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Machine-read from the scan, so expect some errors. Lines that couldn’t be read are left out.
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1.1.1 IIabilrly OI rerponsibdily lor the accuracy. compl rk neu or uufulnerr of any information, apparatur. product or proc ~ uducloud. or rc prev n tri hati owe would no1 Infringe privalcly owned ril hls. B. ADDITIONAL OSJECTIVES AND CHANGES IN STATUS OF X-RAY IRRADIATED SUIMECTS 4 GEIERAL STATE ENT I ACCOMPLISHMENTS WITH SPECIAL REFERENCE TO CONTINUATIO;.I OF PRESENT OBJECTIVES 8 81. Cyto logical Objectives 2. t o 1ou i ca 1Acc omp 1i s h me nt s Relevant to Testes, New Nethods of Approach and New !?E- elin eating the Limits of itcrmal Human a.qila nt it ation of germinal epithelium b. Ultra structural studies c. Seminal fluid examination 4. Hormonal Accomplishments Relevant t o Delineating Norm1 Human Test tcu lar physiology; Ik th cds Used and New Methods b . Urinary interstitial cell - stimulating c . Urinary follicle - stimulating hormone f . Plasma ter to ster one 49
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5. Su mary of Specific Highlights of Work fhb repart W U prepared as r n accornl of work sponsored by the United Strlcr Government. Nelthcr (he United Stalea nor the United Stales Atomic Energy Commission, nor any of their employees, nor rny or a r b mntraclorr. iubconuactorr, or their employees, nub1 any wuranty. crpreu or implied, or muumu a n y 1.1.1 liability Q ruponsibillly for ( h e accuracy. cornpldenru or wcfulncu of any Information, apparatus, moduct or ptoceu disclosed, or represents that lis Y I C Wuld not Infringe privrlcly owned rilhts.
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In the initial grant request we proposed t o apply known amounts of ionizing rnd-iation directly to the testes of normal men i n order to ascertain specific cyto logical and hormonal information. With respect to the cyto logical information, w e proposed: (1) t o determine the exact nature of the cyto logical defect produced inthe development of the germinal epithelium and to relate the extent of the defect to dosage and time; determine dosage tolerance)that will affect the germinal epithelium; ( 3 ) t o determine t h e time of recovery from any given dosage; (2)to find the minimal dosage ( and thereby determine the minimal dosage that leads t o permanent damage of spermatoupon Leydig cell cytology. (5)to determine the simultaneous effects of any dosage With respect to hormonal information, we proposed t o determine the influence of any given radiation-produced testicular alteration upon other parameters such as st it i al cell - stimulating hormone (ICSH)excretion, (8) estrogen excretion, and (9) androgenic hormone excretion. B. ADDITIONAL OBJECTIVES AND CHANGES I N TECHNIQUES As the work progressed and as suggestions were made by the Advisory Committee of the AEC ( meeting in Seattle, November, 1963, December, 1965, March, 1967 and December, 1967 ), some parameters were added and some dropped.
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Subsequently it was propoced t o emphasize the delineation of the cyto logical defect : by quant it a ting the number of recognizably damaged cells during the first16 hours following radiation ; by determining the number of remaining cells of each cell type during the denuding period(the first 46 days ); and by assessing the problem of spermatogon i al renewal i n man. Cytogenetics was introduced as a new parameter in order to harvest the greatest amount of information from the continuing invest - abnormalities f ollow*ing irradiation during meiosis and during mitos is, The purpose of the cytogenetics was t o analyze chromosomal Early in the investigation we observed arise in total urinary gonadotropins following any radiation dose causing denuding of the germinal epithelium. Concurrently no change in urinary ICSH was observed. In order to confirm which gonadotropin was involved we began measuring urinary FSH separately using the Steelman-Pohley epithelium utilized FSH, since urinary ICSH did not change and urinary total gonadotropins increased. ( 1 ). The objective was to affirm whether the germinal We have begun measuring plasma FSH by radioimmunoassay. and add to our data obtained from urinary Observations of FSH. This method has enabled usto confirm observations on the reduction of urinary testosterone suggested that Leydig cells were affected by radiation,additional parameters were added t o measure this effect. These included quant it ation of Leydig
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cells, radioimmunoassay of plasma ICSH and measuring plasma test os - ter one by a competitive protein binding method. Currently an ultra structural study of Lcydig cells i s Underway t o determine any ultra structural changes i n morphology of the ze cells following F a two years we conducted exploratory investigations on the mapping of mei o tic( pachytene ) chromosomes in order to establish a basis for the evaluation of irradiation effects. Neither sufficient precision nor quantitative data resulted. Observing the usual parameters o r chromosomal .breaks, bridging ,translocation and other gross chromosomal defects confirmed that, in man, as in other species, radiation at each dose level (inc iud ing 10r ) caused damage. nothing new was being revealed, and following discussion with the AEC Advisory Committee team (Seattle, March, 1967 ), this approach The same committee, being intrigued with the finding of lowering of urinary testosterone values following irradiation, suggested that metabolic defects in the testicular production of testosterone may be involved. It was proposed that studying urinary pregnanediol and pre gn a net riol might reveal the metabolic defect as was found in rats by Berliner,etal. Hence these two parameters 'were measured. Finding no change,this pursuit has also been abandoned.
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A . STATUS OF X-MY Im DIA TED SUBJECTS Seven subjects were irradiated again following complete recovery from the initial dosage. Of these,three received the identical dosage 03 each of two occasions,three received two different dosages, and one received three different dosages. Each dosage is listed separately above. All proposed irradiation s have been completed. Number of subjects i n whom serial biopsies were taken throughout Number of subjects in whom biopsies wer? avoided following irradiation in order to z pec if ic ally evaluate sperm and Number of subjects in whom biopsies were avoided only during the first 90 day cell depletion period following irradiation ------ 1 9
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Number of subjects having had pre - irradiation vasectomies ---------- 1 2 Number of subjects vasectomized before release ..................... 42 Number of subjects not vasectomized before release ----------------- 0 Number of subjects who completed recovery from irradiation Number of subjects released before complete recovery from Number of subjects who returned after release but before recovery was completed and the investigation continued -------- 5 Number of subjects currently under investigation ------------------- 2 1 For each parameter studied we have found that each subject must serve as his own control. This has been confirmed as results have been analyzed statistically. upon statistical bases, that the number of control observations necessary for each parameter will depend largely upon the nature of the parameter. For example, a single control testicular biopsy usually suffices, whereas a minimum of 18 control serial weekly seminal fluid examinations are necessary to establish a base line. Moreover it has been established, For each method of analysis and for each approach much time and effort has therefore been devoted to delineating the limits and
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variations of the normal physiolo5y of reproduction. As a result, new methods of approach have been worked out and new physiological principles have been uncovered or more precisely defined. As a consequence of this attention to the normal, many of our initial publications deal with such problems. Examples are : "The testicular biopsy : surgical procedure, fixation and staining technics", "Decreases i n sperm concentration due to testicular biopsy procedure duration of each cell association and of each cell type". Heller, C. G., national Congress of Endocrinology, Excerpta Medica International Series, 184, 1012 . "Duration of transit of spermatozoa through "The, ultra structure of four types of human spermatogonia", Rowley, the cells of the seminiferous epithelium of the human testis the Sertoli cell as a constant ff. Rowley, M. J. and Heller, Another developmental aspect, regarding the radiation prog-ram, was t o bolve the problem of delivering as uniform an amount of radiation as possible to all depths of testicular tissue of both testes without
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exposing the subject t o any extraneous radiation. We rejected the conventional X-ray therapy units on the market as unsuitable because of lack of means of uniform coverage of the testes,lack of assurance that uniformity from subject to subject could be established and lack of (or awkward )shielding protection to the subject ls body. A simple portable box was designed by Feter Wootton (physicist) that allowed the scrotum and testes to drop into a plastic box of water at scrota l temperature and then be irradiated by two X-ray tubes. The tubes delivered measured amounts of known quantities of irradiation tothe two submerged testes simultaneously from two directions without ( or with the most minimal ) exposure to the pelvic region. This has been summarized a s "The ef.fects of graded doses of ionizing radiation on the testicular function of man. I. A portable device far delivery of unif o m doses of X-ray irradiation throughout external i zed or~6.iis7', Wootton, P. and Heller, C. G., and i s unpublished to date( 10 1 The accuracy of delivery of this X-ray irradiation device has been confirmed by physical as well as biological dosimetry. The physical check was seen by a team from the Hanford Washington AEC group under the supervision of Dr.William Roesch. The biological check was run by Dr. Eugene Oakberg, Oakridge, using well standardized male mice. These were submerged (in specially designed containers ) into the plastic box inthe same position as the scrota l testes. were exposed to a similar series of graded doses of radiation as the subjects ' testes. The results were comparable to similar studies at
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See Fourth Yearly Progress Report for details( 1966-67 ). Our past data has been statistically evaluated with the view t o adding observations to each parameter, where required,in order to assure a statistically acceptable result. consolidate information regarding hormonal changes resulting from For example, in order to collections for hormonal analyses must be made in eight day pools repeated six times during the control period i n order to establish a statistically valid base line for each subject for comparison with post - irradiation results. number of post - irradiation observations were also pre-determined. Some It was decided that urine The number of subjects per dose and the of the studies have been completed, others are currently being performed and others are projected. statistically significant insight into the hormonal changes. same goal i n mind we have similarly applied statistical analysis to our germinal and Leydig cell quant it ation and sperm count evaluations. Inthe end, w e expect t o have a C. ACCOMPLISHMENTS WIT&SPECIAL REFERENCE TO ORIGINALLY STATED OBJECTIVES AND PLANS FOR CONTINUATION OF PRESENT OBJECTIVES. 1. Cyt o lo q ical Objectives a. Objective: "To determine the exact nature of the cyto logical defect produced inthe development of the germinal epithelium and to relate the extent of the defect to dosage and time." Inthe following discussion dosage has been divided into "low", " interned i ate " , and "high", according to the cyto logical response elicited by exposure t o each dosage.