Letter to DR P Henshaw, Subject: Project Site Visit Dealing with the Effect of Ionizing Radiation on the Male Gonads

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Medical Research Branch Division of Biology and Medicine Atomic Energy Commission Enclosed please find some thoughts in relation to the Project Site Visit of the two projects dealing with the Effect of Ionizing Radiation on the Male Gomds, at Seattle, Washington. I am sure I have not incorporated all that could be said, and if there are any specific questions or points of information you think I might be of help with, please d o not hesitate to contact me. I wish to mention, at this time, that I enjoyed meeting you and spending those few days with you in Seattle. With best personal regards, I am

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The two projects are constructed for the purpose of determining the effect of ionizing radiation, administered under controlled conditions, on the male gonads. Both projects were to run simultaneously and parallel, utilizing similar approaches, although perhaps somewhat different techniques and ideas concerning the details. Basically, however, both investigators approached the problem thus far, from similar points of view, using the following parameters: the testicular tissue, quantitative analysis of germinal epithelium, cytologic analysis of the Leydig c e l l s , determination of spermatozoa numbers in the seminal fluid, analysis of the steroid production, analysis of the gonadotropin output, . and the study of the cytogenetic changes in the testes. The importance of this work and the reasons why it needs to be performed are self-evident. The unique opportunity afforded both Drs. Heller and Paulsen 'J to have willing subjects available and the proper research facilities, as well as the interest and research experience in ,the field of reproductive physiology, is quite unique and should be taken into consideration. It should be noted that the two groups perform their work separately. The experimental subjects come from two different penal institutions. ferently and the technique of delivery differs. separate institutions and there is very little contact between them. its advantages and disadvantages. The radiation exposure equipment is designed difThe two research teams work in The design of the experiments revolves around three main problems: 1-The level or dose of radiation. 3-The parameters utilized to determine the effect of radiation. Apparently, Dr. Paulsen has effective equipment and competent help of a

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physicist from the I versity of Washington, who supr ises' .le dosimetry and operational performance of the radiation equipment. The radiation doses were selected empirically and encompass several levels, ranging from a very low dose of perhaps seven roentgens all the way up to several hundred roentgens. The radiation is administered in air, an important point to be keptin mind when comparing the results with Heller's studies. In all instances, a.single radiation exposure was utilized. The time intervals after the exposure, when the various studies were performed, were again selected arbitrarily and encompassed from a few hours, up to many months after the exposure. In addition, different parameters were measured at different time intervals after the exposure. Histologic studies on testicular tissue were performed by Dr. Paulsen with the help of one of his assistants, MI-. Donald Moore. This segment of study is essentially completed and the information sought obtained. It has been shown that in humans, similar to that found in lower species, l o w and moderate levels of radiation induce damage to early stages of spermatogenesis, namely to the spermatogonial population. Following destruction of these cells, a true "maturation depletion" takes place, resulting in the disappearance of the entire germinal epithelium. Since a number of dose levels were used, a crude idea about the sensitivity of human germinal epithelium to x-irradiation was gained. While the histologic study revealed damage; it did not provide precise information concerning specific sensitivity of the germinal cells to radiation. In addition, an interesting phenomenon, also observable in lower species, was encountered; namely that when recovery occurred, it was spotty andlor markedly delayed. cause for this delay remains unlak. A specific quantitative study of the germinal epithelium was performed in order to obtain precise information of subtle changes and changes that might

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occur with very low ses. This study may also shed qht I the cytodynamic mechanism of action of x-rays on the germinal epithelium. A n approach was utilized which involves an assumption that the Sertoli cell count does not change after radiation. Making this assumption, the germinal cell count changes were expressed a s ratios to Sertoli cells. concerned with precise changes in the various cell types following radiation were obtained. This analysis was performed primarily by Dr. Barr and Mr. Moore. gn.electronic counter for sperm counts. They, as well as others, investigated this instrument for the purpose of sperm counting and demonstrated its adequacy Using this technique preliminary results quite.wel1. I strongly feel that actually, it makes very little difference as to:which- -- technique of counting is used; possibly the electronic counter provides a slightly more accurate result. It definitely cuts down on the number of techoician-hours necessary to do the counts, thus affording the opportunity of doing terest being an indication that a sperm count drop may occur following the procedure of biopsy even in cases where the biopsy did not induce obvious inflammatory reaction, hematoma or other gross pathological changes. This finding is of extreme importance, particularly for this project, since the design of experiments in Dr. Heller's group is vertical one in that they use the same individual for his own control, biopsying him repeatedly, first prior to the exposure and then at time intervals after exposure. Since the biopsy itself may have an affect on the spermatogenesis and subsequently on the sperm count, this experimental design will have to be re-evaluated. done by a steroid chemist at a different institution. Dr. Paulsen was involved The steroid studies were performed in blood. The actual determinations were

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and the individual in charge of this procedure is well known to the community of steroid chemists as an accurate'and reliable worker. , The laboratory in which these studies were performed is highly regarded I'think that the blood testosterone levels as reported by Dr. Paulsen's group are reliable and important Paulsen's group varied from those obtained by Dr. Heller's group. into the details of this problem in the "Remarks" portion of this report. . The gonadotropin studies are a problem since the results obtained by Dr. phase-chromosomes from cultures of testicular tissue. Considerably large body of studies will be necessary before a definite picture emerges, but the procedure is well in hand. rather acid criticism, I feel that Dr. Paulsen's group is quite capable and technically proficient in this particular type of cytogenetic studies. criticism of this work which is also a constructive one, is the fact-chatwhen the qhromosomal analysis is performed on cultures of testicular tissue, -one never knows from which type of cells the metaphase originates. the metaphase originate from a germinal line of cells such as the spermatogonia, or from interstitial cells o r endothelial cells? of such results would be quite different from that of damage occurring in interIt is a valuable study, In spite of Dr.-The obvious This obviously creates a problem If the damage occurred in the germinal cell, the interpretation stitial cells. To my knowledge, at this time no technique is available-to separate germinal cells from the others for such study. - . The basic design was similar to that of Dr. Paulsen's group, but detailsvaried. The range of radiation doses and intervals following radiation differed.

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Heller's group ne to 'changes in testes following ticularly nice preparations similar conclusions concc ing tne histologic radiation as the Paulsen group did. to show degenerative changes in spermatogonia, as 'early as four hours after radiation. -these cells has to be kept in mind. ire spermatogonia one can only assume from their position in the germinal epiObviously, a problem with ident,ificationof They are badly lysed and whether they really.. . The quantitative analysis of the geminal epithelium was done by two differ5nt individuals, Miss Rowley and Dr. -, using different techniques. Rowley used an approach similar to that used by Dr. Paulsen's group; she counted all the germinal cells and related them to the Sertoli cell counts. The results are quite similar to Paulsen's although not identical. Dr. Oakberg makes an attempt to stage the seminiferous epithelium and count only those areas which appear t o show intact stages. Again, there appears to be correlation between- -..- - The seminal fluid sperm count results were similar to Paulsens' group, but Paulsen's group obtained much greater drop in counts and more rapdily; Heller's group got better and more rapid recovery. The steroid studies were performed in Dr. Heller's group by Dr. Donald Brusca who is a young steroid chemist, apparently competent in his work. The approach here is different from Paulsens' group; they study the levels of free i n d conjugated testosterone in the urine, using gas chromatography. Interestingly enough, even after a l o w dose of 30r, they get an initial rise followed by a drop, and then after several weeks, a return to normal. This finding is different from Paulsen's group who observed no change in plasma testosterone levels at -1OOr and only a slight drop at 400r. The gonadotropin studies in Dr. Heller's group apparently under the direction

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immediate rise in gonadotropiv 'is ei.-&y as one day after This does not agree with Paulsen's results, which show either no rise or a rise late after radiation. The cytogenetic studies: Dr. Heller is interested in the meiotic chromosomes; consequently, they study the pachytene spermatocytes on direct preparation from testicular tissue. Dr. Norby, who is a cytogeneticist, is in charge of this work and works closely with M r . Nestor Cachiero. They have already surveyed a rather large number of cells in meiotic prophase. They hope to be able to set up a map of meiotic chromosomes. The work is promising but is still in its infancy. They do show gross changes in the chromosomes following radiation and I will have to make a comment here which mays mirror Dr. bc on un ents, and is based on our own experience with meiotic chromosomes in spermatocytes. The changes shown after radiation, appear to be real; the fragmentation is there. I agree with Dr. Cooper that it will take a lot of work to set up a map of normal chromosomes in meiotic prophase in order to see specifically where the damage occurs. the gross fragmentation and abnormalities, as shown on some of the preparations of the meiotic prophase chromosomes, appear to be real and definitely warrant I would like to reiterate that this project affords unique opportunities and that it has already given important basic information and data which can be utilized in applied work. Both projects merit further support and the fact that these are parallel studies makes the project more desirable. In the parallelism of the projects lies their strength. confirmatory strength, but also minimizes errors in procedures and interpretations, thus adding more credence to the final results. The parallelism not only provides important Also, because of variations in approach and techniques, the parallelism affords additional value. However, a number of conrments are i n order and several points of criticism, which should be

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1) A l l data, v' *I the possible exception of the ~ isto/.'.,ic observations, suffer from heterogeneity of techniques and statistical approach. While in certain areas, this heterogeneity, as mentioned above, is of benefit, in others it definitely is detrimental. Specifically, the gonadotropin assays are performed by different methods and the design of the gonadotropin assays is not satisfactory. part of the study should be carefully re-evaluated. I strongly feel that this important should get together on a common technique and common design. Otherwise, the data cannot be compared and their values are markedly decreased. In addition to the fact that the gonadotropin results are not comparable, more recent developments in the field of gonadotropin &termination suggest that the approach used, particularly the "total" gonadotropin ap- ' proach, could be improved. The seminal fluid counts also could use a good statistician. Since great variations occur from a technical as well as a biological reason, proper statistical approach to this problem is imperative. A major problem which needs attention is the basic design of the experiments, namely the vertical versus horizontal design. design, e.g. using the same individual as control and experimental subjects, consequently having the same indificual biopsied repeatedly. k.Paulsen's findings Dr. Heller prefers the vertical that biopsy is detrimental to the germinal epithelium and also the sperm production and possibly to the hormonal parameters, may change following biopsy, question this approach. Probably, a parallel study where groups of individuals are used for each t h e interval and for the different doses, would be more beneficial. The general impression from the results presented leads one to comment upon the fact that one of the major deficiencies of the project is the fact that many parameters were studied and many approaches used on too few individuals. that this study should be tightened up, cleaned up, perhaps so that fewer para-

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meters were studied this particular time, but larl num,rs of subjects employed per each point. The material that cannot be studied at this time probably could be saved and investigated at a later time. This criticism applies particularly to the parameters which involve quantitative analysis of germinal epithelium, sperm counts and hormonal changes. Since the biological variations and the variance due to the method itself are great in these parameters, it is absolutely necessary to have a large enough number (N) per point t o obtain conAs far as the quantitative analysis of germinal epithelium, which incidentally I think is still the major aim of the project, most probably, fa%ly adequate methods of analysis have evolved. anisms of normal spermatogenesis, the final analysis of the data will have to wait until this is clear. However, since we do not understand the mechFortunately? samples can be saved and re-examined when the time comes. I feel that the unique opportunity of both groups to obtain normal tissues should be utilized to its utmost. be extended to ultrastructural studies involving electronmicroscopy. may provide us with information concerning early and/or minor changes in radiated germinal epithelium. Unfortunately, the normal ultrastructural characteristics of the human testes have never been established, and this happens t o be major study electronmicroscopic characteristics of normal human testes and we have learned that it requires an individual with considerable amount of experience in this area, to Similarly, this opportunity could Off the record, our group is engaged at this moment, in the study of make heads or tails of such a study. following radiation? again may have to wait until the normal is established. theless, the material could be collected and saved since it does not spoil. Finally, I would l i k e to raise a point concerning the technique of exposure to x-irradiation. Paulsen irradiates in air, while Heller places the scrotum into a water medium. While I do not think there is a difference of amount of radiation, …