Human Testicular Irradiation (Partial)

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FIG.1. Section through irradintion unit. logical protection. Exposure doses, uniform to within -5% of the stated average n-ere to be dclivcrcd at. thc rate of approxilnately 100 R/min. The equipment providcd (Figs. 1 and 2 ) consisted of two air-cooled industrid up t o 5 mX tube current, having an inhercnt filtration of 0.14 in. glass plus 0.12 in. 31g, equivnlent t o 2 nini -11. Tlieso units were mounted in a lead-lincd wcrc iiicideiit on a block of ti.<sue ecluivalcnt mntcrid 20 X 30 X G c n l tluck, placccl niido.:ljr bcL\vcen t h e tulwj. Iiiscrtcd int.0 this block of mntcrial in the middle of the uppcr edge w:is n trnnspnrcnt 10 X 15 X G cni vcssel of 0.125 in. I n use the Plesiglass veiscl \vas filled lvitll \varl>~(03-9-l"F) xntcr which served two purposes: (a) physiological, to obtain dcscent of t h e testcs into tlic vessel, and (b) physical, niaking the testcs, water vcsscl and surroundilig the iiii it

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DETER MI NED TIG. 3. Spectrnl distribution of the radiation N determined from absorption curve in copper iiiatcrial one tissue cquiv:ilcnt nia.is, tlirrcby making tlic dusc distributions in the testes indcpendciit of tlic natural variations in the geoinctry of the d ) j e c t . Cliaractcri$tics of the X-ray licnds were dctcnnincd Isforc asselubly into die unit u h g R T’ictorcen “R” iiictcr niodcl 70 bcariiig calibrntioiis :igaiilst a irec air cliaiiil~cr,nnd a secondary standard ccrtificd b y the S ation il Bureau of Standards. TJie>. wcrc found to deliver dosc ratcs t h a t \\we witliiii 155 of one aiiotlier ivlicn opcrutcd under tlic sninc conditions. Sarrow’bemil nl~horptioii studies sho\vctl thcni to have first I-XVLS of 5 nim of clluii~iniin~or 0.25 nini copper, and sccond HVls of 7.95 iim aluiniiium or 0.43 m m of copper. The pcak kiiovohagc as dctcrinincd from the absorption curvc i n coppcr coinbined n-ith data from )[organ (6) mas found to be 13.5 kVp. The spcctral dist rih tion as dct ermiiicd fruni the absorption curves in aluminurn and copper analyzed by the inethod of Greening (7) is shown in Fig. 3. T h e X-ray heads x c r e thcn asseiiiI)lcd into tlic unit and tlic dosc distrilxition previously described (8). The chnmbcr built for tlijs project 1x1s wave length indepcndciit a t the energies of interest and calibratcd by comparison with t h e Victnrem “R” nictcr. The irradintinii of a large voluinc compared u k h thc volunic of intcrcst reduced t h e “wasting cffcct” usually observed in lllrdi3 irrndintctl by parallel opposing firlds. The total size of the irradintcd volme, the point of incidence ol the central axis of the X-my bcums and tlic focus t o subject distance were manipulnted to obtain uniformity of cxposurc .dose througliout the volume of the Plesiglnss vcsscl, t o witliiii 1.7 cnj ol tlic uppermost surfnce of the vessel. Grentcr ~ ~ i f o n i i i t yinay bc achieved by tlic IISC of \vctlgc filters if nccdcd. In use, howcvcr, irradi:ition was only performed

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FIG.4. Co:nparisun of Inowe spermatogonial survival with dnta from type .4 and inlerniedinte spernintognriin pooled. Surviving fractions niesured as nn experimental: rontrul ratio. The mdincradlcs which were now locked in positioli and the heads co\~idbe rcturnetl in one location onIy, if \vitIidra\~nfor servicing. The box housing t h e X-ray tubcs n’as coinpletely liiicd with 1/16 in. lead rcinforced nt strategic points as dctermincd by direct radiation survey. T h e exposure dose rate a t gonadal height, 8 ft from the unit, was reduced t o 10 mR/hr at maximum operation..Thc control panel was a t the end of 20 ft of cable, and therefore this lcvcl of protection \vas considered -adequate in this context. The midbody esposurc dose ratc to the subject \vas of the ordcr of 20 mR/hr also. This was achicvcd by hcaiicr protection of the top and-imier faces of t h e unit. 3Iensureriieiits were made at a point in the middle of t h e vessel in a place 5 cm bclow the upper surfacc (a) in air, and (b) in water. From this data all dose rates throughout tlic cup could be rclnted t o a siniple “in air” calibration carried out a t the sitc of the experiment. I n use, t h e X-r3y units \\-ere operated until-.thcrnial equilibrium was reached. T h e Yictorcen chamber was used t o mcnsllrc the output of each tubc hcad separntely t o check performance. Calibration of both units operating togcthcr was then lnade in t h e place, RS dcscribed bclow. From this the avcragc dose rate in the water-fillcd cup and the time for dclivery of t h e desired dose could be computed. The timcr \\-as of the Liebcl Florschcinl typc ; if thc esposure times wcrc Tess than 1 min the tube currents were redurcd nnd t h c units recalibrated to minimize tinling crrors.

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I n application, t.he cup was then filled with water at scrotal temperature and t h e subject laid prone with testicles suspended in the mater. T h e level of the testicles i n t h e cup was viewed in the mirrors through the view ports. When t h e testicles were below the 1.7 cm level thc mirrors were swung out of the X-ray beam path and the prc-determined dose delivered. The unit was recalibrated a t the termination of the esperiment. Our records show less than 1% change in output during a run. One independent check of the X-ray calibrations was made using R-meters by a physicist of Battelle Sortli\vcst Laboratories. He confirined t h a t “Our independent, calibrations agreed with those of tlic espcrinienters within t h e between our results was about 3YC.” .I second indcpcndent check was macle Laboratory. A special mouse-holding clinmbcr t h a t would fit into the water bos of the X-ray machine was constructcd. Dr. Oakbcrg’s genctically pure strain of mice was used. 3Iicc were irradiated a t various dosage levcls, then autopsicd a t 73 hr alter X-ray. Tlic prcscrved testicular spccitnciis \vwc analyzcd a n d the results conip:ircd to rcsrrlts obtaincd using equipnwnt a t othcr instituSixty-scvcn sirl,jccts rcccivcd acritc tcsticulnr irradiation in doses batwecn 8 and GOO rad. Sis -ulijects n c r c irratliatcd a sccoiid timc, and one subject ;L third time, folhriiT& ccmplctc rccovcry from the initial dosage. Of thcse, thrce receivcd the identical dosage on each of the two occ:isions, three received two different dosagcs, and onc reccivctl thrce diffcrcnt dosngcs. One subject WLS ’given weekly irratlistioiis or 5 rntI,’wk for 1 1 wIi. Each dosage givcii is listed Seleclion of Subjecls The G i subjects for this study ivcrc Iicnlthy noimmnl men‘ bc t wen the agcs . of 2.5 and 5’2 yr. Tlicir rcproductivc normalcy \vas determined prior to irradiation by phJ.sical rsamination, examination of tcsticulnr biopsy specimenr, sperm conccntration and sperm morphology. Lcvcls of urinary total gunadotropins, estrogen and/or plnsinn LH, FSII, and testostcronc werc also di.termined. ‘The men n-ere rolunteen who were farniliaiired with the project and its goals over 1 1 1 extended pre-irr.diation otservntion period to ennble the investigators to ensure informed consent. \Vhere applicnble, the participant’s K i l e ”9s also required to give written consent. E&cmive review of this program was carried out nt 3-mo intervals by an appro\-ed human miiseiit review board. Three additional reviews were provided by ad hoc committees whose meriibrs were selected nationally. Please contact the senior auhor for further information.

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Dose grouping Aditd Ir radio1 ion subjed for dufn analg;ris X - r a y doje dale we

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Dose grouping Adtral Irrodialwn Subjd j o r dafa onalyais .Y-roy dosc dale age dose of 2-13-6.5 to Superscript numbers 1-6 refer to the sis subjects who received more than one S-r:iy dose, e.g., both 25 rad doses mere received by the snnie subject. Additional routine tcsts such as blood cytcJlOg)-, urinalysis slid -various blood chemistry tests wcrc evaluated during t!ic p r c and postirradintioii period.

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FIG.5 . The rcsponse of unn s ry totcrl gonndotropin levels and sperm concentrstion to n single dose of 200 r : d of X-irradiation to the tests. Pod i77.arlia Lio t i Ecaliiafior t For each pararncter stu did cach subjcct send as his o ~ icontrol. Postirradiation chwr\-ations ivcre niadc on cadi paralncter in ent i old n bo ~ c .Scrinl ev duat c speriit and honnonnl cvnluntions (9). Aftcr 19 irradintio~~s,hiop-iics were avoided only during tlic first OO-day ccll dcplction pcriod (9) (suhject receiving 3 rad/\& tabulated LIS onc irradiation only). All hiopsics were prepared for light niicroscopy (10) and, in the latcr yean of the study, for clectron I microscopy (11). Germinal cell and Lcgdig ccll qunrltitation methods wcre d e velopecl (3, 11).and utilizcd on material fised for light microscopy. Sperm counts were performed using t h e h~emocytometcrmctllod on samples submitted \vcckly by each voluntcer. Spernl morpllology (13) \vas esanlined b y Dr. John IIacLeod of Coriicll Xcdicnl School. During the first \cars of this study urinary steroid and hormone cmluations were uscd exclusively. As plaslna methods bccanle nvailablc, urinary cvduations filter and k:ioliii adsorption method of Albcrt (14). Total gonadotropins \vcrc bioassayed by tlic nietliod of Lauson, Hcllcr and 'sevringhaus (15). Luteillizing hormone bioassay was conducted using ttic mnlc hypophysccto1nizcd rat nwthod were hioassnycd using thc method of Lailson, IIcllcr, Goldc~land Scvringllaus (IS). Urinary testosterone and cpitcstosteronc levels wcre deterlnincd usiug glc were phased out. Thc urinary gonadotropills wcre concentrated usin, thc SpcCCl-

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FIG.6. The response of plasma follicle-stimulating hormone, phsin3 luteinizing hormone and sperm conccntration to a single dose of GOO r : d of X-irradiation to the testes. Plasina tcstozteroiic 11-3s determincd using the method of Nurpliy (21). of niet.hods by Odcll el a l . (22, 23). -I Uriiiary Con a do hp irl s(Fig.5) ~irinarytotal gonadotropin valucs wcrc not dctcrmined), a distinct rise in urinary total gonadotropins \v3s notccl. The rise occurred concomitantly n i t h the first denuding of the gerniinal epithclium. Thc risc \\-as sust:tincd uiiril histologicnl rccovcrg bcgan. Suhscqucnt lo~wringof total gonadot.ropin levels to normal, parallclcd thc repopuhtion of the seminiferous tubulcs. Urinary L1-I lcvcls \vert not chniigcd folloving irradiation esposurc at any tinic during the course of depletion, quiescent pcriod, or during rccovcry. Urinary FSH levels parallclcd the rise and fall of total gonadotropins. The four subjects receiving S rad revealcd no change in FSH. Measuring plasma l3I-I lcvcls confirmed the rcsults obtained froin urinary assays. S o change \\-as found follou-ing S rad, a slight increase was eccn a t 20 rad and highly significant incrrascs were found between 73 and GOO rad. contrasted sharply with valucs ohtaiiicd from urinary assays. Only t\\'o dosages

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Spermatogonia Spertnaf ocyf e l Sperm&& All biopsies were quantitated by one individual I J ~the tubul3r method (3). Spermntognnin increase wns after GOO rad where plasn~nLIT levels were double the prcirra Crinary estrogen 1cveIs were not changed foIIo\ving irradiation. Urinary testosterone levels revealed a minimal but statistically significant lou-enng follo\ving irradiation. Plasma testosterone d etc rd nation sho\rcd no statistical Spermatogonia were found to be t h c moat radiosen4tivc cell type with both S rad where no biopsies were taken). Spcrnratocytcs were overtly damaged a t doses of 300-300 rad as shown by their inability t o complete ninturntion division which caused a decrease in resulting sperriiat id numbers. Spcrmntocytcs were

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Dose Time of beginning Firsf a p m in (rad) hi do logi d recovery seminal fluid or in apenn numbers Histological recovery initiation cnlculited by fint increue in germind cell numbers. Recovery initktion by sperm number increse was determined in ejsculated seminnl fluid. howevcr, after 400-GOO rad, the resultant spermatozoa were significantly decreased in number signifying covert spermatid damage. Following testicular depletion through ccllular inaturntion (and the failure of tlfe spermatogonia to supply replacement cells) which takes 4G days, a n additional 21 Jays nxs required (a total of 67 da\-s follouing irradiation) for t h e sperm conccntr:ition to drop t o azoospermia at all dose levels above 7s rad (.Fjg. 5 ) . Dosca of 78 and 50 rad produced ~ n s r k c doligospermia decreasing sperm concentration from normal to nbout 2 In/cc. Lower doses produced moderate oliguspermia (lowering counts to above 10 m/cc). At 7s rad total azoospermia occurrcd in all subjects but one. At the highcr' doses (400-600 rad) sperm concelrtration fell precipitously prior to the 4G day period bccausc of the damage to spermatids (Fig. 6 ) . -It the intermediate dose lcvcls (200-300 rad) the sperm coriceiitratioll did not ;how a decrease prior to the 43 day postirradintion period even though sorile spermatocytcs faiicd to dcvclop. Tlic mising of geiierations of spermatozoa in the ductulnr systczn and the natural high w e k l y variation in sperni concentration may have obscured the diminution. Preliminary results from subjects a t the 600 rad dose level reveal a n incrcose i n Lcydig cell nuiiibers (counts \\-ere corrected for tissue shrinkage by usiug Leydig cell numbers occurred 90 days after irradintion and remained elevated until the time a t which the gernlinal cpithclium began to recover. These results \\'ere obtained from four subjccts receiving GOO rad and were uot dctcrnlincd on subjects rcceiving the lower doses. The hegjnnirig of recovery s shown by an increme iii gern~inalcell numbers precedes the show of sperm or the increase in spcrm i!umbers in the cjnculates of 300 rad the i n c r c s e in germinal cclls may precede the observation of cjaculated sperni by 4 mo, a t GOO rad histological recovery may be init.iated 17 mo before sperm nrc found.

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Coinplbtc recovery as shown by a return to prc ir rd in tion spcrni concciitratioiis nnd gclniinnl ccll nurnbcrs is within 9-1s mo for doses of 100 fad and bclniv, 30 ;]io for doscs of 200 and 300 rad, and 5 or more yenrs for doses of 400 and GOO rnd (only one subject follo\r-ccl to coniplcte recovery a t thc high I n thc scven subjects t1i;it were irradiated R second or third time folloiving coniplctc rccovcrv, the response to tlic rcpcntcd dose was in every i v a ~ rconiparable to the initial-dose response. The first problem associated ivitli this project uxi to delivcr as uiiiforiii a n aniouiit of radiation as possible to all depths of tcsticular tissue of both testcs coiiventioiial X-ray thcrnpj. units on tlic iiiarkct (iii 1963) as unsuitnhlc bccnusc of lael; of mcans of uniform c o v c ~ i g cof thc testes, lack of assurmcc that uniformity from suhjcct to subject could l,c cstahlishcd and lack of (or awkir-aril) shielding protection to tlic subject's body. Therefore, a siiiiplc port:ihlc 110s was clcsigncd tlmt allon.ed tlic scrotuiii arid testcs to drop into 3 plnstic l m fillctl with ivi1tc.r a t scrotal teiupcraturc arid …