Attachment 9 EFFECTS OF IONIZING RADIATION ON THE TESTICULAR FUNCTION OF MAN AT (45-1) 1780 9 YEAR PROGRESS REPORT-May 1972 Carl G. Heller, M.D., Ph.D. Division of Reproductive Physiology Pacific Northwest Research Foundation TABLE OF CONTENTS Page I. OBJECTIVES 1 A. ORIGINAL OBJECTIVES 1 B. ADDITIONAL OBJECTIVES AND CHANGES IN TECHNIQUES 1 C. ABANDONED OBJECTIVES 3 II. MAIN RESEARCH ACCOMPLISHMENTS 4 A. STATUS OF X-RAY IRRADIATED SUBJECTS 4 1. Subjects Irradiated 4 2. Biopsy Date 4 3. Vasectomy Date 5 4. Status of Program 5 B. GENERAL STATEMENT 5 C. ACCOMPLISHMENTS WITH SPECIAL REFERENCE TO ORIGINALLY STATED OBJECTIVES AND PLANS FOR CONTINUATION OF PRESENT OBJECTIVES 8 1. Cytological Objectives 8 2. Cytological Accomplishments Relevant to Delineating the Limits of Normal Human Testes, New Methods of Approach and New Physiological Principles 20 a. Quantitation of germinal epithelium 20 b. Ultrastructural studies 24 c. Seminal fluid examination 31 d. Leydig cells 34 3. Hormonal Objectives 38 4. Hormonal Accomplishments Relevant to Delineating Normal Human Testicular Physiology: Methods Used and New Methods of Approach 42 a. Total genadotropins 42 b. Urinary interstitial cell-stimulating hormone (ICSH) 42 c. Urinary follicle-stimulating hormone (PSH) 43 d. Urinary testosterone 44 e. Plasma PSH and ICSH 44 f. Plasma testosterone 49 5. Summary of Specific Highlights of Work to Date 54 a. Cytological 54 b. Hormonal 56 III. REFERENCES 58 I. OBJECTIVES A. ORIGINAL OBJECTIVES In the initial grant request we proposed to apply known amounts of ionizing radiation directly to the testes of normal men in order to ascertain specific cytological and hormonal information. With respect to the cytological information, we proposed: (1) to determine the exact nature of the cytological defect produced in the development of the germinal epithelium and to relate the extent of the defect to dosage and time; (2) to find the minimal dosage (and thereby determine dosage tolerance) that will affect the germinal epithelium; (3) to determine the time of recovery from any given dosage; (4) to determine the minimal dosage that leads to permanent damage of spermatogenic cells; (5) to determine the simultaneous effects of any dosage upon Leydig cell cytology. With respect to hormonal information, we proposed to determine the influence of any given radiation-produced testicular alteration upon other parameters such as (6) total gonadotropin and (7) inter-stitial cell-stimulating hormone (ICSH) excretion, (8) estrogen excretion, and (9) androgenic hormone excretion. B. ADDITIONAL OBJECTIVES AND CHANGES IN 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. 1 Subsequently it was proposed to emphasize the delineation of the cytological defects by quantitating the number of recognizably damaged cells during the first 16 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 spermatogonial renewal in man. Cytogenetics was introduced as a new parameter in order to harvest the greatest amount of information from the continuing investigation. The purpose of the cytogenetics was to analyze chromosomal abnormalities following irradiation during meiosis and during mitosis, if possible. Early in the investigation we observed a rise 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 assay method (1). The objective was to affirm whether the germinal epithelium utilized FSH, since urinary ICSH did not change and urinary total gonadotropins increased. WE have begun measuring plasma FSH by radioimmunoassay. This method has enabled us to confirm and add to our data obtained from urinary observations of FSH. Since observations on the reduction of urinary testosterone suggested that Leydig cells were affected by radiation, additional parameters were added to measure this effect. These included quantitation of Leydig cells, radioimmunoassay of plasma ICSH and measuring plasma testosterone by a cooperative protein binding method. Currently an ultrastructural study of Leydig cells is underway to determine any ultrastructural changes in morphology of these cells following irradiation. 2 C. ABANDONED OBJECTIVES For the years we conducted exploratory investigations on the mapping of meiotic (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 or chromosomal breaks, bridging, translocation and other gross chromosomal defects confirmed that, in man, as in other species, radiation at each dose level (including 10r) caused damage. Since nothing new was being revealed, and following discussion with the AEC Advisory Committee team (Seattle, March, 1967), this approach was abandoned. 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 pregnanetriol might reveal the metabolic defect as was found in rats by Berliner, et al. (2). Hence these two parameters were measured. Finding no change, this pursuit has also been abandoned. 3 II. MAIN RESEARCH ACCOMPLISHMENTS A. STATUS OF X-RAY IRRADIATED SUBJECTS 1. Subjects Irradiated Total numbers of subjects irradiated 74 8r 4 78r 7 10r 3 100r 10 15r 1 200r 13 20r 8 235r 1 25r 2 300r 2 50r 5 400r 2 5r/11 weeks=55r 1 600r 15 Seven subjects were irradiated again following complete recovery from the initial dosage. Of these, there received the identical dosage on each of two occasions, three received two different dosages, and one received three different dosages. Each dosage is listed separately above. All proposed irradiations have been completed. 2. Biopsy Date Number of subjects in whom serial biopsies were taken throughout the irradiation period 42 Number of subjects in whom biopsies were avoided following irradiation in order to specifically evaluate sperm and hormonal alterations 13 Number of subjects in whom biopsies were avoided only during the first 90 day cell depletion period following irradiation 19 4 3. Vasectomy Data Number of subjects having had pre-irradiation vasectomies 12 Number of subjects vasectomized before release 42 Number of subjects not vasectomized before release 0 4. Status of Program Number of subjects who completed recovery from irradiation before release 22 Number of subjects released before complete recovery from irradiation 26 Number of subjects who returned after release but before recovery was completed and the investigation continued 5 Number of subjects currently under investigation 21 B. GENERAL STATEMENT 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. Moreover it has been established, 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. For each method of analysis and for each approach much time and effort has therefore been devoted to delineating the limits and variations of the normal physiology of reproduction. As a result, new methods of approach have been worked out and new physiological principles have been uncovered or more precisely defined. 5 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", Rowley, M. J. and Heller, C. G., Fertil. Steril., 17:177, 1966 (3), "Decreases in sperm concentration due to testicular biopsy procedure in man", Rowley, M. J., O'Keefe, K. B. and Heller, C. G., J. Urol., 101:347, 1969 (4). "Human spermatogenesis: An estimate of the duration of each cell association and of each cell type". Heller, C. G., Heller, G. V. and Rowley, M. J., Progress in Endocrinology, III International Congress of Endocrinology, Excerpta Medica International Series, 184, 1012 (5). "Duration of transit of spermatozoa through the human male ductular system", Rowley, M. J., Teshima, F. and Heller, C. G., Fertil. Steril., 21:390, 1970 (6). "A method for the quantification of Leydig cells in man", Heller, C. G., Lalli, M. F., Pearson, J. E. and Leach, D. R., J. Reprod. Fert., 25:177, 1971 (7). "The ultrastructure of four types of spermatogonia", Rowley, M. J. and Heller, C. G., Z. Zellforsch., 112:139, 1971 (8). "Quantitation of the cells of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant". Rowley, M. J. and Heller, C. G., Z. Zellforsch., 115:461, 1971 (9). Another developmental aspect, regarding the radiation program, was to solve the problem of delivering as uniform an amount of radiation as possible to all depths of testicular tissue of both testes without exposing the subject to any extraneous radiation. We rejected the conventional X-ray therapy units on the market as unsuitable because of lack of meant 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's body. A simple portable box was designed by Peter Wootton (physicist) that allowed the scrotum and testes to drop into a plastic box of water at scrotal temperature and then be irradiated by two X-ray tubes. The tubes delivered measured amounts of known quantities of irradiation to the two submerged testes simultaneously from two directions without (or with the most minimal) exposure to the pelvic region. This has been summarized as "The effects of graded doses of ionizing radiation on the testicular function of man. I. A portable device for delivery of uniform doses of X-ray irradiation throughout externalized organs", Wootton, P. and Heller, C. G., and is unpublished to date (10). The accuracy of delivery of this X-ray irradiation device has bene 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 in the same position as the scrotal testes. They were exposed to a similar series of graded doses of radiation as the subjects 1 testes. The results were comparable to similar studies at Oakridge, Berkeley, and M.I.T., and verified the accuracy of exposure. 7 See Fourth Yearly Progress Report for details (1966-67). Our past data has been statistically evaluated with the view to adding observations to each parameter, where required, in order to assure a statistically acceptable result. For example, in order to consolidate information regarding hormonal changes resulting from X-ray exposure to the testes, a low (8r), a medium (78r) and a high dose (600r) were selected. It was decided that urine collections for hormonal analyses must be made in eight day pools repeated six times during the control period in order to establish a statistically valid base line for each subject for comparison with post-irradiation results. The number of subjects per dose and the number of post-irradiation observations were also pre-determined. Some of the studies have been completed, others are currently being performed and others are projected. In the end, we expect to have a statistically significant insight into the hormonal changes. With the same goal in mind we have similarly applied statistical analysis to our germinal and Leydig cell quantitation and sperm count evaluations. C. ACCOMPLISHMENTS WITH SPECIAL REFERENCE TO ORIGINALLY STATED OBJECTIVES AND PLANS FOR CONTINUATION OF PRESENT OBJECTIVES 1. Cytological Objectives a. Objective: "To determine the exact nature of the cytological defect produced in the development of the germinal epithelium and to relate the extent of the defect to dosage and time." In the following discussion dosage has been divided into "low", "intermediate", and "high", according to the cytological response elicited by exposure to each dosage. 8 Low dosage effects (10 - 100r): Examination of serial testicular biopsies following exposure to irradiation, revealed that the spermatogonia were primarily affected, and that more mature cells were allowed to complete normal development. The overt damage to the spermatogonia was revealed by pyknosis and other signs of degeneration. The concealed damage to otherwise normally appearing spermatogonia became overt as revealed by the failure of the cells to undergo mitosis and produce preleptotene spermatocytes. At the same time (for dosages of 100r or less) the preleptotene, other spermatocytes and the spermatids failed to reveal either overt or concealed damage. The latter was deduced from their ability to undergo development and maturation (including undergoing maturation-division) to become normal mature spermatozoa and to appear in the ejaculate as normal spermatozoa in regard to numbers as well as morphology. Since the preleptotene spermatocytes were not replaced by the spermatogonia, however, the production of sperm ceased when depletion was completed. Thus the three consequences of "low" dose irradiation are: 1) denuding of the germinal epithelium, 2) damage to spermatogonia, and 3) reduction of number of sperm in the ejaculate to azoospermia at the 100r dose. To evolve these conclusions serial testicular biopsies were obtained at intervals of four to six hours, 16, 24 , 40, 46, 60 and 72 days following irradiation. The germinal cells were studied by light microscopy. The first time of visualizing total depletion in the testes was after 46 days. This is exactly the period of time needed for development of the preleptotene spermatocyte into a mature spermatozoa about to leave the Sertoli cell cytoplasm (11). The time of visualizing the first effect following irradiation was four to six hours. The germinal cells damaged at this time were the spermatogonia. The time of the first effect of X-ray as revealed by the seminal fluid was after 46 days. This is accounted for by the normal development of spermatocytes (46 days) plus the transit time (21 days) during transport of sperm through the ductular system to the ejaculate. Hence azoospermia was not revealed prior to 67 days. 9 The intermediate dosages of X-ray irradiation (less than 400r, greater than 100r) cause degeneration in an additional group of cells, the spermatocytes. In contrast to the lower dosages where the spermatocytes develop normally, at intermediate doses, these cells are covertly injured and not all are allowed to proceed through normal maturation-division. As a result spermatids arising from irradiated spermatocytes are decreased in number. Spermatogonia are overtly injured and their numbers are decreased even more than at the lower doses. This ultimately results in a longer recovery time. Therefore, we have found that at intermediate dosages of between 100 and 400r: 1. Spermatogonia show overt damage but no decrease in numbers within the first 24 hours. 2. Spermatocytes are covertly affected and degenerate while proceeding through maturation-division. 3. The result of spermatocyte injury is a significant decrease in spermatids. 4. The sperm count regularly falls to azoospermia after approximately 67 days. 5. The seminal fluid fails to reveal the reduction in normal spermatid numbers during the first 46 days. This is possibly accounted for by the time of residence of spermatozoa in the ductular system. This varies from one to 21 days (6). Thus mixing of generations of spermatozoa may obscure the reduction in testicular production of spermatozoa. High doses of irradiation (400 - 600r) yield a further response. All cells of the germinal series are injured. Spermatogonia and spermatocytes are overtly damaged and spermatids are covertly damaged. The additional damage to spermatids (revealed by quantitation of germinal cells and sperm count) and the overt spermatocyte damage are found only at this high irradiation dose. As a result of the severe decimation of cell numbers prior to 46 days, the sperm count also falls sharply prior to 46 days. Thus, at high dosages of irradiation, 400r and above, we have found: 10 1. Spermatogonia and spermatocytes are overtly damaged. 2. Spermatids are covertly damaged as revealed by quantitation of testicular cytology and sperm count and morphology. 3. Significant decreases in all cells of the germinal series are observed prior to 46 days. 4. A significant decrease occurs in sperm count prior to 46 days. 5. The sperm count falls sharply to azoospermia and stays there many months. 6. The first visualization of spermatogonial degeneration was 16 minutes after irradiation. The observations were made ultrastructurally. We conclude from the combination of morphological and quantitative data from all dosages that the spermatogonia are the most radiosensitive cell type and spermatids are probably the most radio-resistant cells. Our conclusions are summarized in the following table: [Table] FOR REFERENCE SEE (8bb17.gif) 11 A striking aspect of this data is that cells literally next to each other both in development and spacial placement within the tubule have such different radioresistance. For instance, the type B spermatogonium is the most radiosensitive cell. The cells preceding it (the Ad and Ap) are also radiosensitive. However, the cell arising from the B spermatogonium, the preleptotene spermatocyte, requires ten-fold the amount of X-ray irradiation that the B requires to be damaged. The next major difference in cell radiosensitivity is between the pachytene spermatocyte and the Sa spermatid. Again these cells are adjacent both developmentally and spatially, yet the spermatid requires four times the amount of radiation to be damaged. Thus the spermatids are 40 times as resistant to irradiation damage as are the spermatogonia. In the past year we have completed all proposed irradiations of subjects and thus have an outline of the cytological defect in relation to dose and time. At present we are analyzing the effects at various dose levels in order to have statistically valid dose-response curves for histological quantitation and sperm count. b. Objective: "To find the minimal dosage that will affect the germinal epithelium." The foregoing discussion has revealed the cytological defects were observed at many dose levels. The minimal dose level that produces azoospermia, for example, is 100r. Marked oligospermia was produced following irradiation doses of 78r (seven subjects studied) and 50r (four subjects studied). Sperm counts were decreased from normal to circa 2 m/cc in each of these eleven subjects. Moderate oligospermia was produced at 20r (eight subjects) and 15r (one subject). 12 On the basis of the effects upon sperm counts, testicular cytology, and hormonal studies, the 50 and 78r subjects can be grouped in a single dosage class, and the 15, 20, and 25r groups can be consolidated as giving a single biological effect. Of the three receiving 10r, insufficient seminal fluid data (or none) was obtained because of: 1) sudden parole, 2) prior vasectomy and 3) one subject was subjected to too frequent biopsy operations to be certain that the fall in sperm count was indeed due to irradiation. Of the four receiving 8r, two failed to reveal a drop in sperm count and two revealed a minimal or equivocal drop. In the latter two the drop at best was transient, however, the lowest anticipated point in time for these subjects occurred during the riots at the Oregon State University, and no observations were possible during this critical period. In the 8r group, testicular biopsies were avoided in order to observe "pure" seminal fluid effects. Of the three receiving 10r, only one had serial biopsies for cytological study, another had biopsies for chromosomal study only. The cytological study, however, did reveal irradiation induced changes. Assuming "no change" in sperm count for the four subjects receiving 8r, does not rule out the possibility of transient or minimal cytological damage. The later could be obscured in the seminal fluid analysis due to mixing of generations of spermatozoa during the 21-day transit time through the ductular apparatus. 13 The status of "the minimal dosage affecting cytology" thus is that irradiation doses as low as 10r result in damage to the germinal epithelium in one instance, and doses of 15, 20 and 25r regularly result in damage. We now have added the additional subjects at the required dosages to study both seminal fluid observations and testicular biopsies to more clearly define the minimal dose of X-ray irradiation to the human testis. C. Objective: "To determine the time of recovery from any given dose." Recovery time is being determined using two parameters: sperm count and testicular cytology. The two endpoints being applied are the first appearance of spermatozoa in the seminal fluid (if the subject was at azoospermia) or the first increase noted (if the subject was oligospermia) and the first resurgence of maturation of the spermatogonial cells of the testis. We are finding a large dichotomy in the starting time of testicular recovery versus the increase in seminal fluid sperm count, most pronounced at the intermediate and high doses. Beginning testicular recovery precedes the appearance of sperm in the seminal fluid. The status of this aspect of the investigation is at the completion of the probing stage, which has revealed the dichotomy mentioned above. Hence the listing of the beginning of recovery of the germinal cells is now only a rough approximation. Now that the times that biopsies should be obtained for a given dose to yield the maximal information is better understood, we can obtain precise data on recovery. The above will, of course, apply as well to the objective "spermatogonial renewal". 14 Two other facets of the mechanisms of the investigation upon cytological recovery have kept the number of observations minimal. These are that in approximately one-half of the subjects, biopsies have been avoided in order not to interfere with sperm counts. The second reason, is that no matter how carefully a subject is selected, from the point of view of time to be served in the penitentiary, the attrition rate after some years is extremely high. Attrition is due to unexpected pardons or parole, transfer to the Forest Camp, violation of prison regulations and removal from the general population (and from the experiments) or voluntary withdrawal. Some recovery data has been gathered from each dosage studied and is tabulated as follows: 15 [Table] [FOR REFERENCE SEE 8bb18.gif] 16 The preliminary observations suggest that at low doses (10 - 100r) beginning recovery of sperm count occurs at about six months, for intermediate doses (200 - 300r) at ten months, and for high doses (400 - 600r) at about two years. d. Objective - Complete recovery: "To determine whether recovery is complete or incomplete. To be certain that the same level of sperm count is attained following irradiation as existed before irradiation." (not explicitly stated under I. OBJECTIVES, but often discussed with the Committee). For each subject, many, many control seminal fluid evaluations had to be made to establish a normal value that is statistically acceptable. After the recovery phase, the same number of observations must be made to establish a value that is statistically acceptable. The preliminary result is that in those subjects where sufficient time has elapsed and sufficient observations have been available, recovery appears to be complete, i.e., the same sperm count levels have been attained. The preliminary observations on complete recovery as seen from the table suggest that for the low doses, it is nine to 18 months, for intermediate doses, 30 months, and for high doses, longer than 57 months (none recovered to date). e. Objective: "To determine whether subsequent irradiation following depression and full recovery will lead to a more severe reaction, postpone recovery, or lead to incomplete recovery." (another objective not stated, but discussed with, and by, Dr. Paul Henshaw). 17 In seven subjects that were irradiated a second or third time following complete recovery, the response to the repeated dose was in every way comparable to the initial dose-response. f. Objective: "To assess the problem of spermatogonial renewal in man." he observations were made ultrastructurally. We conclude from the combination of morphological and quantitative data from all dosages that the spermatogonia are the most radiosensitive cell type and spermatids are probably the most radio-resistant cells. Our conclusions are summarized in the following table: [Table] FOR REFERENCE SEE (8bb17.gif) 11 A striking aspect of this data is that cells literally next to each other both in development and spacial placement within the tubule have such different radioresistance. For instance, the type B spermatogonium is the most radiosensitive cell. The cells preceding it (the Ad and Ap) are also radiosensitive. However, the cell arising from the B spermatogonium, the preleptotene spermatocyte, requires ten-fold the amount of X-ray irradiation that the B requires to be damaged. The next major difference in cell radiosensitivity is between the pachytene spermatocyte and the Sa spermatid. Again these cells are adjacent both developmentally and spatially, yet the spermatid requires four times the amount of radiation to be damaged. Thus the spermatids are 40 times as resistant to irradiation damage as are the spermatogonia. In the past year we have completed all proposed irradiations of subjects and thus have an outline of the cytological defect in relation to dose and time. At present we are analyzing the effects at various dose levels in order to have statistically valid dose-response curves for histological quantitation and sperm count. b. Objective: "To find the minimal dosage that will affect the germinal epithelium." The foregoing discussion has revealed the cytological defects were observed at many dose levels. The minimal dose level that produces azoospermia, for example, is 100r. Marked oligospermia was produced following irradiation doses of 78r (seven subjects studied) and 50r (four subjects studied). Sperm counts were decreased from normal to circa 2 m/cc in each of these eleven subjects. Moderate oligospermia was produced at 20r (eight subjects) and 15r (one subject). 12 On the basis of the effects upon sperm counts, testicular cytology, and hormonal studies, the 50 and 78r subjects can be grouped in a single dosage class, and the 15, 20, and 25r groups can be consolidated as giving a single biological effect. Of the three receiving 10r, insufficient seminal fluid data (or none) was obtained because of: 1) sudden parole, 2) prior vasectomy and 3) one subject was subjected to too frequent biopsy operations to be certain that the fall in sperm count was indeed due to irradiation. Of the four receiving 8r, two failed to reveal a drop in sperm count and two revealed a minimal or equivocal drop. In the latter two the drop at best was transient, however, the lowest anticipated point in time for these subjects occurred during the riots at the Oregon State University, and no observations were possible during this critical period. In the 8r group, testicular biopsies were avoided in order to observe "pure" seminal fluid effects. Of the three receiving 10r, only one had serial biopsies for cytological study, another had biopsies for chromosomal study only. The cytological study, however, did reveal irradiation induced changes. Assuming "no change" in sperm count for the four subjects receiving 8r, does not rule out the possibility of transient or minimal cytological damage. The later could be obscured in the seminal fluid analysis due to mixing of generations of spermatozoa during the 21-day transit time through the ductular apparatus. 13 The status of "the minimal dosage affecting cytology" thus is that irradiation doses as low as 10r result in damage to the germinal epithelium in one instance, and doses of 15, 20 and 25r regularly result in damage. We now have added the additional subjects at the required dosages to study both seminal fluid observations and testicular biopsies to more clearly define the minimal dose of X-ray irradiation to the human testis. C. Objective: "To determine the time of recovery from any given dose." Recovery time is being determined using two parameters: sperm count and testicular cytology. The two endpoints being applied are the first appearance of spermatozoa in the seminal fluid (if the subject was at azoospermia) or the first increase noted (if the subject was oligospermia) and the first resurgence of maturation of the spermatogonial cells of the testis. We are finding a large dichotomy in the starting time of testicular recovery versus the increase in seminal fluid sperm count, most pronounced at the intermediate and high doses. Beginning testicular recovery precedes the appearance of sperm in the seminal fluid. The status of this aspect of the investigation is at the completion of the probing stage, which has revealed the dichotomy mentioned above. Hence the listing of the beginning of recovery of the germinal cells is now only a rough approximation. Now that the times that biopsies should be obtained for a given dose to yield the maximal information is better understood, we can obtain precise data on recovery. The above will, of course, apply as well to the objective "spermatogonial renewal". 14 Two other facets of the mechanisms of the investigation upon cytological recovery have kept the number of observations minimal. These are that in approximately one-half of the subjects, biopsies have been avoided in order not to interfere with sperm counts. The second reason, is that no matter how carefully a subject is selected, from the point of view of time to be served in the penitentiary, the attrition rate after some years is extremely high. Attrition is due to unexpected pardons or parole, transfer to the Forest Camp, violation of prison regulations and removal from the general population (and from the experiments) or voluntary withdrawal. Some recovery data has been gathered from each dosage studied and is tabulated as follows: 15 [Table] [FOR REFERENCE SEE 8bb18.gif] 16 The preliminary observations suggest that at low doses (10 - 100r) beginning recovery of sperm count occurs at about six months, for intermediate doses (200 - 300r) at ten months, and for high doses (400 - 600r) at about two years. d. Objective - Complete recovery: "To determine whether recovery is complete or incomplete. 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The following reagents are then added to the vial using standard preparations for handling large amounts of radioactive iodine: 1. 25 of a 0.4 M PO4, pH 7.5. 2. 10 of 2 mg of ICSH or FSH in a 0.1 M PO4, 0.15 M NaCl, pH 7.8 buffer. 3. 10 of chloramine I (25 mg in 10 ml of above buffer). 4. 25 of Na2 S205 (25 mg in 10 ml of above buffer). The entire reaction mixture is applied to the exposed surface of the Sephadex G-75 column. The column is prepared in a soft glass tube previously equilibrated in 0.01 M PO4 NaCl, pH 7.8 at room temperature, and washed with 1 ml of 2% bovine serum albumin to coat the glassware and Sephadex with albumin, thereby preventing absorption of radioiodinated hormone onto the glass tube. Separation of ICSH 131I or FSH-131I from inorganic 131I is achieved by passing this solution through the Sephadex column. The eluants are collected ten drops per tube for 20 tubes, and counted in the gamma counter. In our previous studies, two peaks of radioactivity are obtained; an early peak beginning at tube three to five and trailing off by tube six; and a second peak containing free 131I beginning at about tube seven. The ICSH-131I or FSH-131I and is used in the assay. The specific activity of ICSH-131I or FSH-131I and is used in the assay. The specific activity of ICSH-131I is between 200 to 500 uc per ug, and is 250 to 650 uc per ug for FSH-131I. 36 The radioimmunoassays will be carried out using Odell's methods with slight modifications. All reagents will be added to 10 x 75 mm tubes in the following order: 1. buffer (as mentioned earlier) to make a total volume of 1.0 ml. 2. 100 (ul) of 0.1 MEDTA, pH 7.8. 3. 200 of plasma to be assayed (or of "standard hormone"). 4. 100 containing 0.05 to 0.15 mug ICSH-131I or FSH-131I. 5. 100 of antisera suitably diluted usually 1:10,000 (final dilution 1:100,000 for anti-HCG) or 1:40,000 (final dilution 1:400,000 for anti-FSH). Complete standard dose-response curves are fun in all assays, and plotted on a semilog paper. For this purpose known amounts of ICSH or FSH are added instead of plasma. The range of standard we are using is from 0.1 mIU to 100 mIU (1mIU = 2.08 mug of LER-907 for ICSH and 5.0 mug for FSH). All tubes are incubated for four days at 40C at which time 50 of anti-RGG (2nd antibody) is added to each tube and the mixture incubated 24 hours longer at 40C to achieve separation of antibody-bound from free ICSH-131I or FSH-131I. Tubes are then centrifuged at 500g and the supernatant removed by suction. Radioactivity is measured in a gamma spectrometer, and all results expressed as a per cent of counts per precipitate. Zero per cent is defined as no ICSH-131-I (FSH- 131I in the case of FSH) bound to anti-body. In our previous studies, 5 - 10% of iodinated hormone was non-specifically trapped in the precipitate. These counts could be removed by washing the precipitate but this was found not to contribute significantly to the precision of the assay. One hundred per cent is defined as the number of counts precipitated in tubes containing ICSH-131I or FSH-131I and antibody, but no unknown or standard ICSH or FSH. The results are calculated in terms of ug of LER-907 per 100 ml of plasma. Each assay of pooled plasma is run as a control reference along with the unknown plasma. The plasma samples of the same subject are run in the same assay if possible. We have found that the intra-assay variation (2 S.D.) is + 11% for ICSH and + 10% for FSH. The inter-assay variation (2 S.D.) is + 31% for ICSH and + 30% for FSH. These techniques are capable of measuring between 0.8 ng to 30 ng per assay tube for ICSH and between 4 ng to 75 ng per assay tube for FSH. We have found that the average concentration of plasma ICSH taken from 50 healthy men is 9.04 + 2.65 ug LER-907/100 ml. The average concentration of plasma FSH taken from 35 healthy men is 34.4 8.55 ug LER-907/100ml. 37 Since standard LER-907 was suggested by the National Pituitary Agency in 1968, two laboratories have been compared our results with are Nankin (32) and Paulsen (33). Our results are comparable to these two laboratories in terms of ug LER-907/100ml plasma, as shown in the following table: No. Plasma ICSH* No. Plasma FSH Normal mean range Normal mean range men men _________________________________________________________________ Our Laboratory 50 9.0 4.8 - 15.3 35 34.0 22 - 45. Nankin (32) 79 7.6 3 - 21.0 57 19.0 8 - 41. Paulsen (33) - - - 54 - 23 - 53 * g LER-907/100ml plasma We have also exchanged blinded samples with Dr. Paulsen and found equivalent values. Our previous results are also comparable to mIU 2nd IRP-HMG if conversion factors are used. However, there is confusion if mIU of LER-907 is used, because other conversion factors are involved of which, unfortunately, many investigators are not aware. (radioimmunoassay 1mIU LER ILLEGIBLE = 4.6 mIU 2nd IRP-HMG for ICSH, and 1 mIU LER-907 = 1.9 mIU 2nd IRP-HMG for FSH). In addition, we have also measured plasma ICSH and FSH in men exhibiting a variety of abnormal conditions. A few of these are listed in the following table: Plasma ICSH* Plasma FSH* mg LER-907/100ml mg LER-907/100ml Hypogonadotropic hypo- gonadism 4.0 17.5 Functional preuberal castrate syndrome 65.5 222.0 Surgical castrate 52.6 111.5 Sertoli-cell-only syndrome 26.6 126.5 Postmenopausal syndrome 121.3 436.0 Male climacteric syndrome 114.0 194.0 * Normal men (ICSH range 4.8 - 15.3; FSH range 22.0 - 45.4) f. Plasma testosterone Plasma testosterone is measured by competitive protein binding by the method of Murphy(34). This involves extraction of plasma with ether three times, shaking each time on a Vortex mixer. The ether phase is dried down in a water bath at 450C with filtered air and the extract then placed on a column chromatograph of Sephadex LH-20 (column dimensions approximately 3/8" x 17"). The solvent system is chloroform: heptane:ethanol:water in the proportions 50:?50:1:0.12 (saturation). The testosterone fractions, located by running an identical column of tritium-labeled testosterone only, are collected in 2.5 ml aliquot and dried down. 38 To determine the amount of testosterone in these fractions, standard curves are set up for each assay. A series of tubes containing 0, .1, .2, .4, .6, .8, 1.0, 1.2 and 1.5 ng of testosterone in ethanol are dried down. To these standard curve tubes and the fractions containing unknown amounts of testosterone are added .1 ml of "protein tracer" (0.1 ml third trimester pregnancy plasma, 0.09 ml of 10 uc/ml3H-T in ethanol, 10 ml .2M phosphate buffer). The tubes are then incubated five minutes at 450C, then 30 minutes at 40C. All following procedures are carried out at 40C. A 1 ml aliquot of 10% Korenman's suspension (in phosphate buffer) is then added to each tube to absorb free testosterone, and the tubes incubated for five minutes. The tubes are shaken for one minute, then centrifuged at 4000 rpm for five minutes. A 0.5 ml aliquot of the supernatant is pipetted off and counted in a scintillation counter. The curve obtained by plotting cpm vs. ng testosterone in the standard curve tubes is used to determine the amount of testosterone in the plasma sample. Results are reported as ng testosterone/100ml plasma. This assay can be performed by two persons in two and one- half days, running 14 columns - six duplicate samples, one tracer column, and one plasma pool column. We found that all glassware must be washed ultrasonically, then washed with distilled ethanol, and finally washed with double-distilled H2O. Ether used in the extraction must be freshly distilled. We find we can run three assays every two weeks, with fortuitous timing of dishwashing, distillations, etc. We currently do not distill the solvents in our solvent system; instead, we treat the solvent system with Norit A and filter through a Millipore system. One of the biggest problems in all plasma testosterone assays by competitive protein binding has been the solvent blank (34, 35) in the chromatography procedures. It affects the amount of testosterone "seen" by the protein - either by competing with testosterone or somehow altering the site of binding. Besides presenting problems for the standard curve, the solvent blank is often not reproducible. It has been suggested that the effect of the solvent blank be "subtracted" from the standard curve. This is not possible, as the solvent blank effect varies with the level of testosterone (34, 35). 39 This method eliminates both problems. Chromatography on LH- 20 presents a small and reproducible solvent blank. Since an accurate aliquot of 2.5 ml of solvent is collected, it is possible to add 2.5 ml of solvent to each of the tubes of the standard curve, compensating for its effect. With 24 duplicate and triplicate plasma samples over a range of 0.1 to 1.5 ng testosterone, the average % difference larger value - smaller value x 100 is 5.2%, with a range of larger value 0.2 to 16.5% In water blanks testosterone was undetectable. An indication of minimum sensitivity is the detection of testosterone at the female level with a plasma sample of 0.5 ml. From this, and indications from the shape of a "profile" curve, we can obtain by plotting eluate volume vs. ng testosterone equivalent, we can routinely detect 0.5 ng testosterone. We have done the following plasma testosterone determinations: Subject Testosterone (ng/100 ml) Functional prepuberal castrate (age 14) 60 Hypogonadism 46 Male climacteric 208 A female patient had a plasma testosterone level of 42 ng/100ml. Subjects receiving testosterone propionate have testosterone levels exceeding 1100 ng/100ml. Our general agreement with other established methods of plasma testosterone measurement is shown on the following table: [FOR REFERENCE SEE 8bb22.gif] 5. Summary of Specific Highlights of Work to Date a. Cytological i) Development of a method for the quantitation of germinal cells. ii) Development of a method for the quantitation of Leydig cells. iii) Confirmation of the timing of spermatogenesis in normal men as reported by Heller and Clermont 11), with X-ray as the tool (16) iv) An outline of the quantitative response of the various germinal cell types to irradiation at dose levels of approximately 10r to 600r. 40 v) Classification of the dose-response for the various germinal cell types. Low doses (10r - 100r) - spermatogonia affected Intermediate doses (100r - 300r) - spermatogonia affected as well as spermatocytes (but the latter do not appear visibly damaged under the light microscope) High doses (400r - 600r) - all cell types are affected; spermatids, however, are not visibly damaged using the light microscope. vi) Evaluation of the effect of biopsy upon sperm count. vii) Ultrastructural description of four types of human spermatogonia, a first in the field of electron microscopy (8) viii) Preliminary identification of another type of spermatogonium, perhaps radiation-resistant, from data on light microscopic examination of X-ray-depleted biopsies. ix) Human germinal cells embedded in Epon and viewed under the light microscope have been described for the first time. x) Determination of ductular transport time of mature spermatozoa from the time they are released from the Sertoli cell cytoplasm until they appear in the ejaculate(6). xi) Discovery that the morphology of sperm during the recovery is normal, that is, the testis cleans itself of abnormal cells. xii) Quantitative inspection of the recovery of all doses studied following irradiation. The earliest spermatogonial recovery begins at approximately 150 days for all doses. xiii) Individuals given the same dose of X-ray irradiation respond in a slightly different manner, i.e., recovery may take longer in one than another. 41 xiv) Discovery that humans are unique with regard to germinal epithelium recovery, as compared to other mammals studied. Surviving human spermatogonia do not repopulate before differentiation occurs as with mouse, rat, etc. In humans the spermatogonia differentiate rapidly into more mature cells. Thus during the process of depletion in mouse (21), spermatogonia surviving irradiation damage quickly renew themselves and repopulate the seminiferous tubules. Only after such renewal takes place does differentiation occur. In man, following doses of 15 to 50, the surviving spermatogonia do not repopulate the entire seminiferous tubule, but after some slight renewal effort, quickly differentiate. This further denudes the germinal epithelium and further lowers sperm count. Later, as more spermatogonia begin renewal but also begin differentiation, this phenomenon results in a great delay in recovery at all doses (15 to 600r). xv) Determination of the duration of each cell type by evaluation of germinal cell quantitation (5). xvi) Immediate effect of X-ray on sperm morphology. During depletion sperm remain normal following irradiation at doses below 400r; at 40r and above, sperm morphology is severely damaged in the first 67 days after X-ray, indicating damage to cells that were spermatids at the time of irradiation. b. Hormonal i) Accumulation of further evidence toward substantiating the "utilization hypothesis" (43), that is, that gonadotropins are directly related to the functional status of the testes. This assumes that the germinal elements in the testes normally utilize gonadotropin and that following cellular depletion of the tubule less gonadotropin is utilized. This results in more gonadotropin appearing in the venous effluent of the testes, the general circulation and in the urine. This is consistent with our finding that total gonadotropins are increased as the germinal epithelium is denuded following irradiation. ii) Data showing that urinary and plasma follicle-stimulating hormone increases as the germinal epithelium is depleted following irradiation, and decreases as repopulation occurs. Also, data revealing that urinary ICSH does not increase following irradiation. iii) Adaptation of a method for the radioimmunological determination of plasma ICSH, which shows a rise in plasma ICSH following irradiation. iv) Leydig cell function appears to be depressed by higher doses of irradiation as reflected by lowered urinary testosterone levels. Compensatory mechanism seem to be elicited as reflected by increase in plasma ICSH and increase in Leydig cell numbers at high dose levels. 42 v) Administration of exogenous human chorionic gonadotropins following irradiation reveals that the Leydig cells are as capable of responding to this stimulus as are normal Leydig cells. This may explain their response to the elevated endogenous ICSH. III. REFERENCES 1. Steelman, S.L. and Pohley, F.M.: Assay of the follicle- stimulating hormone based on the augmentation with human chorionic gonadotropin, Endocrinology, 53:604, 1953. 2. Berliner, D.L., Ellis, L.C. and Taylor, G.N.: The effects of ionizing radiation on endocrine cells. II. Restoration of androgen production with a reduced nicotinamide adenine dinucleotide phosphate-generating system after irradiation of rat testes, Radiat. Res., 22:345, 1964. 3. Rowley, M.J. and Heller, C.G.: The testicular biopsy: surgical procedure, fixation and staining technics, Fertil Steril., 17:177, 1966. 4. Rowley, M.J., O'Keefe, K.B. and Heller, C.G.: Decreases in sperm concentration due to testicular biopsy procedure in man, J. Urol., 101:347, 1969. 5. Heller, C.G., Heller, G.V. and Rowley, M.J.: human spermatogenesis: An estimate of the duration of each cell association and of each cell type, III International Congress of Endocrinology, June 30-July 5, 1968, Mexico City, Excerpta Medica Foundation, International Congress Series, 184:1012, 1969. 6. Heller, C.G., Teshima, F. and Rowley, M.J.: Duration of transport of spermatozoa through the human male ductular system, Fertil. Steril., 21:390, 1970. 7. Heller, C.G., Lalli, M.F., Pearson, J.E. and Leach, D.R.: A method for the quanitification of Leydig cells in man. J. Reprod. Fertil., 25:177, 1971. 8. Rowley, M.J. and Heller, C.G.: The ultrastructure of four types of human spermatogonia, Z. Zellforsch., 112:139, 1971. 9. Rowley, M.J. and Heller, C.G.: Quantitation of the cells of the seminiferous epithelium of the human testis employing the Sertoli cell as a constant, Z. Zellforsch., 115, 461, 1971. 10. Wootton, P. and Heller, C.G.: The effects of graded doses of ionizing radiation on the testicular function of man. I. A portable device for delivery of uniform doses of x-ray irradiation throughout externalized organs, (in preparation). 43 11. Heller, C.G. and Clermont, Y.: Kinetics of the germinal epithelium in man, Rec. Prog. Hormone Res., 20:545, 1964. 12. Fawcett, D.W.: Changes in the fine structure of the cytoplasmic organelles during differentiation, In: Developmental Cytology, Chap. 8, D. Rudnick (ed.), Ronald Press Co., New York, New York, pp. 161-189, 1959. 13. Andre, J.: Contribution a la connaissance du chondriome, J. Ultrastruc. Res., Supple. 3, 185p, 1962. 14. Clermont, Y.: The cycle of the seminiferous epithelium in man, Amer. J. Anat., 1:35, 1966. 15. Clermont, Y.: Spermatogenesis in man, Fertil. Steril., 17:705, 1966. 16. Rowley, M.J. and Heller, C.G.: An analysis of the effect of one or more testicular biopsies upon sperm count, Proc. Northwest Soc. Clin. Med., Portland, Oregon, 1965. 17. Heller, C.G., Wootton, P., Rowley, M.J., Lalli, M.F. and Brusca, D.R.: Action of radiation upon human spermatogenesis, Excerpta Medica Foundation, International Congress Series, #112, pp. 408-410, 1966. 18. MacLeod, J. and Gold, R.Z.: The kinetics of human spermatogenesis as revealed by changes in the ejaculate, Ann. N.Y. Acad. Sci., 55:707, 1952. 19. Roosen-Runge, E.C.: The process of spermatogenesis in mammals, Biol. Rev., 37:343, 1962. 20. Heller, G.V., O'Keefe, K.B. and Heller, C.G.: Effects of follicle-stimulating hormone (FSH) on Sertoli cells in the hypophysectomized rat, Clin. Res., 16:113, 1968 (abstract). 44 21. Oakberg, E.F.: Initial depletion and subsequent recovery of spermatogonia of the mouse after 20r of gamma rays and 100, 300 and 600r of x-rays, Rad. Res., 11:700, 1959. 22. Clermont, Y. and Morgentaler, H.: Quantitative study of spermatogenesis in the hypophysectomized rat, Endocrinology, 57:369, 1955. 23. Heller, C.G. and Leach, D.R.: Quantification of Leydig cells and measurement of Leydig cell-size following administration of human chorionic gonadotrophin to normal men, J. Reprod. Fertil, 25:185, 1971. 24. Maddock, W.O. and Nelson, W.O.: The effects of chorionic gonadotropin in adult men: increased estrogen and 17- ketosteroid excretion, gynecomastia, Leydig cell stimulation and eminiferous tubule damage, J. Clin. Endocrinol., 12:985, 1952. 25. McRoberts, J.W., Olson, A.D. and Herrmann, W.L.: The determination of urinary testosterone in men, women and children, Clin. Chem., 14:565, 1968. 26. Albert, A.: Procedure for routine clinical determination of urinary gonadotropin, Proc. Staff Meet. Mayo Clin., 30:522, 1955. 27. Greep, R.O., Van Dyke, H.B. and Chow, B.F.: Use of anterior lobe of prostatic gland in assay of metakentrin, Proc. Soc. Exptl. Biol., Med., 46:644, 1941. 28. Thorslund, T. and Paulsen, C.A.: A computer program for the analysis of data rom "Parallel-line" biological assays, Endocrinology, 72:663, 1963. 29. Greenwood, F.C. and Hunter, W.M.: The preparation of I131 labeled human growth hormone of high specific radioactivity, Biochem. J., 89:114, 1963. 30. Odell, W.D., Ross, G.T. and Rayford, P.L.: Radioimmunoassay for luteinizing hormone in human plasma or serum:physiological studies, J. Clin. Invest., 46:248, 1967. 45 31. Odell, W.D., Parlow, A.F., Cargille, C.M. and Ross, G.T.: Radioimmunoassay for human follicle-stimulating hormone: psychological studies, J. Clin. Invest., 47:2551, 1968. 32. Nankin, H.R., Yanaihara, T. and Troen, P.: Response of gonadotropins and testosterone to clomiphene stimulation in a pubertal boy, J. Clin. Endocr. Metab., 33:360, 1971. 33. Paulsen, C.A.: In: Advance in Experimental Medicine and Biology, vol. 10, The Human Testis, E. Rosemberg and C.A. Paulsen (eds.), Plenum Press, 1970. 34. Murphy, B.E.P.: Methodological problems in competitive protein-binding techniques; the use of Sephadex column chromatography to separate steroids, Acta Endocr. Suppl., 147:37, 1970. 35. Mayes, D.M. and Nugent, C.A.: Determination of plasma testosterone by the use of competitive protein binding, J. Clin. Endocr. Metab., 28:1169, 1968. 36. Rivarola, M.A. and Migeon, C.J.: Determination of testosterone and androst-4-ene-3, 17-dione concentration in human plasma, Steroids, 7:103, 1966. 37. Bardin, C.W. and Lipsett, M.B.: Estimation of testosterone and androstenedione in human peripheral plasma, Steroids, 9:71, 1967. 38. Gandy, H.M. and Peterson, R.E.: Measurement of testosterone and 17-ketosteroids in plasma by the double isotope dilution derivative technique, J. Clin. Endocr. Metab., 28:949, 1968. 39. Frick, J. and Kinel, F.A.: The measurement of plasma testosterone by competitive protein-binding assay. Steroids, 13:495, 1969. 40. Maeda, R., Okamoto, M., Wegienka, L.C. and Foesham, P.H.: A clinically useful method for plasma testosterone determination, Steroids, 13:83, 1969. 46 41. Rosenfield, r.L., Eberlein, W.R. and Bongiovanni, A.M.: Measurement of plasma testosterone by means of competitive protein binding analysis, J. Clin. Endocr. Metab., 29:854, 1969. 42. Furuyama, S., Mayes, D.M. and Nugent, C.A.: A radioimmunoassay for plasma testosterone, Steroids, 16:415, 1970. 43. Heller, C.G., Paulsen, C.A., Mortimore, G.E., Jungok, E.C. and Nelson, W.O.: Urinary gonadotropins, spermatogenic activity, and classification of testicular morphology - their bearing on the utilization hypothesis, Ann. N.Y. Acad. Sci., 55:685, 1952. 47