diamines (WIN compounds), progestens, antimetabolites, dinitropyrrole, nitrofurans, thiophene derivatives, and cadmium salts and after elevation of testicular temperature. In none of these cases however has this type of damage been satisfactorily proven.
Cellular damage. Its presence is more clearly defined, and most of the drugs examined, especially when the effects are noted by histological examination, have shown varying degrees of this form of damage. Spermatogonial damage has been observed with certain antiandrogens, although these cells may not be the most sensitive to these agents. Certain ethyleneimine derivatives, notably ethylene urea and ethylene urethane, show gross cell destruction in the testis. Of the alkane sulphonates, Busulphan, dimethyl busulphan, methylene dimethane sulphonate, and isopropyl methane sulphonate show spermatogonial damage. Methylhydrazine derivatives as well as cadmium salts also show gross damage to this cell type. Cellular damage to primary spermatocytes seems to be a characteristic feature of temperature elevation of the testis, and several drugs which are thought to be acting on the testis by inducing this mechanism, such as the dinitropyrrole, nitrofuran, thiophene, bis(dichloroacetyl)diamine derivatives as well as cadmium salts, also exert a major action on this cell type. Bis(dichloroacetyl)diamines also exert some action on spermatids at an early stage. The final proof of cellular damage is the quantitative measurement of the extent of pycnosis and fragmentation, but this is not often observed, either because of the rapid removal of damaged cells or owing to the failure of damaged cells to develop into the normal complement of more mature forms.
Morphological damage. It is a point for discussion as to whether there should be a distinction between "cellular" and "morphological" damage, but if the cell survives for a period and leaves the testis with the spermatozoon, the damage is regarded as more persistent and classified as "morphological." Such changes in spermatid development have been noted after treatment with the alkane sulphonate derivatives, and have been described in detail in human studies after treatment with some of the bis(dichloroacetyl)diamines. Abnormal and bizarre forms of spermatozoal structure have also been described. The possibility exists that minor deformities of spermatozoa may not interfere with their viability but early or late genetic damage could not be ruled out. The most commonly observed form of morphological damage in the testis is the production of large polyploid cells during the late spermatocyte and early spermatid phases of development. A clear-cut example of morphological damage results from treatment with deuterium oxide, which is considered to induce a morphological change in the structure of the acrosome, so that the normal process of fertilization is impaired.
Early genetic damage. The most obvious form of genetic damage sustained by the germinal system is that resulting in deaths during early embryonic life. This may be due either to failure of fertilized eggs to implant or to death after implantation. Many instances of reported sterility after treatment of the male may be the result of this type of damage, and for potential antifertility agents it would be essential to know if this is so, since lower doses may induce late genetic damage. Measurement of dominant lethal action may be made in early embryos. Most of the alkylating agents produce some degree of early genetic damage. In the particular case of the simple alkyl alkane sulphonates such as methyl, ethyl, and n-propyl methane sulphonate, the main type of damage produced is directed towards the adult spermatozoa in such a way that the resulting fertilized egg fails to cleave normally, and both pre- and postimplantation deaths occur. The bis(dichloroacetyl)diamines and all antimetabolites should be suspected of causing some degree of early genetic damage, but except for some work in insect spermatogenesis, there is no satisfactory proof of such damage in the higher animals.
Late genetic damage. Damage to offspring of treated males has been conclusively demonstrated with triethyleneimine, nitrogen mustard, and methyl methane sulphonate. Owing to the very tedious nature of the experiments, very few drugs have been rigorously studied from this point of view. In general, those drugs which produce changes in DNA structure, such as the alkylating agents and some antimetabolites, as well as those which have already shown some early genetic damage at higher dose levels, may be suspected of producing late genetic damage.
Matrix damage. This occurs in some instances in association with extensive damage to the germinal epithelium, as with ethylene urea and ethylene urethane. The progestens, methylhydrazine derivatives, and cadmium salts also show extensive damage to the matrix, and in the case of the last mentioned agent, the testicular structural tissue may be the primary site of action.
An interpretation of the action of any new group of cytotoxic substances must take into account the possibilities that the effects observed may be the result of a direct action of the agent on the cell or an indirect one mediated through some of the many complex hormonal and other regulatory pathways in the whole organism. To unravel this complex question is probably easier in the testis than in any other differentiating system in the mammal, since the cellular kinetics of the spermatogenic epithelium, at least in the rodent, is now well known. The interrelationships of this system with the pituitary-gonadal axis is becoming clearer, and a way is opening to understand the mechanism by which the whole organism may control the functioning of one of its component systems. It is clear that the final definition of the mode of action of a drug must involve basic biophysical and chemical mechanisms at the level of a single molecule or a small group of molecules that are essential biochemical foci of action, whether they be at gene or cytoplasmic level.
An important technical advance in this field would be the separation of the component cells of the testis by some physical method of centrifugation, countercurrent, or filtration, destroying as little as possible of the enzyme activity, so that detailed biochemical investigations involving cell component separations may be effected. Continued study of the spectrum of drug action on spermatogenesis is desirable, however, since apart from recognizing new drug specificities of action, the potential interest to the emergency problem of population control cannot be overstressed.
- 1967 by The Williams & Wilkins Co.